Methods for treating cancer patients with homologous recombination deficiency based on pathognomonic long molecule footprints of backup repair pathways

Abstract

The present disclosure provides methods for determining whether a cancer patient with homologous recombination deficiency will benefit from treatment with PARP inhibitors or platinum agents. These methods are based on screening a cancer patient for the presence of reciprocal structural variants (SVs) that comprise pairs of distant intra- or inter-chromosomal loci that contain exchanged genomic material.

Description

Atty. Dkt. No.115872-3014 METHODS FOR TREATING CANCER PATIENTS WITH HOMOLOGOUS RECOMBINATION DEFICIENCY BASED ON PATHOGNOMONIC LONG MOLECULE FOOTPRINTS OF BACKUP REPAIR PATHWAYS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 508,449, filed June 15, 2023, the contents of which are incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present technology relates to methods for determining whether a cancer patient with homologous recombination deficiency (HRD) will benefit from treatment with PARP inhibitors or platinum agents. These methods are based on screening a cancer patient for the presence of reciprocal structural variants (SVs) that comprise pairs of distant intra- or inter-chromosomal loci that contain exchanged genomic material. STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA247749 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Cancer cells defective in homologous recombination (HR) are responsive to DNA crosslinking chemotherapies, PARP inhibitors, and inhibitors of polymerase theta, a key mediator of the backup pathway alternative end-joining. Such cancers include those with pathogenic bi-allelic alterations in core HR genes and another cohort of cases that exhibit sensitivity to the same agents and harbor genomic hallmarks of HR deficiency (HRD). In advanced epithelial ovarian cancers, the standard of care currently consists of optimal cytoreductive surgery followed by platinum-based chemotherapy (www.nccn.org / professionals / physician_gls / pdf / ovarian.pdf). However, nearly all stage III and all stage IV cancers recur, and thus overall survival following initial surgery and platinum-based chemotherapy is considered highly determined by inherent platinum 1 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 sensitivity (Markman, M. et al., J Clin Oncol 22, 3120-3125 (2004); Bouberhan, S. et al., J Clin Oncol 37, 2424-2436 (2019)). Accordingly, there is an urgent need for developing reliable and accurate methods for predicting whether a HRD cancer patient will benefit from treatment with PARP inhibitors or platinum agents. SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides a method for selecting a cancer patient for treatment with a PARP inhibitor or a platinum agent comprising: (a) detecting levels of reciprocal rearrangements in a biological sample obtained from the cancer patient that are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of a PARP inhibitor or a platinum agent. In another aspect, the present disclosure provides a method for prolonging survival of a cancer patient comprising administering to the cancer patient an effective amount of a PARP inhibitor or a platinum agent, wherein levels of reciprocal rearrangements in a biological sample obtained from the cancer patient are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold. In some embodiments of the methods disclosed herein, the levels of reciprocal rearrangements are detected via next-generation whole-genome sequencing.

[0007] In any of the preceding embodiments of the methods disclosed herein, one or more of the reciprocal rearrangements comprise an orientation that is positive (e.g., reciprocal duplication (rDup)), negative (e.g., reciprocal deletion (rDel)), or mixed (e.g., reciprocal deletion-duplication (rDelDup)).

[0008] In certain embodiments, one or more of the reciprocal rearrangements comprise a negative orientation (rDel), and / or mixed orientation (rDelDup) and the cancer patient harbors a BRCA2 mutation. Additionally or alternatively, in some embodiments, the rDel is a trans interchromosomal reciprocal deletion or a trans intrachromosomal reciprocal deletion, and / or the rDelDup is a cis interchromosomal reciprocal deletion-duplication, a cis intrachromosomal reciprocal deletion-duplication, a trans interchromosomal reciprocal deletion-duplication, or a trans intrachromosomal reciprocal deletion-duplication.

[0009] In other embodiments, one or more of the reciprocal rearrangements comprise a positive orientation (rDup), and / or mixed orientation (e.g., rDelDup) and the cancer patient harbors a BRCA1 mutation. Additionally or alternatively, in some embodiments, the rDelDup is a cis interchromosomal reciprocal deletion-duplication, a cis intrachromosomal reciprocal deletion-duplication, a trans interchromosomal reciprocal deletion-duplication, or 2 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 a trans intrachromosomal reciprocal deletion-duplication and / or the rDup is a cis interchromosomal reciprocal duplication, a cis intrachromosomal reciprocal duplication, a trans interchromosomal reciprocal duplication, or a trans intrachromosomal reciprocal duplication.

[0010] Additionally or alternatively, in some embodiments, the cancer patient is diagnosed with, or is at risk of having a homologous recombination deficiency (HRD). In certain embodiments, the HRD comprises mutations that result in the inactivation of at least one of the following genes: PALP2 / FANCN, BRIP1 / FANCJ, BARD1, RAD51, RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), FANCA, FANCB, FANCC, FANCD2, FANCE, FANCG, FANCI, FANCL, FANCM, FAN1, SLX4 / FANCP and ERCC1. In any and all embodiments of the methods disclosed herein, the cancer patient harbors a mutation in one or more driver mutations selected from among BRCA2, BRCA1, PALB2, RAD51C, RAD51B, IDH1, CDK4, RB1, FIGNL1, MDC1, BARD1, and RBBP8. Additionally or alternatively, in some embodiments, the cancer patient is suffering from a cancer selected from among breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. In certain embodiments, the cancer patient exhibits stage I, stage II, stage III or stage IV cancer. Additionally or alternatively, in some embodiments, the cancer patient has not received at least one prior line of anti-cancer therapy. In other embodiments, the cancer patient has received at least one prior line of anti-cancer therapy. Examples of anti-cancer therapy include chemotherapy, radiation therapy, surgery or any combination thereof.

[0011] In any and all embodiments of the methods disclosed herein, the PARP inhibitor is a PARP-specific inhibitory nucleic acid (e.g., sgRNAs, antisense RNAs, ribozymes, or shRNAs), iniparib, olaparib, niraparib, rucaparib, talazoparib, veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, 3-aminobenzamide, or an anti-PARP neutralizing antibody.

[0012] In any and all embodiments of the methods disclosed herein, the platinum agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, pyriplatin, picoplatin, or satraplatin.

[0013] Additionally or alternatively, in some embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.

[0014] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise determining an additional HRD signature in the cancer patient. 3 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 In some embodiments, the additional HRD signature comprises one or more of loss of heterozygosity (LOH), large scale state transitions (LST), and telomeric imbalance (tAI). In certain embodiments, the additional HRD signature comprises one or more of single base substitution signature 3 (SBS3), single base substitution signature 8 (SBS8), short tandem duplications (RefSig R3), short deletions (RefSig R5), microhomology-mediated indels, and HRD score. In other embodiments, the additional HRD signature comprises ID8 signature, and ID6 signature.

[0015] In another aspect, the present disclosure provides a method for discriminating between BRCA1-deficient and BRCA2-deficient tumors in cancer patients comprising: (a) detecting a plurality of structural variant (SV) features in one or more tumor samples obtained from a plurality of cancer patients, wherein the plurality of SV features comprise homeologous deletions, positive reciprocal rearrangements (rDups), negative reciprocal rearrangements (rDels), and mixed reciprocal rearrangements (rDelDups); (b) for each of the one or more tumor samples, (i) identifying tumor samples having elevated levels of negative orientation reciprocal rearrangements (rDels) and / or homeologous deletions relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and / or (ii) identifying tumor samples having elevated levels of positive orientation reciprocal rearrangements (rDups) relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and (c) classifying the tumor samples of (b)(i) as BRCA2-deficient tumors and the tumor samples of (b)(ii) as BRCA1- deficient tumors. The SV features in the one or more tumor samples may be detected using whole genome sequencing. In some embodiments, the BRCA2-deficient or BRCA1- deficient tumor samples further comprise elevated levels of mixed orientation reciprocal rearrangements (rDelDups) relative to the control biological sample obtained from a healthy subject or a predetermined threshold.

[0016] Additionally or alternatively, in some embodiments, the size of the homeologous deletions ranges from about 1 kbp to about 100 kbp. The size of the homeologous deletions may be about 1 kbp, about 2 kbps, about 3 kbps, about 4 kbps, about 5 kbps, about 6 kbps, about 7 kbps, about 8 kbps, about 9 kbps, about 10 kbps, about 15 kbps, about 20 kbps, about 25 kbps, about 30 kbps, about 35 kbps, about 40 kbps, about 45 kbps, about 50 kbps, about 55 kbps, about 60 kbps, about 65 kbps, about 70 kbps, about 75 kbps, about 80 kbps, about 85 kbps, about 90 kbps, about 95 kbps, or about 100 kbps. 4 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0017] In any of the preceding embodiments of the method disclosed herein, the plurality of SV features further comprise one or more of 1-100 Kbp simple duplications, rearrangement signature 3 (RS3), rearrangement signature (RS5), deletions with microhomology (mhDel), COSMIC single base signature 3 (SBS3), COSMIC single base signature 8 (SBS8), rearrangement signatures 3 and 5, RS3 and RS5, and loss of heterozygosity (LOH). In certain embodiments, the BRCA1-deficient tumor samples further comprise elevated levels of 1-100 Kbps simple duplications relative to the control biological sample obtained from a healthy subject or a predetermined threshold. The size of the simple duplications may be about 1 kbp, about 2 kbps, about 3 kbps, about 4 kbps, about 5 kbps, about 6 kbps, about 7 kbps, about 8 kbps, about 9 kbps, about 10 kbps, about 15 kbps, about 20 kbps, about 25 kbps, about 30 kbps, about 35 kbps, about 40 kbps, about 45 kbps, about 50 kbps, about 55 kbps, about 60 kbps, about 65 kbps, about 70 kbps, about 75 kbps, about 80 kbps, about 85 kbps, about 90 kbps, about 95 kbps, or about 100 kbps.

[0018] Additionally or alternatively, in some embodiments, the cancer patients are suffering from a cancer selected from among breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. In certain embodiments, the cancer patients exhibit stage I, stage II, stage III or stage IV cancer.

[0019] Additionally or alternatively, in some embodiments, the method further comprises identifying the cancer patients from which the BRCA2-deficient tumor samples were obtained and administering to the cancer patients an effective amount of chemotherapy or immunotherapy. In other embodiments, the method further comprises identifying the cancer patients from which the BRCA1-deficient tumor samples were obtained and administering to the cancer patients an effective amount of chemotherapy. In some embodiments, the chemotherapy comprises a PARP inhibitor or a platinum agent. Examples of PARP inhibitors include, but are not limited to, iniparib, olaparib, niraparib, rucaparib, talazoparib, veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, or 3- aminobenzamide. Examples of platinum agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, pyriplatin, picoplatin, or satraplatin.

[0020] Examples of immunotherapy include immune checkpoint blockade agents such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, ipilimumab, cemiplimab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab. 5 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGs.1A-1F: Reciprocal pairs are enriched in BRCA1d and BRCA2d tumors. FIG.1A: Schematic of exact and approximate reciprocality, using translocations as an example. Exactly reciprocal junctions link breakends that adjoin the same breakpoint (schematic on the right) giving rise to a classic reciprocal translocation (or inversion, if intrachromosomal) which is associated with no copy change. Approximately reciprocal junctions are associated with a gap segment (dark blue), undergoes a copy loss (middle left) or gain (right examples) based on the orientation of junctions around it. The gap segment polarity refers to whether the adjoining junctions connect to the gap segment (+ polarity, right two examples) or its adjacent breakends (- polarity, left middle example) and determines whether there is a copy gain (+) or loss (- polarity) of the gap segment. Both (-) and (+) gap segments can give rise to translocations that are balanced; however, (+) gap segments are equally consistent with a templated insertion (right). FIG.1B: Gap segment lengths and polarities of three canonical reciprocal pair patterns (schematic on the right) plotted across BRCA1-deficient (BRCA1d), BRCA2-deficient (BRCA2d), or HR-proficient (HRP) cases (density plots on the left). Density is calculated as a Gaussian kernel normalized by the number of BRCA1d (n = 9), BRCA2d (n = 23), or HR-proficient (HRP, n = 251) cases in each plot. FIGs.1C-1E: Examples (top) of rDups, rDels, and rDelDups with violin plots showing their relative burdens across 15 BRCA1d, 13 BRCA2d or 236 HR-proficient (HRP) samples, which are independent from data in panel b. P values obtained by Wald test on a gamma-Poisson regression model. FIG.1F: Distribution of junction spans associated with different classes of reciprocal pair SVs. Note that junction span is distinct from gap segment length; the former refers to the genomic distance between the two breakends belonging to a junction, while the latter refers to the distance between reciprocal breakends belonging to distinct junctions.

[0022] FIGs.2A-2D: LR WGS reveals cis and trans phases for similar reciprocal pair topologies. FIG.2A: Multiple phased allelic reconstructions are consistent with the reciprocal duplication (rDup) SV pattern observed in short-read WGS. Each derivative allele is represented as a “walk” or oriented sequence of reference genomic segments. A phased reconstruction comprises a set of derivative alleles that together account for junction and segmental copy numbers observed in the short-read WGS genome graph. For an rDup, two of the three possible reconstructions (cis 1 and 2) place junctions adjacently, corresponding to the templated insertion of a distant segment between duplicated copies of 6 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 a gap segment at the source locus. In the third case (trans) the junctions are located on discontiguous or distant alleles consistent with a large translocation or inversion, respectively, where each derivative allele harbors a copy of both gap segments. Each reconstruction has a distinct LR WGS footprint, as visualized by a heatmap whose pixels represent long molecule barcode sharing between rearranged loci (bottom right schematic, also applicable to panels FIGs.2B-2C). FIGs.2B-2C: These panels show two rDups each phased by LR WGS in (FIG.2B) cis and (FIG.2C) in trans. FIG.2D: Counts of 10X- phased reciprocal duplications (rDups), deletions (rDels) and deletion-duplications (rDelDup) from either BRCA1-deficient (BRCA1d, n = 22) or BRCA2-deficient (BRCA2d, n = 14) tumors. Reciprocal pairs are colored by their junction span (either 1-10 Mbp, >10 Mbp, or interchromosomal), see FIG.1F caption for explanation.

[0023] FIGs.3A-3G: Aberrant replication restart model links trans reciprocal pairs to megabase-scale chromosomal alterations. FIG.3A: Derivative haplotypes (middle) for cis and trans rDups in somatic chromosomal coordinates harmonized so that coordinate 0 corresponds to the location of the first junction in the walk. Coloring corresponds to the genome graph pattern (left). Density (right) shows distribution of gap segment lengths within cis- and trans-phased derivative chromosomes resulting from rDups. FIG.3B: Scatter plot of gap lengths for each rDup among BRCA1d (n = 46) and BRCA2d (n = 50) BOPP cases. The shorter and longer gap segments in each pair are plotted on the x and y axes respectively. rDups are phased by LR-WGS (as indicated in the legend). FIG.3C: Schematic illustrating two types of outcomes for inter- and intra-chromosomal trans reciprocal pairs giving rise to translocations and inversions that either maintain centromere dosage (Type I) or result in dicentric or acentric products (Type II). FIG.3D: Fraction of Type I and Type II orientations among observed (left, LR WGS) and imputed (right, short-read WGS) cis and trans reciprocal pairs. P values obtained by Fisher’s Exact Test. FIG.3E: Schematic illustrating potential LOH produced following interchromosomal and intrachromosomal reciprocal pair formation. FIG.3F: LOH length distributions plotted alongside gap segment-to-telomere and gap segment-to-gap segment lengths for inter- and intra-chromosomal reciprocal pairs, respectively, among BRCA1d (n = 46) and BRCA2d (n = 50) BOPP cases. FIG.3G: Proposed replication restart model linking tandem duplications, cis and trans rDups, and LOH. Locus 1 (ABC) undergoes replication fork collapse and may invade Locus 2 (DEF) giving rise to cis and trans rDups, where the latter 7 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 may lead to LOH (following mis-segregation). seDSB = single-ended double-strand break. Variations of this model for rDelDups and rDels are shown in FIG.11.

[0024] FIGs.4A-4F: LR WGS uncovers footprints of SSA in BRCA2-deficient genomes. FIG.4A: Example of homeology (inexact sequence homology) across a deletion junction in a BRCA2-deficient case detected by linked-read WGS sequencing. Heatmap (top track) shows reference sequence similarity in 20 bp bins flanking junction breakends. Each pixel in the heatmap indicates the sequence similarity between a pair of 20-mers (calculated as (1 - Levenshtein edit distance / 40) corresponding to two locations in the reference (top left legend). Homeologous bin pairs are those with >80% sequence similarity. The black line represents a continuous run of homeology, whose length is inferred through image analysis of the heatmap (see Methods). Bottom track shows barcoded LR WGS read alignments supporting the homeologous deletion junction, where each y axis position represents a distinct LR barcode. FIG.4B: Heatmap showing counts of bases with homeology (sequence similarity ≥ 0.8) across all detected LR and standard WGS junctions on a coordinate system defined around the location and orientation of each junction breakend. Pixels are colored according to the fraction of homeologous bases in the corresponding bin pair. FIG.4C: Length of homeology (see Methods) measured across all linked-read (top) and WGS (bottom) deletions. FIG.4D: Fraction of homeologous junctions among simple deletions >1 Kbp among BRCA1d, BRCA2d, and HR-proficient (HRP) tumors. P values obtained by gamma-Poisson regression. FIG.4E: Junction span (distance between breakends) of homeologous 10X LRS and WGS deletions in BRCA2d tumors. FIG.4F: Schematic of SSA mechanism, including dependence on long-range end resection and BRCA1 function. LR = linked read, WGS = whole genome sequencing, SSA = single stranded annealing.

[0025] FIGs.5A-5J: SV features distinguish between BRCA1- and BRCA2- deficiency. FIG.5A: Receiver operating characteristic curves (ROC) and precision recall curves (PRC) comparing SV features highlighted in this study with those used in HRDetect for accurately classifying HR-deficiency (either BRCA1d or BRCA2d). P values denote comparison of AUROC via DeLong test. FIGs.5B-5C: Feature importance of highlighted SV features in an independent set of BOPP and non-BOPP tumors. “Combo” refers to a combination of the B1+2 classifier-specific SV features highlighted in this study. FIG.5D: ROC curves assessing B1+2 and a random forest classifier using only the 6 HRDetect 8 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 features in predicting BRCA1d vs. BRCA2d status. FIGs.5E-5F: Feature importance for BRCA1d vs. BRCA2d classification in an independent set of BOPP and non-BOPP tumors. See FIG.17 and Methods for training and testing dataset summary. FIG.5G: B1 and B2 scores in the B1+2 classifier across all samples analyzed in this study including those excluded from training and testing for harboring VUS or mono-allelic variants. FIG.5H: Counts of cases that are B1- or B2-positive (score >0.5) for genes harboring a rare biallelic germline or somatic mutation in additional (i.e. not BRCA1 or BRCA2) HR pathway genes. FIG.5I: Frequency of HR-deficiency, as defined by B1 or B2 score >0.5, among common cancer types. FIG.5J: clinical vignette illustrating patient with BRCA2d neuroendocrine prostate cancer. Left, Classifier scores (top) and features (bottom) for example patient versus genotype-defined cohorts (BRCA1d, BRCA2d, HR-proficient, HRP). Top right, PSA and CEA response kinetics. Time points 1-4 correspond to axial computed tomography images depicted on bottomright, illustrating favorable response to platinum-based chemotherapy. LOH = burden of large genomic segments harboring loss of heterozygosity. ihDels = count of large deletions (>1 kbp) with homeology. mhDel proportion = proportion of small deletions (<50 bp) with microhomology. RS3, RS5 = proportion of junctions with rearrangement signature 3 or 52. SBS3, SBS8: COSMIC single base signature 3 and 847. PRAD = prostate adenocarcinoma, PACA = pancreas adenocarcinoma, OV = ovarian cancer, BRCA = breast adenocarcinoma. PSA = prostate specific antigen, CEA = carcinoembryonic antigen.

[0026] FIGs.6A-6D: Comparing the burden of SV classes across genotypes. FIGs. 6A-6C: Comparison of the burden (count) of (FIG.6A) simple SV classes, (FIG.6B) total junctions, and (FIG.6C) complex SV classes across BRCA1d (n = 9), BRCA2d (n = 23), and HR-proficient cases (HRP, n = 251). P values obtained by Wald test on a gamma- Poisson regression model. (BFB, breakage fusion bridge. DM, double minute. TIC, templated insertion chain). FIG.6D: Fraction of cases with at least one TIC event across BRCA1-deficient, BRCA2-deficient, and HR-proficient cases.

[0027] FIGs.7A-7E: Characteristics of reciprocal junction clusters. FIG.7A: Quantile-quantile plot of observed -log10P values obtained by evaluating 473,382 observed gap segment lengths from 283 tumor samples against an exponential null model (see Methods). X axis represents -log10transformed quantiles from the uniform distribution. Gap segment lengths with width less than 1 Mbp are shown in red. FIG.7B: Density plot 9 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 showing distribution of gap segment lengths with FDR<0.1 across the sample set, the vast majority of which are less than 1 Mbp. FIG.7C: Examples of 2, 3, and multi way (based on gap segment number) reciprocal SV topologies, including a spectrum of simple and complex SV classes. FIG.7D: Histogram of reciprocal SV cluster lengths across 283 tumor samples. FIG.7E: Violin plots comparing cyclic vs. linear and 2 way vs. higher order (3+ way) reciprocal SV topologies. P values obtained by Wald test on a gamma- Poisson regression model.

[0028] FIGs.8A-8E: Haplotype deconvolution of reciprocal pairs and LR WGS cohort validates reciprocal pairs in BRCA-mutated cancers. FIG.8A: Enumeration of all possible deconvolutions of each of the three reciprocal pair classes (rDups, rDelDups, and rDels). FIG.8B: Molecular and pathological characteristics of the 46 breast cancer cases profiled with LR WGS in this cohort. FIG.8C: Sequencing library characteristics across 92 LR WGS profiled samples. FIG.8D: Concordance between LR WGS and short- read WGS reciprocal pair calls. FIG.8E: Comparison of rDup and rDel counts among BRCA1d (n = 22) and BRCA2d (n = 14) cases within the LRS cohort. P values obtained by Wald test on a gamma-Poisson regression model.

[0029] FIGs.9A-9F: Phasing rDel and rDelDup with linked-read sequencing. FIG. 9A: Accuracy of phasing algorithm across simulated LR data sampled from cis or trans allelic configurations of observed rDel, rDelDup, and rDel loci. FIG.9B: Number of LR barcodes supporting cis vs trans configuration for each of the 186 reciprocal pair loci observed across 46 tumors profiled with LR WGS. FIGs.9C, 9E: Predicted linked-read profiles for rDelDup or rDel. Each plot shows a genome graph (track labeled "CN", copy number) and possible allelic walks in reference coordinates (track labeled "Supported walks") together with the barcode sharing heatmap (middle top) and resulting cis and / or trans haplotypes in the cancer genome. In each plot, the track labeled "Supported walks" represented enumerated paths in reference coordinates through the corresponding graph ("CN" track) with linked-read data supporting the barcode sharing heatmap above ("Linked- reads" track). Each heatmap shows the number of barcodes shared by pairs of bins within and between locus 1 and 2. The "Tumor haplotypes" track show the total phased linear alleles in somatic coordinates inferred based on the LRS data. FIGs.9D, 9F: Examples of cis and trans rDelDup or rDel in BRCA1- and BRCA2-deficient cases. CN, copy number. 10 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 ALT, rearranged haplotype. BX, linked-read barcodes. LRS, 10X linked-read whole genome sequencing.

[0030] FIGs.10A-10F: Links between reciprocal pairs, LOH, tandem duplications. FIG.10A: schematic defining the (+) gap segment of rDelDup reciprocal pairs (left). The (+) gap segment lengths of cis and trans phased haplotypes are shown in the center and right panels, respectively. FIG.10B: (+) gap segment length distribution of rDelDup reciprocal pairs identified as being in cis or trans by 10X linked-reads is shown as vertical lines. The multi-modal distribution of unphased rDelDup (+) gap segment lengths is shown as the black background line. FIG.10C: Width threshold for imputing phase of short-read WGS rDup and rDelDup events. ROC curve for sensitivity and specificity of cis / trans phasing using gap threshold (left panel). The red point shows the selected threshold (cis phase is imputed if the log10 width < 3.5). rDup (center panel) and rDelDup (right panel) gap width distributions with background color showing the width threshold used for phase imputation. FIG.10D: base pairs of microhomology at reciprocal pair junctions in BRCA1d and BRCA2d tumors. FIG.10E: rDup (x-axis) and tandem duplication (y-axis) count per sample across 46 BRCA1d samples. R2and P value obtained by Spearman rank correlation. FIG.10F: Violin plot showing length in log scale of the longer rDup segment, the (+) rDelDup segment, and length of tandem duplications in BRCA1d (red) and HR- proficient (HRP) samples (gray). P values obtained by Wilcoxon rank-sum test.

[0031] FIGs.11A-11B: Aberrant replication restart model of rDelDups and rDels. FIGs.11A-11B: Small extensions of the replication restart model for rDups (FIG.3G) can be used to explain rDelDups (panel FIG.11A) and rDels (panel FIG.11B) around a locus (ABC) that undergoes replication fork collapse and invades a second locus (DEF), resulting in distinct phased outcomes including trans configurations that can lead to LOH in the next cell cycle following mitotic segregation of sister chromatids. As with rDups, fork collapse leads to replication restart with template switch and potential half-crossover leading to cis (non-crossover) versus trans (crossover or half-crossover) repair products. Deletions arise as a result of replication restart downstream (rather than upstream) of the stalled and / or collapsed fork.

[0032] FIGs.12A-12B: Support for homeologous junctions. FIG.12A: Comparison of mapping quality for reads supporting homeologous and non-homeologous junctions. Left, density plot showing mapping quality for junction-supporting reads realigned to hg19, 11 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 from a subsample of 500 non-homeologous junctions and 500 homeologous junctions. Middle, barplot of the fraction of reads with high mapping quality (MQ=60). Error bars represent 95% confidence interval on the Bernoulli trial parameter calculated using the fraction of non-homeologous (73,871 / 95,934) or homeologous (16,954 / 31,078) junction supporting reads that had MQ of 60. Right, barplot of the fraction of reads stacked by intervals of mapping quality. FIG.12B: Left, reference 150-mer BWA mapping quality in the neighborhood of homeologous and non-homeologous breakends. Reference mapping quality determined by realigning sliding window of 150-mers from hg19 stepping by 1 base to hg19 and averaging across each base pair. Right, plot of alternate mappability scores calculated as the average of the reciprocal of the number of unique locations that each 150- mer overlapping a breakend-associated base pair aligns.

[0033] FIGs.13A-13E: Comparing B1+2 and HRDetect performance. FIG.13A: ROC and precision-recall curves comparing B1+2 and HRDetect performance on detecting HR-deficiency (BRCA1d or BRCA2d) in an independent (HMF) test cohort. P value denotes comparison of AUROC via DeLong test. FIGs.13B-13C: B1+2 feature importance in non-BOPP and BOPP for detecting HR-deficiency. See FIG.6 and Methods for training and testing dataset summary. FIG.13D: Scatterplot of B1+2 vs HRDetect scores across all 7918 tumors, including those excluded from training and testing. FIG. 13E: Scatter plot of scores from a random forest model trained only on SV features from the B1+2 (y axis) or HRdetect classifier (n = 7918).

[0034] FIGs.14A-14F: Distinguishing between BRCA1 and BRCA2 deficiency. FIG.14A: ROC (receiver operating characteristic) and PR (precision-recall) curves for discriminating BRCA1d from BRCA2d tumors by CHORD and by a random forest trained on CHORD features augmented with the new SV introduced in this manuscript. FIGs.14B- 14C: Feature importance for BRCA1 versus BRCA2 deficiency classification. FIG.14D: tumor type and pan-cancer prevalence of B1+2 positivity. The fraction of B1+2 positive (B1 + B2 score > 0.5) cases per tumor type is shown on the left; only tumor types with at least twenty examples are shown. Orange bar denotes the pan-cancer B1+2 positivity rate. FIG. 14E: The fraction of cases with B2>B1 out of the cases that were B1+2 positive; only tumor types with at least five B1+2 positive examples are shown. Tumor types significantly enriched for B2 positivity relative to the reference class (breast and ovarian cancer, highlighted in orange) are indicated with stars. Error bars show 95% confidence intervals 12 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 and stars indicate relative enrichment of B2 positivity within each tumor type. Significance levels: **** (P < 1E-4), *** (P < 1E-3), ** (P < 0.01), * (P < 0.05), obtained by Fisher’s Exact Test. FIG.14F: number of B1+2-positive (B1+ B2 score >0.5) cases with B1>B2 or B2>B1 and harboring biallelic or mono-allelic and pathogenic or VUS variants in HR- associated genes(see Results and Methods for more detailed definitions). LOH = burden of large genomic segments harboring loss of heterozygosity. HomeoDel = count of large deletions (>1 kbp) with homeology. del-MH = proportion of small deletions (<50 bp) with microhomology. RS3, RS5 = proportion of junctions with rearrangement signature 3 or 52. SBS3, SBS8: COSMIC single base signature 3 and 8.

[0035] FIGs.15A-15B: Clinical courses of pancreas cancers with elevated B1+2 scores. Panels on the left show clinical features, while panels on the right show B1+2 classifier features and B1+2, HRDetect, and CHORD classifier scores for each patient relative to BRCA1d, BRCA2d, and HRP cohorts. FIG.15A: metastatic pancreas adenocarcinoma case (WCM1341) with high B1 score (0.962), with CA19-9 response kinetics (left) and serial axial computed tomography (CT) (blue triangles and corresponding right panels) demonstrating excellent response to chemotherapy. FIG.15B: high risk stage IIb pancreas adenocarcinoma case (WCM1462) with high B2 score (0.31), with CA19-9 and CEA response kinetics (top) and axial CT (bottom).

[0036] FIG.16: Clinical characteristics of cases with elevated B1+2 scores.

[0037] FIG.17: Case datasets and analysis cohorts overview. Schematic illustrating cases included for analysis in the present manuscript. MSK, Memorial Sloan Kettering Cancer Center. PCAWG, Pancancer Analysis of Whole Genomes / International Cancer Genome Consortium. HMF, Hartwig Medical Foundation. Cohorts are mapped to key analyses in the study, denoted by main and extended data figure (EDF) panels. DETAILED DESCRIPTION

[0038] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0039] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure 13 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0040] It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0041] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology.

[0042] Cancer genomes provide a durable record of the genetic alterations that are acquired from DNA damage and DNA repair defects during normal cell development and carcinogenesis. Algorithmic whole genome sequencing (WGS) biomarkers of HR deficiency can help identify patients that respond to genotoxic therapy, including PARP 14 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 inhibition, even in the absence of canonical BRCA1 or BRCA2 mutations. Given the limitations of previous WGS approaches in assessing long-range DNA structure, it was hypothesized that as yet uncharacterized SV features may reveal the scars of specific backup repair pathways in HR deficient human cancers.

[0043] The present disclosure demonstrates that the presence of elevated reciprocal pairs in advanced cancers are predictive of patient responsiveness to HRD targeted therapies such as platinum agents or PARP inhibitors. Moreover, HR deficiencies (either BRCA1d or BRCA2d) in patient tumors were more accurately predicted using the structural variant (SV) features described herein (homeologous deletions, reciprocal pairs, 1-100 Kbp simple duplications) compared to the SV features used in conventional classifiers for predicting HR deficiency (e.g., HRDetect). See FIG.5A. Further, BRCA1 and BRCA2 deficiencies differentially affect responsiveness to checkpoint blockade immunotherapy, thereby impacting therapeutic decision making in cancer patients.37As demonstrated in the Examples herein, classifers including the SV features described herein substantially outperformed (AUC=0.92, AUPRC = 0.91) the HRDetect-like classifier (AUC=0.8, AUPRC = 0.83) in distinguishing BRCA1d from BRCA2d tumors (P = 0.005, DeLong test; FIG.5D), particularly in non-BOPP cancers (FIGs.5E-5F). Definitions

[0044] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0045] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0046] The term “adapter” refers to a short, chemically synthesized, nucleic acid sequence which can be used to ligate to the end of a nucleic acid sequence in order to facilitate attachment to another molecule. The adapter can be single-stranded or double- 15 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 stranded. An adapter can incorporate a short (typically less than 50 base pairs) sequence useful for PCR amplification or sequencing.

[0047] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0048] As used herein, the terms “amplify” or “amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods are well known to the skilled artisan and include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two- step multiplexed amplifications, rolling circle amplification (RCA), recombinase- polymerase amplification (RPA)(TwistDx, Cambridge, UK), transcription mediated amplification, signal mediated amplification of RNA technology, loop-mediated isothermal amplification of DNA, helicase-dependent amplification, single primer isothermal amplification, and self- sustained sequence replication (3SR), including multiplex versions or combinations thereof. Copies of a particular nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or “amplification products.”

[0049] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.

[0050] The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in “antiparallel 16 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 association.” For example, the sequence “5'-A-G-T-3’” is complementary to the sequence “3’-T-C-A-5.” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0051] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0052] “Detecting” as used herein refers to determining the presence of a mutation or genetic alterations in a nucleic acid of interest in a sample. Detection does not require the method to provide 100% sensitivity. Analysis of nucleic acid markers can be performed using techniques known in the art including, but not limited to, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam- Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol, 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); Drmanac et al., Nat. Biotechnol, 16:54-58 (1998). Non- limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be 17 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 performed using commercially available kits and instruments from companies such as the Life Technologies / Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche / 454 next generation sequencing system.

[0053] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0054] As used herein, the expression “DNA homologous recombination (HR) pathway” has its general meaning in the art. It refers to the cellular pathway through which Double Stranded DNA breaks (DSB) are repaired by a mechanism called Homologous Recombination. Inside mammalian cells, DNA is continuously exposed to damage arising from exogenous sources such as ionizing radiation or endogenous sources such as byproducts of cell replication. All organisms have evolved different strategies to cope with these lesions. One of the most deleterious forms of DNA damage is DSB. In mammalian cells, there are two major pathways to repair DSB: Homologous recombination (HR) and Non Homologous End Joining (NHEJ). HR is the most accurate mechanism to repair DSB because it uses an intact copy of the DNA from the sister chromatid or the homologous chromosome as a matrix to repair the break.

[0055] “Deficiency in the HR pathway” or “HRD” as used herein, refers to a condition in which the HR pathway for repairing DNA is deficient or inactivated. Exemplary proteins involved in the HR pathway can encompass, but are not limited to, inactivation of at least one of the following genes: BRCA1, BRCA2, PALP2 / FANCN, BRIP1 / FANCJ, BARD1, 18 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 RAD51, RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), FANCA, FANCB, FANCC, FANCD2, FANCE, FANCG, FANCI, FANCL, FANCM, FAN1, SLX4 / FANCP and ERCC1. See also US20170260588.

[0056] As used herein, “HRD score” refers to the number of loss of heterozygosity (LOH) regions >15 Mb, but less than a whole chromosome in length, observed within a tumour genome.

[0057] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15-100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al.1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0058] As used herein, “homeology” refers to approximate (>80%) similarity between a pair of genomic sequences.

[0059] “Large scale state transitions” or “LST” as used herein refer to breakpoints along a genome that result in genomic DNA segments of at least 3, 4, 5, 6, 7, 8, 9, 10, 1112, 13, 14, 15, 16, 17, 18, 19 or 20 megabases in length. 19 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0060] As used herein, the term “library” refers to a collection of nucleic acid sequences, e.g., a collection of nucleic acids derived from whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof. In one embodiment, a portion or all of the library nucleic acid sequences comprises an adapter sequence. The adapter sequence can be located at one or both ends. The adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector.

[0061] The library can comprise a collection of nucleic acid sequences, e.g., a target nucleic acid sequence (e.g., a tumor nucleic acid sequence), a reference nucleic acid sequence, or a combination thereof. In some embodiments, the nucleic acid sequences of the library can be derived from a single subject. In other embodiments, a library can comprise nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects). In some embodiments, two or more libraries from different subjects can be combined to form a library having nucleic acid sequences from more than one subject.

[0062] A “library nucleic acid sequence” refers to a nucleic acid molecule, e.g., a DNA, RNA, or a combination thereof, that is a member of a library. Typically, a library nucleic acid sequence is a DNA molecule, e.g., genomic DNA or cDNA. In some embodiments, a library nucleic acid sequence is fragmented, e.g., sheared or enzymatically prepared, genomic DNA. In certain embodiments, the library nucleic acid sequences comprise sequence from a subject and sequence not derived from the subject, e.g., adapter sequence, a primer sequence, or other sequences that allow for identification, e.g., “barcode” sequences.

[0063] “Loss of heterozygosity (LOH)” may result from several mechanisms. For example, in some embodiments, a region of one chromosome is deleted in a somatic cell. The region that remains present on the other chromosome (the other non-sex chromosome for males) is an LOH region as there is only one copy (instead of two copies) of that region present within the genome of the affected cells. The LOH region can be any length (e.g., from a length less than about 1.5 Mb up to a length equal to the entire length of the chromosome). In some embodiments, an LOH event results in a copy number reduction. In other embodiments, a region of one chromosome (one non-sex chromosome for males) in a somatic cell can be replaced with a copy of that region from the other chromosome, thereby eliminating any heterozygosity that may have been present within the replaced region. In such cases, the region that remains present on each chromosome is an LOH region and can 20 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 be referred to as a “copy neutral LOH” region. Copy neutral LOH regions can be any length (e.g., from a length less than about 1.5 Mb up to a length equal to the entire length of the chromosome).

[0064] As used herein, “microhomology” or “MH” refers to short regions of DNA sequence homology. In some embodiments, microhomology occurs directly at a breakpoint junction of a genomic rearrangement (e.g., germline or somatic breakpoint junction). Although definitions of breakpoint microhomology vary with respect to the length of the homologous region, it can be defined as a series of nucleotides (<70) that are identical at the junctions of the two genomic segments that contribute to the rearrangement. In other embodiments, microhomology occurs adjacent to the junction of a genomic rearrangement without any overlap.

[0065] As used herein, “mhDels, ” “del-MH” or “MH-dels” refer to small deletions (<50 bp) with microhomology.

[0066] “Next-generation sequencing or NGS” as used herein, refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 103, 104, 105or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. Nature Biotechnology Reviews 11:31-46 (2010).

[0067] As used herein, the term “overall survival” or “OS” means the observed length of life from the start of treatment to death or the date of last contact.

[0068] As used herein the term “PARP inhibitor” refers to a compound which is capable of inhibiting the activity of the enzyme polyADP ribose polymerase (PARP), a protein that is important for repairing single-strand breaks (‘nicks’ in the DNA). If such nicks persist unrepaired until DNA is replicated (which must precede cell division), then the replication itself will cause double strand breaks to form. Drugs that inhibit PARP cause multiple double strand breaks to form in this way, and in tumors with BRCA1, BRCA2 or PALB2 mutations these double strand breaks cannot be efficiently repaired, leading to the death of the cells. 21 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0069] As used herein, a “sample” refers to a substance that is being assayed for the presence of a mutation or alterations in a nucleic acid of interest. Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation. A biological sample may be a body fluid or a tissue sample. In some cases, a biological sample may consist of or comprise blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, and the like. Fresh, fixed or frozen tissues may also be used. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample. Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant are suitable.

[0070] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0071] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0072] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0073] The term “stringent hybridization conditions” as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42oC. overnight; washing with 2x SSC, 0.1% SDS at 45oC; and washing with 0.2x SSC, 0.1% SDS at 45oC. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases. 22 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0074] As used herein, the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.

[0075] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0076] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0077] “Reciprocal SV” or “reciprocal pairs” or “reciprocal rearrangements” as used herein refer to structural variants (SVs) / rearrangements that comprise pairs of distant intra- chromosomal loci or inter-chromosomal loci that contain exchanged genomic material. Within a reciprocal pair, a segment that connects two proximal breakends is known as a “gap”, and is associated with a polarity determined by orientation of the flanking breakends. The number of base pairs in the gap is the gap length and the gap length of the RPs disclosed herein has a length ranging from 100 bps to 1.3 Mbp. In some embodiments, the RP has a gap length ranging from 100 bps to 1 kbp, 1 kbp to 10 kbps, or 10 kbps to 100 kbps or 100 kbps to 1.3 Mbp. A gap has (+) polarity if the breakends are facing towards each other (the adjoining junctions connect to the gap segment (+ polarity, right two examples of FIG 1A). A gap has (-) polarity if breakends are facing away from each other, (the adjoining junctions connect to adjacent breakends (- polarity, left middle example of FIG 1A).

[0078] The reciprocal rearrangement may be a reciprocal duplication (rDup), a reciprocal deletion (rDel), or a reciprocal deletion-duplication (rDelDup). In cis reciprocal rearrangements, junctions are placed adjacently, corresponding to the templated insertion of a distant segment between duplicated copies of a gap segment at the source locus (FIG. 2A). In trans reciprocal rearrangements, the junctions are located on discontiguous or 23 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 distant alleles consistent with a large translocation or inversion, respectively, where each derivative allele harbors a copy of both gap segments (FIG.2A). Conventional HRD Signatures

[0079] Inherited germline mutations in BRCA1 and BRCA2 are associated with an elevated risk of breast, ovary, pancreas, and prostate cancer. During early carcinogenesis, the second allele often becomes defective due to loss of heterozygosity leading to cancers with two defective copies of core components of the homologous recombination (HR) pathway. HR deficient cancers constitute a clinically important subgroup.

[0080] As carcinogenesis proceeds over many cell divisions, genetic insults typically repaired through HR are instead shunted to backup repair pathways such as alternative end- joining and non-homologous end-joining, leaving behind characteristic genomic DNA repair scars. In 2012, three similar signatures were reported: loss of heterozygosity (LOH), large scale state transitions (LST), and telomeric imbalance (tAI), each characterized by large MBp intra- and interchromosomal rearrangements. These three tests were combined into one genomic readout known commercially as Myriad myChoice CDx HRD score, which is now FDA approved as a companion diagnostic test to select ovarian cancers patients eligible for two PARP inhibitors, Olaparib and Niraparib. While the Myriad myChoice HRD test is currently used to predict PARP inhibitor sensitivity, there is a large overlap between platinum and PARP inhibitor sensitivity as both depend upon inactive HR pathways. Another FDA-approved diagnostic test, FoundationOne CDx, uses LOH and BRCA-status to determine patients eligible for treatment with Olaparib or Rucaparib.

[0081] Other HRD signatures subsequently discovered include a base substitution pattern (SBS3) characterized by an even distribution of substitutions without a contextual bias, small deletions with microhomology around flanking the breaksite, and small and large tandem duplications and deletions (RefSig R3 / R5). A composite score of SBS3, HRD, indels with microhomology, RefSig R3, and RefSig R5, known as HRDetect (Davies et al., Nat Med 23(4):517-525 (2017); US20200126635), is highly predictive of cases with BRCA1 / 2 mutations in breast and ovarian cancer genomes. In addition, the small deletion signature was further refined as composed of the ID6 signature, consisting of small deletions of ≥ 5bp with small stretches of microhomology in their flanking sequences, and the ID8 signature, primarily associated with germline BRCA1 mutated cases, exhibits deletions of similar size but without microhomology. 24 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0082] The ID6 signature is consistent with repair via alternative end-joining and its predominant mediator polymerase theta (Pol q, gene name POLQ). Repair through Pol θ is also termed Theta Mediated End-joining (TMEJ), a pathway highly used in the absence of functional NHEJ or HR. Loss of POLQ is synthetically lethal with BRCA1 and BRCA2 loss and Pol θ inhibitors are preferentially active in BRCA1 and BRCA2 deleted cell lines. The enzyme typically searches for microhomology within 15 base pairs on either side of the break and utilizes mainly 3 or more base pairs of microhomology. Other pathways, including canonical non-homologous end-joining, can utilize up to 2 bp of microhomology (MH). Thus, a challenge in evaluating TMEJ scars is the degree of overlap between features suggestive of NHEJ and those suggestive of TMEJ. Both NHEJ and TMEJ can utilize short stretches of microhomology up to 2bp, leaving behind identical deletions. NGS Platforms

[0083] In some embodiments, high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators. In other embodiments, sequencing is performed via sequencing-by-ligation. In yet other embodiments, sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing etc.

[0084] The Ion TorrentTM(Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication. For use with this system, a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated. A proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. The pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. 25 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0085] The 454TM GS FLXTMsequencing system (Roche, Germany), employs a light- based detection methodology in a large-scale parallel pyrosequencing system. Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. For use with the 454TMsystem, adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR). Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate. The four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.

[0086] Sequencing technology based on reversible dye-terminators: DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.

[0087] Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template- dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.

[0088] Sequencing by synthesis (SBS), like the "old style" dye-termination electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence. A DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip. Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by 26 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 repeated removal of the blocking group to allow polymerization of another nucleotide. The signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate-driven light reactions and hydrogen ion sensing having all been used. Examples of SBS platforms include Illumina GA and HiSeq 2000. The MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.

[0089] In contrast to the sequencing by synthesis method, the sequencing by ligation method uses a DNA ligase to determine the target sequence. This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand. This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo). This method is primarily used by Life Technologies’ SOLiDTMsequencers. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.

[0090] SMRT ^ sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. PARP Inhibitors

[0091] In one aspect, the present disclosure provides inhibitory nucleic acids (e.g., sgRNAs, antisense RNAs, ribozymes, or shRNAs) that inhibit PARP expression and / or activity. The mammalian nucleic acid sequences of PARP are known in the art (e.g., NCBI Gene ID: 142). The inhibitory nucleic acids of the present technology may comprise a nucleic acid molecule that is complementary to a portion of a PARP nucleic acid sequence. In some embodiments, the inhibitory nucleic acids (e.g., sgRNAs, antisense RNAs, 27 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 ribozymes, or shRNAs) target at least one exon and / or intron of PARP. An exemplary nucleic acid sequence of Homo sapiens PARP1 is provided below: 1 agcaatctat cagggaacgg cggtggccgg tgcggcgtgt tcggtggcgg ctctggccgc 61 tcaggcgcct gcggctgggt gagcgcacgc gaggcggcga ggcggcagcg tgtttctagg 121 tcgtggcgtc gggcttccgg agctttggcg gcagctaggg gaggatggcg gagtcttcgg 181 ataagctcta tcgagtcgag tacgccaaga gcgggcgcgc ctcttgcaag aaatgcagcg 241 agagcatccc caaggactcg ctccggatgg ccatcatggt gcagtcgccc atgtttgatg 301 gaaaagtccc acactggtac cacttctcct gcttctggaa ggtgggccac tccatccggc 361 accctgacgt tgaggtggat gggttctctg agcttcggtg ggatgaccag cagaaagtca 421 agaagacagc ggaagctgga ggagtgacag gcaaaggcca ggatggaatt ggtagcaagg 481 cagagaagac tctgggtgac tttgcagcag agtatgccaa gtccaacaga agtacgtgca 541 aggggtgtat ggagaagata gaaaagggcc aggtgcgcct gtccaagaag atggtggacc 601 cggagaagcc acagctaggc atgattgacc gctggtacca tccaggctgc tttgtcaaga 661 acagggagga gctgggtttc cggcccgagt acagtgcgag tcagctcaag ggcttcagcc 721 tccttgctac agaggataaa gaagccctga agaagcagct cccaggagtc aagagtgaag 781 gaaagagaaa aggcgatgag gtggatggag tggatgaagt ggcgaagaag aaatctaaaa 841 aagaaaaaga caaggatagt aagcttgaaa aagccctaaa ggctcagaac gacctgatct 901 ggaacatcaa ggacgagcta aagaaagtgt gttcaactaa tgacctgaag gagctactca 961 tcttcaacaa gcagcaagtg ccttctgggg agtcggcgat cttggaccga gtagctgatg 1021 gcatggtgtt cggtgccctc cttccctgcg aggaatgctc gggtcagctg gtcttcaaga 1081 gcgatgccta ttactgcact ggggacgtca ctgcctggac caagtgtatg gtcaagacac 1141 agacacccaa ccggaaggag tgggtaaccc caaaggaatt ccgagaaatc tcttacctca 1201 agaaattgaa ggttaaaaaa caggaccgta tattcccccc agaaaccagc gcctccgtgg 1261 cggccacgcc tccgccctcc acagcctcgg ctcctgctgc tgtgaactcc tctgcttcag 1321 cagataagcc attatccaac atgaagatcc tgactctcgg gaagctgtcc cggaacaagg 1381 atgaagtgaa ggccatgatt gagaaactcg gggggaagtt gacggggacg gccaacaagg 1441 cttccctgtg catcagcacc aaaaaggagg tggaaaagat gaataagaag atggaggaag 1501 taaaggaagc caacatccga gttgtgtctg aggacttcct ccaggacgtc tccgcctcca 1561 ccaagagcct tcaggagttg ttcttagcgc acatcttgtc cccttggggg gcagaggtga 1621 aggcagagcc tgttgaagtt gtggccccaa gagggaagtc aggggctgcg ctctccaaaa 28 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 1681 aaagcaaggg ccaggtcaag gaggaaggta tcaacaaatc tgaaaagaga atgaaattaa 1741 ctcttaaagg aggagcagct gtggatcctg attctggact ggaacactct gcgcatgtcc 1801 tggagaaagg tgggaaggtc ttcagtgcca cccttggcct ggtggacatc gttaaaggaa 1861 ccaactccta ctacaagctg cagcttctgg aggacgacaa ggaaaacagg tattggatat 1921 tcaggtcctg gggccgtgtg ggtacggtga tcggtagcaa caaactggaa cagatgccgt 1981 ccaaggagga tgccattgag cacttcatga aattatatga agaaaaaacc gggaacgctt 2041 ggcactccaa aaatttcacg aagtatccca aaaagttcta ccccctggag attgactatg 2101 gccaggatga agaggcagtg aagaagctga cagtaaatcc tggcaccaag tccaagctcc 2161 ccaagccagt tcaggacctc atcaagatga tctttgatgt ggaaagtatg aagaaagcca 2221 tggtggagta tgagatcgac cttcagaaga tgcccttggg gaagctgagc aaaaggcaga 2281 tccaggccgc atactccatc ctcagtgagg tccagcaggc ggtgtctcag ggcagcagcg 2341 actctcagat cctggatctc tcaaatcgct tttacaccct gatcccccac gactttggga 2401 tgaagaagcc tccgctcctg aacaatgcag acagtgtgca ggccaaggtg gaaatgcttg 2461 acaacctgct ggacatcgag gtggcctaca gtctgctcag gggagggtct gatgatagca 2521 gcaaggatcc catcgatgtc aactatgaga agctcaaaac tgacattaag gtggttgaca 2581 gagattctga agaagccgag atcatcagga agtatgttaa gaacactcat gcaaccacac 2641 acaatgcgta tgacttggaa gtcatcgata tctttaagat agagcgtgaa ggcgaatgcc 2701 agcgttacaa gccctttaag cagcttcata accgaagatt gctgtggcac gggtccagga 2761 ccaccaactt tgctgggatc ctgtcccagg gtcttcggat agccccgcct gaagcgcccg 2821 tgacaggcta catgtttggt aaagggatct atttcgctga catggtctcc aagagtgcca 2881 actactgcca tacgtctcag ggagacccaa taggcttaat cctgttggga gaagttgccc 2941 ttggaaacat gtatgaactg aagcacgctt cacatatcag caagttaccc aagggcaagc 3001 acagtgtcaa aggtttgggc aaaactaccc ctgatccttc agctaacatt agtctggatg 3061 gtgtagacgt tcctcttggg accgggattt catctggtgt gaatgacacc tctctactat 3121 ataacgagta cattgtctat gatattgctc aggtaaatct gaagtatctg ctgaaactga 3181 aattcaattt taagacctcc ctgtggtaat tgggagaggt agccgagtca cacccggtgg 3241 ctctggtatg aattcacccg aagcgcttct gcaccaactc acctggccgc taagttgctg 3301 atgggtagta cctgtactaa accacctcag aaaggatttt acagaaacgt gttaaaggtt 3361 ttctctaact tctcaagtcc cttgttttgt gttgtgtctg tggggagggg ttgttttggg 3421 gttgtttttg ttttttcttg ccaggtagat aaaactgaca tagagaaaag gctggagaga 29 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 3481 gattctgttg catagactag tcctatggaa aaaaccaagc ttcgttagaa tgtctgcctt 3541 actggtttcc ccagggaagg aaaaatacac ttccaccctt ttttctaagt gttcgtcttt 3601 agttttgatt ttggaaagat gttaagcatt tatttttagt taaaaataaa aactaatttc 3661 atactattta gattttcttt tttatcttgc acttattgtc ccctttttag ttttttttgt 3721 ttgcctcttg tggtgagggg tgtgggaaga ccaaaggaag gaacgctaac aatttctcat 3781 acttagaaac aaaaagagct ttccttctcc aggaatactg aacatgggag ctcttgaaat 3841 atgtagtatt aaaagttgca tttgaaattc ttgactttct tatgggcact tttgtcttcc 3901 aaattaaaac tctaccacaa atatacttac ccaagggcta atagtaatac tcgattaaaa 3961 atgcagatgc cttctcta (SEQ ID NO: 7)

[0092] The present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of a PARP mRNA. The antisense nucleic acid may be antisense RNA, or antisense DNA. Antisense nucleic acids based on the known nucleic acid sequences of PARP can be readily designed and engineered using methods known in the art.

[0093] Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire PARP coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame). In some embodiments, the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the coding region of PARP mRNA. In certain embodiments, an antisense nucleic acid molecule can be complementary to a noncoding region of the PARP coding strand. In some embodiments, the noncoding region refers to the 5′ and 3′ untranslated regions that flank the coding region and are not translated into amino acids. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PARP. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[0094] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological 30 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thouridine, 5-carboxymethylaminometh-yluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-metnylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-cxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0095] The antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and / or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and / or translation. The hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.

[0096] In some embodiments, the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. In some embodiments, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res.15:6625- 6641(1987)). The antisense nucleic acid molecule can also comprise a 2′-O - 31 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 methylribonucleotide (Inoue et al., Nucleic Acids Res.15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0097] The present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of a PARP mRNA, thereby reducing or inhibiting PARP expression. In some embodiments, the shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length. Double-stranded RNA (dsRNA) can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA. For example, a double-stranded siRNA or shRNA molecule is engineered to complement and hybridize to an mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA- induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as PARP mRNA).

[0098] The present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of a PARP mRNA, thereby reducing or inhibiting PARP expression. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single- stranded nucleic acid, such as an mRNA. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))) can be used to catalytically cleave PARP transcripts, thereby inhibiting translation of PARP.

[0099] A ribozyme having specificity for a PARP-encoding nucleic acid can be designed based upon a PARP nucleic acid sequence. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PARP-encoding mRNA. See, e.g., U.S. Pat. No.4,987,071 and U.S. Pat. No.5,116,742. Alternatively, PARP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411- 1418, incorporated herein by reference.

[0100] The present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of a PARP nucleic acid sequence. Guide RNAs for use in CRISPR-Cas 32 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 systems are typically generated as a single guide RNA comprising a crRNA segment and a tracrRNA segment. The crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules. The crRNA segment comprises the targeting sequence that binds to a portion of a PARP nucleic acid sequence, and a stem portion that hybridizes to a tracrRNA. The tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme. In some embodiments, the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, the crRNA segment and the tracrRNA segment are provided as separate RNAs. The combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO2015 / 089465.

[0101] In some embodiments, a synthetic guide RNA is a single RNA represented as comprising the following elements: 5ʹ-X1-X2-Y-Z-3ʹ where X1 and X2 represent the crRNA segment, where X1 is the targeting sequence that binds to a portion of a PARP nucleic acid sequence, X2 is a stem sequence that hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence. In some embodiments, the linker sequence comprises two or more nucleotides and links the crRNA and tracrRNA segments. In some embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA.

[0102] In some embodiments, a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5ʹ-X1-X2-3ʹ where X1 is the targeting sequence that binds to a portion of a PARP nucleic acid sequence, X2 is a stem sequence that hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.

[0103] Exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO / 2015 / 089465, which is incorporated by reference herein. In general, a stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to 33 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 the tracrRNA. In general, degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the stem sequence or the complementary sequence in the tracrRNA. In some embodiments, the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the tracrRNA has additional complementary sequences that form hairpins. In some embodiments, the tracrRNA has at least two or more hairpins. In some embodiments, the tracrRNA has two, three, four or five hairpins. In some embodiments, the tracrRNA has at most five hairpins.

[0104] In a hairpin structure, the portion of the sequence 5ʹ of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3ʹ of the loop corresponds to the tracrRNA sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5ʹ to 3ʹ), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator: (a) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 1); (b) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 2); (c) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 3); (d) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt 34 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 gaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 4); (e) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT (SEQ ID NO: 5); and (f) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT

[0105] Selection of suitable oligonucleotides for use as a targeting sequence in a CRISPR Cas system depends on several factors including the particular CRISPR enzyme to be used and the presence of corresponding proto-spacer adjacent motifs (PAMs) downstream of the target sequence in the target nucleic acid. The PAM sequences direct the cleavage of the target nucleic acid by the CRISPR enzyme. In some embodiments, a suitable PAM is 5'- NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively. Generally, the PAM sequences should be present between about 1 to about 10 nucleotides of the target sequence to generate efficient cleavage of the target nucleic acid. Thus, when the guide RNA forms a complex with the CRISPR enzyme, the complex locates the target and PAM sequence, unwinds the DNA duplex, and the guide RNA anneals to the complementary sequence on the opposite strand. This enables the Cas9 nuclease to create a double-strand break.

[0106] A variety of CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site. In some embodiments, the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.

[0107] In another aspect, the present disclosure provides pharmacological inhibitors of PARP including, but not limited to iniparib, olaparib, niraparib, rucaparib, talazoparib, 35 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, or 3-aminobenzamide. Anti-PARP neutralizing antibodies may also be employed in the methods disclosed herein. Pharmaceutical Compositions Including the PARP Inhibitors and Platinum Agents

[0108] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.

[0109] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0110] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are 36 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.

[0111] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0112] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case 37 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.

[0113] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0114] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Modes of Administration and Effective Dosages

[0115] Any method known to those in the art for contacting a cell, organ or tissue with one or more PARP inhibitors and / or platinum agents disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more PARP inhibitors and / or platinum agents to a mammal, suitably a human. When used in vivo for therapy, the one or more PARP inhibitors and / or platinum agents described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular PARP inhibitor or platinum agent used, e.g., its therapeutic index, and the subject’s history. 38 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0116] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more PARP inhibitors and / or platinum agents useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The PARP inhibitor or platinum agent may be administered systemically or locally.

[0117] The one or more PARP inhibitors and / or platinum agents described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disease or condition described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0118] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[0119] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous 39 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0120] The pharmaceutical compositions having one or more PARP inhibitors and / or platinum agents disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0121] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0122] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. 40 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0123] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

[0124] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[0125] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable 41 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[0126] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly α-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic / glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0127] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99 / 15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96 / 40073 (Zale, et al.), and PCT publication WO 00 / 38651 (Shah, et al.). U.S. Pat. Nos.5,674,534 and 5,716,644 and PCT publication WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0128] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0129] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn 42 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.

[0130] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0131] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0132] Typically, an effective amount of the one or more PARP inhibitors and / or platinum agents disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, 43 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more PARP inhibitor / platinum agent concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0133] In some embodiments, a therapeutically effective amount of one or more PARP inhibitors and / or platinum agents may be defined as a concentration of inhibitor at the target tissue of 10-32to 10-6molar, e.g., approximately 10-7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0134] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0135] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human. Methods for Treatment Selection in HRD Cancer Patients

[0136] In one aspect, the present disclosure provides a method for selecting a cancer patient for treatment with a PARP inhibitor or a platinum agent comprising: (a) detecting levels of reciprocal rearrangements in a biological sample obtained from the cancer patient that are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of a PARP inhibitor or a platinum agent. In another aspect, the present disclosure provides a method for prolonging survival of a cancer patient comprising administering to the cancer 44 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 patient an effective amount of a PARP inhibitor or a platinum agent, wherein levels of reciprocal rearrangements in a biological sample obtained from the cancer patient are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold. In some embodiments of the methods disclosed herein, the levels of reciprocal rearrangements are detected via next-generation whole-genome sequencing.

[0137] In any of the preceding embodiments of the methods disclosed herein, one or more of the reciprocal rearrangements comprise an orientation that is positive (e.g., reciprocal duplication (rDup)), negative (e.g., reciprocal deletion (rDel)), or mixed (e.g., reciprocal deletion-duplication (rDelDup)).

[0138] In certain embodiments, one or more of the reciprocal rearrangements comprise a negative orientation (rDel), and / or mixed orientation (rDelDup) and the cancer patient harbors a BRCA2 mutation. Additionally or alternatively, in some embodiments, the rDel is a trans interchromosomal reciprocal deletion or a trans intrachromosomal reciprocal deletion, and / or the rDelDup is a cis interchromosomal reciprocal deletion-duplication, a cis intrachromosomal reciprocal deletion-duplication, a trans interchromosomal reciprocal deletion-duplication, or a trans intrachromosomal reciprocal deletion-duplication.

[0139] In other embodiments, one or more of the reciprocal rearrangements comprise a positive orientation (rDup), and / or mixed orientation (e.g., rDelDup) and the cancer patient harbors a BRCA1 mutation. Additionally or alternatively, in some embodiments, the rDelDup is a cis interchromosomal reciprocal deletion-duplication, a cis intrachromosomal reciprocal deletion-duplication, a trans interchromosomal reciprocal deletion-duplication, or a trans intrachromosomal reciprocal deletion-duplication and / or the rDup is a cis interchromosomal reciprocal duplication, a cis intrachromosomal reciprocal duplication, a trans interchromosomal reciprocal duplication, or a trans intrachromosomal reciprocal duplication.

[0140] Additionally or alternatively, in some embodiments, the cancer patient is diagnosed with, or is at risk of having a homologous recombination deficiency (HRD). In certain embodiments, the HRD comprises mutations that result in the inactivation of at least one of the following genes: PALP2 / FANCN, BRIP1 / FANCJ, BARD1, RAD51, RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), FANCA, FANCB, FANCC, FANCD2, FANCE, FANCG, FANCI, FANCL, FANCM, FAN1, SLX4 / FANCP and ERCC1. In any and all embodiments of the methods disclosed herein, the cancer patient harbors a mutation in one or more driver mutations selected from among BRCA2, BRCA1, PALB2, 45 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 RAD51C, RAD51B, IDH1, CDK4, RB1, FIGNL1, MDC1, BARD1, and RBBP8. Additionally or alternatively, in some embodiments, the cancer patient is suffering from a cancer selected from among breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. In certain embodiments, the cancer patient exhibits stage I, stage II, stage III or stage IV cancer. Additionally or alternatively, in some embodiments, the cancer patient has not received at least one prior line of anti-cancer therapy. In other embodiments, the cancer patient has received at least one prior line of anti-cancer therapy. Examples of anti-cancer therapy include chemotherapy, radiation therapy, surgery or any combination thereof.

[0141] In any and all embodiments of the methods disclosed herein, the PARP inhibitor is a PARP-specific inhibitory nucleic acid (e.g., sgRNAs, antisense RNAs, ribozymes, or shRNAs), iniparib, olaparib, niraparib, rucaparib, talazoparib, veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, 3-aminobenzamide, or an anti-PARP neutralizing antibody.

[0142] In any and all embodiments of the methods disclosed herein, the platinum agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, pyriplatin, picoplatin, or satraplatin.

[0143] Additionally or alternatively, in some embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.

[0144] Once treated for a particular period of time (e.g., between one to six months), the patient can be assessed to determine whether or not the treatment regimen has an effect. If a beneficial effect is detected, the patient can continue with the same or a similar cancer treatment regimen. If a minimal or no beneficial effect is detected, then adjustments to the cancer treatment regimen can be made. For example, the dose, frequency of administration, or duration of treatment can be increased. In some cases, additional anti-cancer agents can be added to the treatment regimen or a particular anti-cancer agent can be replaced with one or more different anti-cancer agents. The patient being treated can continue to be monitored as appropriate, and changes can be made to the cancer treatment regimen as appropriate.

[0145] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise determining an additional HRD signature in the cancer patient. In some embodiments, the additional HRD signature comprises one or more of loss of heterozygosity (LOH), large scale state transitions (LST), and telomeric imbalance (tAI). In certain embodiments, the additional HRD signature comprises one or more of single base 46 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 substitution signature 3 (SBS3), single base substitution signature 8 (SBS8), short tandem duplications (RefSig R3), short deletions (RefSig R5), microhomology-mediated indels, and HRD score. In other embodiments, the additional HRD signature comprises ID8 signature, and ID6 signature.

[0146] In another aspect, the present disclosure provides a method for discriminating between BRCA1-deficient and BRCA2-deficient tumors in cancer patients comprising: (a) detecting a plurality of structural variant (SV) features in one or more tumor samples obtained from a plurality of cancer patients, wherein the plurality of SV features comprise homeologous deletions, positive reciprocal rearrangements (rDups), negative reciprocal rearrangements (rDels), and mixed reciprocal rearrangements (rDelDups); (b) for each of the one or more tumor samples, (i) identifying tumor samples having elevated levels of negative orientation reciprocal rearrangements (rDels) and / or homeologous deletions relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and / or (ii) identifying tumor samples having elevated levels of positive orientation reciprocal rearrangements (rDups) relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and (c) classifying the tumor samples of (b)(i) as BRCA2-deficient tumors and the tumor samples of (b)(ii) as BRCA1- deficient tumors. The SV features in the one or more tumor samples may be detected using whole genome sequencing. In some embodiments, the BRCA2-deficient or BRCA1- deficient tumor samples further comprise elevated levels of mixed orientation reciprocal rearrangements (rDelDups) relative to the control biological sample obtained from a healthy subject or a predetermined threshold.

[0147] Additionally or alternatively, in some embodiments, the size of the homeologous deletions ranges from about 1 kbp to about 100 kbp. The size of the homeologous deletions may be about 1 kbp, about 2 kbps, about 3 kbps, about 4 kbps, about 5 kbps, about 6 kbps, about 7 kbps, about 8 kbps, about 9 kbps, about 10 kbps, about 15 kbps, about 20 kbps, about 25 kbps, about 30 kbps, about 35 kbps, about 40 kbps, about 45 kbps, about 50 kbps, about 55 kbps, about 60 kbps, about 65 kbps, about 70 kbps, about 75 kbps, about 80 kbps, about 85 kbps, about 90 kbps, about 95 kbps, or about 100 kbps.

[0148] In any of the preceding embodiments of the method disclosed herein, the plurality of SV features further comprise one or more of 1-100 Kbp simple duplications, rearrangement signature 3 (RS3), rearrangement signature (RS5), deletions with microhomology (mhDel), COSMIC single base signature 3 (SBS3), COSMIC single base 47 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 signature 8 (SBS8), rearrangement signatures 3 and 5, RS3 and RS5, and loss of heterozygosity (LOH). In certain embodiments, the BRCA1-deficient tumor samples further comprise elevated levels of 1-100 Kbps simple duplications relative to the control biological sample obtained from a healthy subject or a predetermined threshold. The size of the simple duplications may be about 1 kbp, about 2 kbps, about 3 kbps, about 4 kbps, about 5 kbps, about 6 kbps, about 7 kbps, about 8 kbps, about 9 kbps, about 10 kbps, about 15 kbps, about 20 kbps, about 25 kbps, about 30 kbps, about 35 kbps, about 40 kbps, about 45 kbps, about 50 kbps, about 55 kbps, about 60 kbps, about 65 kbps, about 70 kbps, about 75 kbps, about 80 kbps, about 85 kbps, about 90 kbps, about 95 kbps, or about 100 kbps.

[0149] Additionally or alternatively, in some embodiments, the cancer patients are suffering from a cancer selected from among breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. In certain embodiments, the cancer patients exhibit stage I, stage II, stage III or stage IV cancer.

[0150] Additionally or alternatively, in some embodiments, the method further comprises identifying the cancer patients from which the BRCA2-deficient tumor samples were obtained and administering to the cancer patients an effective amount of chemotherapy or immunotherapy. In other embodiments, the method further comprises identifying the cancer patients from which the BRCA1-deficient tumor samples were obtained and administering to the cancer patients an effective amount of chemotherapy. In some embodiments, the chemotherapy comprises a PARP inhibitor or a platinum agent. Examples of PARP inhibitors include, but are not limited to, iniparib, olaparib, niraparib, rucaparib, talazoparib, veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, or 3- aminobenzamide. Examples of platinum agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, pyriplatin, picoplatin, or satraplatin.

[0151] Examples of immunotherapy include immune checkpoint blockade agents such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, ipilimumab, cemiplimab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab. EXAMPLES

[0152] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill 48 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 in the art with preparing or using the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology. Example 1: Materials and Methods

[0153] Short-read whole genome sequencing cohort

[0154] BAM alignments for primary tumor genome sequencing data were obtained as previously described in17. Briefly, 989 tumor / normal (T / N) pairs were obtained from The Cancer Genome Atlas (TCGA) Research Network (Genomic Data Commons at portal.gdc.cancer.gov / legacy-archive, accession: phs000178.v11.p8). Additional WGS data was obtained for 874 T / N pairs from the International Cancer Genome Consortium (ICGC) from multiple studies publicly available through European Genome-Phenome Archive (EGA; https: / / ega-archive.org). These cohorts include: 124 breast cancers published in24(EGA: EGAS00001001178), 179 melanomas published in48(EGA: EGAS00001001552), 49 lung adenocarcinomas published in49(EGA: EGAS00001002801), 422 esophageal adenocarcinomas published in50(EGA: EGAD00001004417), and 100 malignant lymphomas (EGA: EGAD00001002123).

[0155] Additional BAMs for 121 T / N pairs from a pan-cancer cohort obtained as part of a New York City-based multi-institution collaborative research effort comprised of Memorial Sloan Kettering Cancer Center, New York University, Stony Brook University Hospital, Lenox Hill, Northwell Health, Columbia University, Montefiore, Cornell, and led by the New York Genome Center were included here and were previously described in17. Study approval was obtained via a central institutional review board (IRB), Biomedical Research Alliance of New York, and by local IRBs, including Stony Brook University and Northwell Health. Also, 55 prostate cancers previously published were obtained via dbGaP with accession, phs000447.v1.p119. BAMs for 80 T / N pairs were obtained from a collaborative precision oncology effort between the Weill Cornell Englander Institute for Precision Medicine (EIPM) and the New York Genome Center (NYGC) This study was approved by an institutional Review Board (WCM IRB #1305013903).340 T / N pairs across 80 cases across longitudinally / spatially distinct biopsies from Barrett’s esophagus tumors were obtained as part of a previous study51. 49 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0156] Callsets were obtained from 1,484 additional T / N pairs contributing additional primary tumor whole genomes from the Pan-Cancer of Whole Genomes Analysis52(FIG. 17: “PCAWG” dataset) and 3,957 T / N pairs from metastatic whole genomes from the Hartwig Medical Foundation which included germline, somatic SNV / InDel, and somatic structural variant calls53(FIG.17: “HMF” dataset). All calls were generated from alignments to variants of hg19.

[0157] Linked-read whole genome sequencing cohort

[0158] To validate observed structural variant patterns associated with homologous recombination deficiency from this larger dataset, a new cohort of 46 cases biopsied for ductal carcinomas of breast with predicted BRCA1 (n = 28) or BRCA2 (n = 18) deficiency was collected as part of a prospective radiation oncology research study at Memorial Sloan Kettering Cancer Research Center (MSKCC) (New York, NY) and sequenced with Illumina short-read WGS and also with linked-read WGS (see below for protocol details). Raw sequencing data is available under NCBI BioProject accession: PRJNA746293. (FIG.17: “MSK” dataset).

[0159] Pipelines

[0160] For all alignments that were obtained or generated, harmonized pipelines were run across 2,489 T / N pairs using previously described pipelines (TCGA, ICGC, EIPM, and MSKCC datasets) (the dataset hereafter referred to as "in-house", combined “Hadi” + “MSK” datasets in FIG.17)17.

[0161] SV calling

[0162] Briefly, genome-wide, high-density binned (200 bp) coverage was calculated (github.com / mskilab / fragCounter) and corrected for GC and mappability content. Primary coverage segmentation was generated using a modified implementation of Circular Binary Segmentation54. Somatic structural variant calls were obtained with SvAbA55and were filtered using a generated panel-of-normals that consisted of all germline SVs detected across 2,489 tumor / normal pairs. Any somatic SV found within 500 bp of a junction within the germline SV PON with matching orientations was discarded. PCAWG consensus SVs were obtained using SV release 1.6 and read depths obtained were also processed with CBS to obtain tumor / normal coverage ratios.

[0163] HMF SVs were obtained from the Hartwig Medical Foundation through data sharing agreement with the proprietors53. Briefly, junction calls from GRIDSS56and 1 kbp tumor:normal coverage ratios57corrected for GC content were obtained for 3,957 50 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 tumor / normal pairs. Primary segmentation was generated by modified CBS upon the coverage ratios.

[0164] Junction Balance Analysis (JaBbA)

[0165] This high-confidence set of junctions and primary segmentation were used to perform Junction Balance Analysis (JaBbA, github.com / mskilab / JaBbA) to generate junction-balanced genome graphs for 7,918 and 46 samples from the short-read and linked- read WGS cohorts, respectively, across all datasets in which junction and segment copy number are accounted for in an integrated model with purity and ploidy set as hyperparameters. For a detailed treatment of the formulation behind JaBbA, see17. Heterozygous germline SNP data was used to infer allelic copy number after total copy number inference is performed genome-wide as described17.

[0166] Purity and ploidy estimation

[0167] Across in-house cases, tumor purity and ploidy inputs to JaBbA were generally calculated with ppGrid (available through JaBbA) and ASCAT (Van Loo et al.2010) respectively. For the 46 cases from the MSKCC cohort, a review of purity / ploidy was conducted to enhance downstream genotyping accuracy; ultimately, alternative manual purity / ploidy estimates from MSKCC were chosen for 4 out of 46. For PCAWG and HMF data, purity and ploidy estimates were obtained from the respective PCAWG (https: / / dcc.icgc.org / releases / PCAWG / ) and HMF (https: / / www.hartwigmedicalfoundation.nl / en / database / ) portals53.

[0168] Linked-read WGS SV calling

[0169] For the linked-read WGS sequences generated from the MSKCC cohort of 46 cases, structural variants were called using LinkedSV58. These junctions were merged with SvAbA junctions called on the corresponding short-read WGS for each case, and were input into JaBbA using short-read coverage profiles to generate genome graphs. Merging was performed using the gGnome R package (github.com / mskilab / gGnome) to determine junctions that were uniquely detected by linked-read WGS (LinkedSV).

[0170] Analysis of gap segment topology

[0171] Gap segments were defined as short genomic segments joining reference- consecutive breakends, each belonging to distinct junctions and occurring on opposite strands. Each gap segment was additionally associated with a polarity (+ or -) based on the strand of the right most (highest reference coordinate) breakend; (+) polarity corresponded to a gap segment with breakends facing each other, (-) polarity to a gap segment with 51 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 breakends facing away from each other on the reference. The length threshold to define gap segments was visually chosen as 1 Mbp after inspection of a density plot of bridge candidate (i.e. segments satisfying the above criteria) length. This threshold was confirmed through the application of a background model, where the bridge candidate length distribution in each sample was fit with an exponential distribution and each bridge candidate was assigned a p value according to the left tail of the exponential cumulative distribution function. Short (<1 Mbp) bridge candidates were found to significantly deviate from expectation (FDR < 0.10) under this model.

[0172] Applying this definition, gap segments with shared junctions were next clustered together (applying "eclusters" gGraph method in the gGnome R package). Bridge clusters in which every junction was connected to two gap segments was labeled as "cyclic". Bridge clusters were annotated with number of cluster-associated junctions and gap segments. A quasi-reciprocal pair (rPair) is a special case of a cyclic bridge cluster that contains 2 gap segments of either orientation. rDups, rDels, and rDelDups each contain 2 (+) gap segments, 2 (-) gap segments, or 1 (+) and 1 (-) gap segment, respectively.

[0173] Annotating known structural variant events

[0174] Classes of previously described17simple and complex SV were annotated in junction-balanced genome graphs derived by JaBbA for both the short-read (n = 2,489) and linked-read WGS cohort (n = 46). The following simple events were classified within each graph: deletions, duplications, translocations, inversions, and inverted duplications. The following complex events were classified: breakage-fusion-bridge cycles (BFBC), double minutes (DM), tyfonas, chromoplexy, chromothripsis, and templated insertion chains (TIC). Implementation of each event classifier can be found in the "events" function in the gGnome R package.

[0175] Variant calling and genotyping

[0176] For the 2,489 in-house WGS tumor / normal pairs, somatic mutation calls were generated with Strelka1 for SNVs and InDels. Germline mutation calls were obtained strictly with Strelka2 run on alignments from blood or adjacent normal samples and filtered for common variants above a population allele frequency of 1% (ExAC population: ftp.broadinstitute.org / pub / ExAC_release / release0.3.1 / subsets / ). SNVs and indels were filtered through a universal genome-wide mask for hg19 (https: / / github.com / lh3 / CHM-eval) to remove artifacts due to low-mappability, as described in59. All germline and somatic SNVs and indels were annotated with ClinVar status 52 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 (ftp.ncbi.nlm.nih.gov: / pub / clinvar / vcf_GRCh37, database date 2022-07-30). Impacts of protein-coding SNVs and indels were also annotated via SnpEff (GRCh37.75 database). SNVs and indels were considered pathogenic if annotated as “Pathogenic” or “Likely_Pathogenic” via ClinVar CLNSG or if their SnpEff annotation fell within the following classes: “frameshift variant”, “start_lost”, “stop_gained”, “stop_lost”, “splice_acceptor_variant”, or “splice_donor_variant”. ClinVar annotation took precedence over SnpEff.

[0177] Loss-of-heterozygosity (LOH) was determined by allele-specific copy number (CN) using allele counts across germline heterozygous SNP sites. Specifically, LOH was called in regions where minor allele CN = 0 and major allele CN > 0, using allelic copy number as inferred from short-read sequencing data (see Junction Balance Analysis (JaBbA). Similarly, homologous deletions (homdels) were called in regions where total copy number (sum of major and minor allele CN = 0).

[0178] Genotype was determined across samples for 48 HR-related genes (Table 1), including BRCA1, BRCA2, PALB2, and RAD51*.11 of these genes were highlighted in60. Table 1 Curated list of HR-related genes AUNIP BABAM1 BARD1 BRCA1 BRCA2 BRCC3 BRIP1 EME1 EME2 EXO1 FAM175A FIGNL1 GEN1 H2AFX HELQ MCM8 MCM9 MDC1 53 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 MRE11A MUS81 NBN PALB2 RAD50 RAD51 RAD51AP1 RAD51B RAD51C RAD51D RAD52 RAD54B RAD54L RBBP8 REV1 RMI1 RMI2 RPA1 RPA2 RTEL1 SFR1 SHFM1 SLX1A SLX1B SLX4 SWSAP1 UIMC1 XRCC2 XRCC3 ZSWIM7

[0179] Biallelic loss was called for genes if they harbored any of the following: 1) two or more germline and / or somatic pathogenic mutations (including SNVs, indels, and SVs), 2) one germline or somatic pathogenic mutation along with LOH, or 3) a homdel. Within the linked-read WGS cohort, 22 cases were found to harbor bi-allelic loss of BRCA1, 14 cases found with bi-allelic loss of BRCA2, and 1 case with bi-allelic loss of both BRCA1 and BRCA2. 54 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0180] For PCAWG dataset cases, somatic SNV and indel calls were obtained from ICGC (2016 data freeze), and annotated driver mutations were obtained from the PCAWG consortium52. HMF data proprietors provided the following for cases in their dataset: germline SNVs and indels (via GATK HaplotypeCaller), somatic SNVs and indels (via Strelka1 and annotated by SnpEff).

[0181] Short-read whole genome sequencing

[0182] Short-read whole genome sequencing for the 46 MSKCC donors was performed at the New York Genome Center (NYGC) (New York, NY) to a target of 80X tumor and 40X normal coverage. Library preparation from genomic DNA for these new cases was performed using the NEBNext Ultra II End Repair / dA-Tailing Module, NEBNext Ultra Ligation Module (New England Biolabs, Ipswich, MA) and KAPA Dual-Indexed Adapter Kit (Roche, Switzerland) following manufacturer’s protocols. Quality control was performed on the finished libraries with the Agilent 2100 Bioanalyzer on the High Sensitive DNA Chip platform (Agilent Technologies, Santa Clara, CA) and KAPA Library Quantification Kit (Roche, Switzerland). QC determined that libraries contained an average peak height (fragment size) of at least 400 base pairs. Libraries were sequenced on an Illumina NovaSeq 6000 System (Illumina, San Diego, CA) to generate paired-end 2x150 base pair reads. Reads were aligned to the GRCh37 / hg19 reference using Burrows-Wheeler aligner software61, bwa mem, 0.7.10-r789). Read post-processing was done in accordance with best practices for post-alignment data processing with Picard tools (https: / / broadinstitute.github.io / picard / ) to mark duplicates, the GATK (v.2.7.4) (https: / / gatk.broadinstitute.org / hc / en-us) IndelRealigner module, and GATK base quality recalibration.

[0183] 10X linked-read whole genome sequencing

[0184] Each of the 46 MSKCC BRCA1 or BRCA2 mutated cases was subject to additional linked-read WGS. High molecular weight (HMW) genomic DNA (gDNA) was extracted using a QIAGEN MagAttract HMW DNA Kit (QIAGEN, Germany) according to the suggested protocol. Simply, approximately 2 million fresh cells were lysed, HMW gDNA was captured by magnetic particles. Then the magnetic particles with HMW gDNA was washed in wash buffer and eluted in EB Buffer (10 mM Tris-HCl, pH 8.5). The HMW gDNA had a mode length of 50 kbp and max length 200 kbp, as estimated on a separate 75V pulse-field gel electrophoresis (PFGE) using a BluePippin 5-430kbp protocol (Sage Science, Beverly, MA).10X sequencing library preparation was performed using a 55 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 Chromium Genome Library Kit v2 (10X Genomics, Pleasanton, CA) following the Chromium Genome Reagent Kits v2 User Guide. Briefly, 1 ng of extracted HMW gDNA was used to prepare a library, with an average fragment length of 625 bp (ranging from 300 to 2000 bp, measured with the Agilent Bioanalyzer High Sensitivity DNA Chip). Quality control for the finished libraries was performed as the same as the above of general WGS library preparation. The prepared libraries were sequenced on an Illumina NovaSeq 6000 Sequencing System (Illumina, San Diego, CA) with S4 flow cell, to an average read depth of about 33X. All linked-reads were aligned to GRCh37 / hg19 with the EMerAld aligner (v0.6.2)62. Germline haplotypes were obtained from Strelka2 germline SNV calls processed using HapCut2 (github.com / vibansal / HapCUT2)63.

[0185] Phasing rearranged haplotypes with LR WGS

[0186] Our specific goal in somatic phasing was to distinguish SVs that placed both reciprocal junctions on the same molecule (cis) from those that placed junctions at distant loci (including distinct derivative chromosomes) in the cancer genome (FIG.8A). Somatic phasing is distinct from parental phasing, which determines whether reciprocal breakends arose on the same or distinct parental homologue.

[0187] We approached somatic phasing by assessing LR WGS molecule support for derivative rearranged haplotypes. Derivative rearranged haplotypes can be deconvolved from junction balanced genome graphs as walks17. Walks were derived from JaBbA graphs on the LR WGS cohort for which both LR and short-read WGS were available using the "walks" gGraph method in the the gGnome package (https: / / github.com / mskilab / gGnome). Barcoded reads were matched against each possible walk within a 100 kbp window of the junctions to be phased (gGnome score.walks function). The walk (or set of walks) that carried the largest number of junction-supporting barcodes were considered the likeliest haplotype explaining the rearrangement.

[0188] Specifically with respect to the rDup and rDelDup patterns, two sets of possible derivative haplotypes exist: cis and trans. Cis haplotypes for rDup / rDelDup patterns are walks that harbor both involved rearrangements consecutively, or in other words, contain a single segment that is flanked by both junctions. Trans haplotypes are two separate walks that each harbor one of the two rDup or rDelDup rearrangements and would have to exist simultaneously, and thus are considered as a single set of walks. Junction-supporting LR barcodes were counted for each possible walk across every rDup / rDelDup instance in the LR WGS cohort. To assess whether the rDup or rDelDup patterns existed with the junctions 56 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 in cis or trans, walk-supporting LR barcode counts were compared among the individual cis walks and the trans walks summed together. The walk (or set of walks in the trans case) was taken to be the derivative haplotype underlying the rDup or rDelDup pattern. Haplotypes were also validated by visually assessing the barcode sharing patterns for each rDup / rDelDup present in the dataset to confirm the haplotype as labeled by this heuristic.

[0189] Imputing SRS reciprocal pair haplotypes

[0190] To impute the haplotype phase of reciprocal pairs identified by short-read WGS, we applied a threshold of 3.5 to the log10 gap width. Specifically, for rDup reciprocal pairs, the imputed haplotype phase was cis if the minimum of the two log10 gap widths was below 3.5, and trans otherwise. For rDelDup pairs, the imputed haplotype phase was cis if the log10 width of the (+)-polarity gap was less than 3.5, and trans otherwise. The imputed phase of all rDel pairs was trans because this is the only phase possible given the junction topology.

[0191] Sequence homeology

[0192] Homeology refers to approximate (>80%) similarity between a pair of genomic sequences. To assess sequences at junction-associated breakends, we applied a sliding bin approach. For every base within a 200 bp window around each breakend pair, a "kernel" of size 20 bases was queried for the corresponding hg19 reference sequence consisting of each base and the adjacent 19 bases. The kernels between each breakend were aligned to one another exhaustively, constructing a 200 by 200 matrix and the Levenshtein edit distance was calculated for each matrix cell. Each matrix cell thus represents a similarity between two 20 bases-wide sequences at a given distance from junction breakends. The matrix was converted into a binary image by converting each cell into a pixel representing whether the sequence pair represented in each cell has at least 80% similarity. Connected components in the image are ascertained and labeled with a linearity measure based on the Pearson's correlation of the pixel indices. Each junction is then annotated with the size of the largest connected component with a linearity of at least r2> 0.9. This value thus represents the longest contiguous stretch of bases with at least 80% sequence similarity. This procedure was run using the "homeology" function using the GxG R package (see Code availability).

[0193] Discordant and split reads supporting junctions with homeology were realigned to hg19 using bwa mem (implemented using an R wrapper in the package RSeqLib), to obtain uniform mapping quality scores for those cases harboring junction homeology within the in-house dataset in which alignments were present. Reference mappability was 57 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 determined using two orthogonal means. In the first, sliding 150-mers stepping by 1 base were queried across hg19 and aligned to the full reference using bwa mem to determine mapping quality scores. Average mapping quality was determined for each base for hg19. The second method used GEM mappability score with a sliding 150-mer across hg19 as described

[0194] Mutational signatures

[0195] Mutational signatures were derived from the signature.tools.lib R package suite for implementing the HRDetect algorithm2. Briefly, SNV signatures were deconvolved using the known signature weights from COSMIC SNV signature version 2 (https: / / cancer.sanger.ac.uk / signatures / signatures_v2 / , available through signature.tools.lib R package)65) with an implementation of non-negative least squares ("SignatureFit" function from the signature.tools.lib package). With the same approach, JaBbA-derived SVs were classified into the 32 SV types classified based on size, topology, and junction clustering as previously described24and were fit to rearrangement signatures derived from 560 breast cancers. Microhomology in small deletions was searched in 3' flanking sequence for up to 25 bases. The HRD-LOH index was determined by the number of segments per genome larger than 15 Mbp (but under the span of an entire chromosome) harboring loss of heterozygosity.

[0196] Classifying HR, BRCA1-, and BRCA2-deficiency

[0197] To build classifiers distinguishing overall HR-deficiency, BRCA1-deficiency and BRCA2-deficiency, random forests, RFs, (from the randomForest R package) were trained on a cohort of pan-cancer primary tumors consisting of 62 BRCA1-deficient, 66 BRCA2-deficient, and 2,536 controls that were confidently HR-proficient (lacking CLINVAR pathogenic, CLINVAR VUS, truncating, or missense mutation in BRCA1, BRCA2, RAD51, RAD51B / C / D, and PALB2 and LOH in BRCA1 or BRCA2). The following six features were counted for each case using the R package signature.tools.lib: COSMIC SNV signatures 3, 8, rearrangement signatures 3, 5, HRD-LOH index, and proportion of deletions with microhomology. rDups, rDels, and rDelDups were additionally counted after annotation upon each genome graph.

[0198] To evaluate performance of random forests, receiver-operating characteristic (ROC) curves and corresponding areas under the curve (AUCs) were computed on an independent test cohort of pan-cancer metastatic tumors (HMF cohort) consisting of 40 BRCA1-deficient, 92 BRCA2-deficient, and 1,834 HR-proficient controls using the pROC 58 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 R package (v1.18.0, https: / / cran.r-project.org / web / packages / pROC / ). Feature importance was determined by resampling the test cohort across 30 bootstraps with permutation. Decrease in accuracy upon permuting each feature on the test cohort was calculated.

[0199] In the following two comparisons, classifier skill to discriminate overall HR- deficiency from HR-proficiency was analyzed by using the full training cohort and evaluating the resultant models on the full testing cohort. An SV only RF was trained on rDups, rDels, rDelDups, homeologous deletions, duplications with length 10-100kbp, RS3, and RS5 as features and compared against an RF trained on rearrangement signatures 3 and 5 as features to compare the efficacy of the classes of SVs described in this manuscript against the established SV types previously used in HRDetect2. A full RF consisting of currently described features and previously established features (rDups, rDels, rDelDups, homeologous deletions, duplications with length 10-100kbp, RS3, RS5, MH-dels, SNV3, SNV8, and LOH score) was trained and then tested against the published HRDetect model (consisting of MH-dels, SNV3, SNV8, RS3, RS5, and LOH score as features) using ROC curves and feature importance metrics. HRDetect scores were obtained by running the function "applyHRDetectDavies2017" from the signature.tools.lib R package on a feature matrix composed of test samples.

[0200] The third comparison evaluated classifier skill to discriminate BRCA1- deficiency from BRCA2-deficiency. For this test, the full RF trained with current and previous features was used to compare against a RF trained with HRDetect-only features. In contrast to the above, ROC and feature importance evaluation was performed on only the 40 BRCA1d and 92 BRCA2d cases from the test cohort.

[0201] Statistical information

[0202] All statistics were performed as stated in figure legends using the R programming language (v.4.0.2). P values obtained that are smaller than 2.2E-16 are not accurately estimated in R and are listed as such ("P value < 2.2E-16"). Generalized linear modeling was performed using "glm" or "glm.nb" function from the stats or MASS R package. Wilcoxon rank sum testing performed using "wilcox.test" function from the stats R package. Fisher's exact test was performed using the function "fisher.test" from the stats R package. Receiver-operator curves (ROC) were generated using the function "roc" from the R package "pROC". Comparison ROC curves was done using the function "roc.test" from R package "pROC" with argument "method = 'delong'".

[0203] Code availability 59 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0204] Analyses were performed using R-4.0.2 with R packages available from CRAN (https: / / cran.r-project.org / ). The following lists R packages developed by authors to perform the described analyses. Genome-wide coverages for samples for which a BAM alignment was present were calculated with the fragCounter R package (github.com / mskilab / fragCounter). Fitting of junction-balanced genome graphs was carried out using JaBbA R package (github.com / mskilab / jabba)17. Analysis of junction links and link clusters as well as classification of complex event types within each genome graph was performed with the function "eclusters" in the package gGnome (github.com / mskilab / gGnome). Walk deconvolution on genome graphs was also performed using gGnome.10X LR barcodes supporting junctions were queried using the "score.walks" function in the skitools R package (github.com / mskilab / skitools). Visualization of genomic tracks were made with the gTrack R package (github.com / mskilab / gTrack). Analysis of sequence homeology across junction breakends is implemented with the function "homeology" in the package GxG (github.com / mskilab / GxG). Custom tools for miscellaneous data manipulation tasks were implemented using the package khtools (github.com / kevinmhadi / khtools). Example 2: Genome graph analysis of HR-deficient and HR-proficient cancers

[0205] To investigate the role of complex SVs in HR-deficient cancers, we assembled a cohort of 979 predominantly primary (95%) cancer WGS profiles from four tumor types commonly associated with HR-deficiency (breast, ovary, prostate, and pancreas cancer; referred to as “BOPP” moving forward, see Methods and FIG.17)20. We next sought to confidently identify BRCA1d, BRCA2d, and HR-proficient cases in this BOPP cohort; we required biallelic inactivation of BRCA1 or BRCA2 for a tumor to be classified as BRCA1d (n=24) or BRCA2d (n=36) respectively21(see Methods). We also identified 487 HR proficient BOPP samples that lacked pathogenic or rare variants in any HR-associated gene (e.g. BRCA1, BRCA2, PALB2, RAD51C; see Table 1 for full list). We excluded the remaining 432 BOPP cases, which comprised tumors with monoallelic alterations and / or variants of unknown significance (VUSs) in BRCA1 or BRCA2 or mutations in other HR- associated genes. We then compared SV patterns between BRCA1d, BRCA2d, and HR- proficient tumors using state of the art methods that integrate copy number changes and rearrangements across genome graphs17.

[0206] Analyzing the burden of individual simple SV classes between BRCA1d, BRCA2d, and HR-proficient tumors, we confirmed the previously observed enrichment of short (1-100 Kbp) SV duplications (P = 1.49E-11, Relative Risk (RR) = 27.4) in BRCA1d 60 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 cancers and deletions (BRCA1: P = 1.49E-6, RR=3.52, BRCA2d: P = 2.87E-21, RR = 7.82) in both BRCA1d and BRCA2d cancers (FIG.6A). Although BRCA1d and BRCA2d cancers had higher SV burdens than HR-proficient cancers (BRCA1: RR 5.00, p = 5.29E-9, BRCA2: RR = 2.31, 1.50E-4, FIG.6B), we found no significant difference in the burden of simple translocations and inversions (FIG.6A) as has been previously noted3.

[0207] We next asked whether HR-deficient tumors were enriched in specific classes of complex SVs. We found no significant difference in the burden of seven previously characterized complex SV categories (including breakage fusion bridge cycles, chromothripsis, and double minutes17in BRCA1d or BRCA2d relative to HR-proficient tumor samples (FIG.6C). Contrary to the commonly held assumption that HR-deficient cancers are exceptionally rearranged compared to HR-proficient cancers, we found that they harbor similar burdens of most SV classes, including complex SVs. Templated insertion chains (TICs), however, were significantly enriched among both BRCA1d and BRCA2d relative to HR-proficient tumors (BRCA1d, P < 2.2E-16, RR = 6.56; BRCA2d, P = 2.5E-12, RR = 2.96, Wald test on gamma-Poisson regression; FIG.6C). Example 3: HR-deficient cancers harbor distinct patterns of reciprocal SVs

[0208] TICs are a broad class of complex rearrangements whose junctions are approximately reciprocal, forming complex chains or cycles in the genome15. They are thought to arise from the copying and pasting of smaller (<10kb) and genomically dispersed DNA segments in between larger (megabase-scale) segments15. TICs are distinct but topologically related to chromoplexy (chains of balanced translocations), of which classic balanced translocations and inversions are a special case19. Despite their enrichment in BRCA1d and BRCA2d cancers, TICs were still found in a substantial fraction (36%) of HR-proficient tumors (FIG.6D) and thus were not pathognomonic for HR-deficiency. We hypothesized that a more comprehensive analysis of reciprocal junctions might yield uniquely HR-deficiency specific patterns.

[0209] A classic reciprocal rearrangement (i.e. balanced translocation or inversion) occurs without loss or gain of genetic material and involves a pair of DNA junctions with breakends that adjoin the same breakpoint (FIG.1A, left). However, many rearrangements, including translocations and inversions, are approximately reciprocal with breakends that are nearby but not adjacent on the genome. While these events are mostly balanced, they harbor a copy loss or gain at the intervening segment, which we call a gap segment (FIG. 1A, middle). The direction of copy loss vs. gain at the gap segment is determined by its polarity, which by conservation of mass yields a copy gain when breakends join the gap 61 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 segment (+ polarity) and copy loss when breakends join the flanking segments (- polarity). For a (+) gap segment, the identical locus topology and copy number profile may additionally be equally consistent with a translocation or a simple templated insertion, where a gap segment is copied to a distant locus and leaves the source locus unaltered (FIG. 1A, right).

[0210] In expanding this definition of reciprocality, we sought to identify clusters of junctions that were likely to have occurred together in tumor evolution. While junctions with breakends that are a few bases apart (short gap segments) are likely to have occurred at the same time, breakends that are several megabases apart (long gap segments) may have occurred independently, particularly in highly rearranged genomes. To screen our data for new reciprocal SV patterns, we divided our cohort of 547 BRCA1d, BRCA2d, or HR- proficient BOPP tumors into a discovery (n = 283 tumors with 9 BRCA1d, 23 BRCA2d, 251 HR-proficient) and validation set (n = 264 with 15 BRCA1d, 13 BRCA2d, and 236 HR-proficient tumors, FIG.17). Analyzing 473,382 gap segments across the discovery set, we identified 1 Mbp as a gap segment length that was consistently (FDR<0.1) unlikely to have occurred independently under an exponential null model (FIGs.7A-7B, see Methods). Linking rearrangement junctions under this reciprocal gap segment length threshold revealed simple paired patterns (e.g. balanced translocations with short 6-7 bp (-) polarity gap segments) as well as more complex cyclic and non-cyclic reciprocal SV topologies comprising (+) and / or (-) polarity gap segments (FIGs.7C-7D).

[0211] Comparing reciprocal SV topologies across genotypes, we found that paired and cyclic patterns were most significantly enriched in BRCA1d ( ^^^^ = 10.17 events per case, RR 4.72, P = 3.6E-12, Wald test on gamma-Poisson regression) and BRCA2d cancers ( ^^^^ = 5.59, RR 4.73, P = 5.7E-11) relative to HR-proficient ( ^^^^ = 1.13) (FIG.7E). We call these relatively simple patterns reciprocal pairs. Notably, we did not find genotype-specific differences in non-cyclic or higher-order reciprocal SVs, comprising higher-order templated insertion events and chromoplexies (FIGs.7C-7E). Visualizing reciprocal pair gap segment lengths and polarities revealed three distinct BRCA1- and / or BRCA2-deficiency specific subpatterns (FIG.1B). The first was an enrichment in BRCA1d tumors of reciprocal pairs with two 100-1000 bp (+) polarity gap segments, which we called reciprocal duplications (rDups, FIG.1C, left). The second was an enrichment in BRCA2d tumors-of reciprocal pairs with two 1 bp - 10 Kbp (-) gap segments, which we called reciprocal deletions (rDels, FIG.1D, left). The third was an enrichment in BRCA1d and BRCA2d cases of reciprocal 62 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 pairs comprising 1bp - 100 Kbp gap segments of opposite (+) and (-) polarity, which we call reciprocal deletion-duplications, (rDelDups, FIG.1E, left). Inspection of individual reciprocal pair loci (FIGs.1C-1E) confirmed that these occurred in genomic regions that otherwise did not harbor other rearrangements within a 1 Mbp vicinity.

[0212] Analysis of left-out BOPP cohort samples (15 BRCA1d, 13 BRCA2d, 236 HR- proficient, n = 264 tumors) revealed significantly higher burdens of rDups in BRCA1d ( ^^^^ = 6.87 per case) relative to BRCA2d ( ^^^^ = 1.08, P = 2.0E-7, RR = 9.90, Wald test on gamma- Poisson regression) and HR-proficient ( ^^^^ = 0.13, P = 6.7E-14, RR = 44.81) tumors (FIG. 1C, right). Conversely, rDels were significantly enriched in BRCA2d ( ^^^^ = 1.92 per case, P = 7.3E-5, RR = 4.29) relative to HR-proficient ( ^^^^ = 0.67) tumors (FIG.1D, right). Finally, both BRCA1d (P = 5.0E-9, RR = 8.18) and BRCA2d (P = 1.4E-9, RR = 6.55) tumors harbored significantly higher burdens of rDelDups ( ^^^^ = 2.20 and 2.00 per case, respectively) than HR-proficient cancers ( ^^^^ = 0.32, FIG.1E, right). Example 4: Linked-read WGS confirms BRCA1- and BRCA2-deficiency specific reciprocal patterns

[0213] Nearly identical rearrangement topologies can, in theory, have very distinct chromosomal outcomes, or phases (FIG.1a). We noted that the topology and copy number profile of reciprocal pairs was consistent with either of two outcomes: (1) the templated insertion of a small (around 1 bp - 100 Kbp) segment to an ectopic site or (2) a larger balanced rearrangement such as a translocation or inversion (FIG.8A). We additionally observed that most reciprocal pairs comprised long-range (either interchromosomal or >10 Mbp intrachromosomal) junctions (FIG.1F). This indicated that, depending on the outcome, reciprocal pairs could have either a minimal or major impact on chromosomal structure.

[0214] To resolve this aspect of reciprocal pairs, we performed linked-read (LR) and standard WGS for 46 tumors originally found to have inherited or somatic mutations in BRCA1 (27 cases) or BRCA2 (19 cases; Table 1, FIG.17, FIG.8B) on panel sequencing and their matched normal samples. Re-analysis of these cases with WGS identified 22 BRCA1d and 14 BRCA2d cases, as well as one double-deficient (BRCA1d and BRCA2d) case, based on the LOH and allelic status of pathogenic alterations in BRCA1 and BRCA2. The remaining 9 cases were found to have only monoallelic mutations in BRCA1 (n=5) BRCA2 (n=4) upon WGS analysis and were excluded from BRCA1d, BRCA2d, and HR- proficient comparisons. LR WGS provided deep (median 149X) genome-wide physical 63 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 coverage of tumor and normal samples through barcoded short-read sequencing of long DNA molecules (median length 24.4 Kbp; FIG.8C). We reasoned that long molecules would help resolve reciprocal pairs into phased somatic haplotypes and provide insight into the mechanistic origin and outcome of these SVs.

[0215] We identified 186 reciprocal pairs among BRCA1d and BRCA2d cases ( ^^^^ = 5.17 per case). Comparison of standard and LR WGS profiles showed concordant rDel, rDup, and rDelDup calls (83.5% overlap, FIG.8D) though LR WGS identified 29 additional reciprocal pairs. Confirming results from the BOPP short-read WGS cohort, BRCA1d tumors had higher burdens of rDups than BRCA2-deficient tumors (P = 1.95E-4, RR = 49.50, Wald test on gamma-Poisson regression), and rDups were found in most (82%) BRCA1d tumors but only 1 BRCA2d tumor. Similarly, rDels were present in most (71%) BRCA2-deficient tumors but absent in all but 4 BRCA1d tumors (P =1.85E-7, RR = 11.67, FIG.8E). Example 5: Somatic phasing of reciprocal pairs reveals both cis and trans configurations

[0216] To assess whether reciprocal pairs could be responsible for large scale rearrangements in HR-deficient cancers, we inferred their derivative chromosomal structure, or phase (FIG.8A). Our specific goal in these analyses was to distinguish between cis (copy-paste, templated insertion) outcomes and trans (balanced translocation or inversion) outcomes on the basis of LR WGS alignment patterns (FIG.2A, FIG.8A, see Methods). Somatic phasing of cancer variants is distinct from germline phasing; germline cis refers to variants that arose on the same parental homologue, while somatic cis refers whether variants are located next to each other on a cancer chromosome. Benchmarking analyses showed that we were able to distinguish between cis vs. trans configurations with high accuracy (>98%) across the range of LR molecule sizes and locus structures observed in our data (FIG.9A, see Methods). Applied to our data, we found that the raw LR WGS alignment patterns were generally unambiguous in their support for a cis vs. trans allelic configuration and consistent with the inferred calls (FIGs.2B-2C, FIG.9B).

[0217] Phasing of 94 rDups in BRCA1d samples revealed a predominance (67 / 94, 71%) of cis phases (FIGs.2B, 2D), each resulting in the copy-pasting of a gap segment in between the tandem duplicated gap segments of a distant locus. For example, given two loci ABC and DEF, this would yield an ABEBC haplotype harboring the variant BE and EB junctions in tandem, while leaving the other DEF haplotype un-rearranged (FIG.2B). The remaining (29%) of loci harbored trans configurations (FIGs.2C-2D) where the BE and EB junctions were placed on discontiguous (for interchromosomal rDups) or distant (for 64 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 intrachromosomal rDups) rearranged alleles. In these outcomes, two distinct derivative alleles ABEF and DEBC shared the duplicated B and E segments. This included balanced translocations with up to ~20 Kbp of duplicated sequence at the junction (FIG.2C).

[0218] Similarly, we used LR WGS to phase 46 rDelDups across 37 cases (22 BRCA1d, 14 BRCA2d, and 1 both BRCA1d and BRCA2d). As with rDups we found both cis and trans phases at various loci (FIGs.9C-9D), though with a ~2:1 trans predominance in BRCA1d tumors and ~4:1 cis predominance in BRCA2d tumors (FIG.2D).

[0219] Given ABC and DEF loci, cis rDelDups comprised a “cut, copy, and paste” outcome with an additional copy of E replacing B on a derivative AEC allele, with an unrearranged DEF locus harboring the other E copy; in contrast, trans loci showed the same (1-241 Kbp) E segment duplicated across two distinct DEC and AEF derivative loci. Finally BRCA2d tumor-specific rDels were predicted by short-reads to give rise to strictly trans outcomes, which was confirmed on LR WGS (FIG.2D, FIGs.9E-9F). These results demonstrate that trans reciprocal pairs are frequent among rDups, rDels, and rDelDups and may serve as a source of large scale rearrangements in BRCA1d and BRCA2d tumors. Example 6: Chromosomal impact of cis and trans reciprocal pairs

[0220] Given the distinct chromosomal outcomes of cis vs. trans reciprocal pairs, we sought features that would distinguish these loci in short-read WGS and thus enable the study of reciprocal pair phase across a larger cohort. Deeper analysis and visualization of the phased structure of cis rDups (FIG.3A, top) revealed that a short (50bp-1kbp) E segment was predominantly interleaved between two copies of a long (1-300 Kbp) B segment in an ABEBC configuration. Trans rDups, however, comprised pairs of longer (1kbp-300 Kbp) B and E segments in ABEF and DEBC haplotypes (FIG.3A, bottom).

[0221] Plotting these LR WGS phased cis and trans loci alongside unphased data from the short-read WGS BOPP cohort, oriented to the length of the longer gap segment in each pair (y axis), revealed two distinct rDup clusters. The first “long-short” rDup cluster involved the linking of longer (1-100 Kbp) and shorter (10bp - 1Kbp) gap segments, and comprised exclusively cis events. In contrast, trans rDups were entirely contained in the second “long-long” cluster (FIG.3B). A similar pattern was seen with cis rDelDups where short (10bp - 1Kbp) ectopic E segments were incorporated in cis while longer (1kb - 100 Kbp) E segments were incorporated in trans (FIG.10A). This mirrored the multi-modal distribution of (+) gap segment lengths in unphased rDelDups (FIG.10B). These results indicated that simple thresholds on gap segment lengths might be used to impute reciprocal pair phase in short-read WGS. Specifically, we found that a threshold of 3,162 bp on the 65 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 length of the smaller (+) polarity segment gave approximately 73% specificity and 75% sensitivity for imputing rDel and rDup phase (FIG.10C, see Methods).

[0222] Since trans reciprocal pairs may engender long range SVs (e.g. balanced translocations and inversions), we predicted that trans but not cis events would be constrained in their chromosomal orientation. Specifically, trans reciprocal pairs can occur in one of two centromeric orientations; the first (Type I) orientation generates only monocentric chromosomes, while the second (Type II) generates one or more acentric derivatives (FIG.3C). Since acentric DNA fragments are prone to loss in subsequent cell divisions, a junction residing on an acentric fragment will be preferentially lost and the remaining junction will not be detected as a reciprocal pair. This will result in a Type I bias for trans reciprocal pairs. Conversely, since templated insertions do not alter the chromosomal dosage of centromeres, cis loci should be agnostic to Type I vs. Type II orientation.

[0223] Indeed, analyzing our LR WGS phased data, we found that trans loci had an over 9:1 Type I vs. Type II bias while cis loci were equally likely to be in either Type I or Type II orientation (P = 3.9E-6, OR = 8.57, Fisher’s exact test, FIG.3D, left). We next analyzed unphased reciprocal pairs in the short-read WGS BOPP cohort, imputing cis and trans phase on the basis of size and orientation (FIGs.3A-3B, FIGs.10A-10C, see Methods). We found the same bias in these data (P = 1.4E-4, OR = 1.96, FIG.3D, right), despite having only approximate phases. In particular, these analyses showed that intrachromosomal trans reciprocal pairs yield megabase-scale inversions similarly constrained by centromere dosage. Taken together, these results suggest that trans reciprocal pairs have large-scale chromosomal consequences, and thus can be implicated in cytogenetically visible aberrations classically associated with BRCA1 and BRCA2 inactivation.

[0224] A non-specific genomic hallmark of HR-deficiency is LOH22. We surmised that trans reciprocal pairs might be a source of LOH in two scenarios: (1) If a balanced translocation was followed by loss of one of the derivatives leading to telomeric LOH (e.g. via mitosis) and (2) if a type II intrachromosomal reciprocal pair gave rise to a circular acentric segment resulting in an interstitial LOH (FIG.3E). To assess whether reciprocal pairs may serve as LOH precursors, we assessed gap segment to telomere distances for interchromosomal reciprocal pairs and gap segment to gap segment distances for intrachromosomal pairs and found a close correspondence between these distributions and 66 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 observed LOH length distributions (FIG.3F). Although such megabase-scale alterations are not unique to HR-deficient cancers, their quantitative enrichment in such genomes is the basis of several currently employed clinical assays of HR-deficiency14. Our data suggest that trans reciprocal pairs likely underpin a substantial fraction of such LOH events. Example 7: Replication restart model of reciprocal pairs

[0225] The observation of distinct cis and trans phases arising from nearly identical junction topologies (e.g. rDups) suggested that they may represent distinct outcomes of a shared DNA repair intermediate. Notably, most reciprocal pairs joined distant genomic locations (FIG.1F, FIG.2D) and had minimal (<3bp) sequence homology (FIG.10D), suggesting a possible homology-independent repair mechanism. The aberrant restart of a broken replication fork23has been implicated in the genesis of ~10-100 Kbp tandem duplications in BRCA1d tumors24. Indeed, a key role of HR in human cells is in the repair of single-ended DSBs (seDSB), which can arise at stalled replication forks25–28. This suggests that additional HR-deficiency specific SV scars, such as reciprocal pairs, may arise as an aberrant HR-independent response to a seDSB.

[0226] Exploring the possibility of a shared mechanism between tandem duplications and reciprocal pairs, we analyzed their distributions in our data. Notably, tandem duplications (tDups), rDups, and rDelDups frequently co-occurred in BRCA1d cases (rDups, found in 82% of cases with tDup, R2= 0.32, P = 5.5E-5, Correlation test with linear model; FIG.10E; rDelDups, 80% of cases with tDup). In addition, the size distribution of the longer (+) gap segments in rDups (median 8.0 Kbp) or rDelDups (median 9.0 Kbp), but not the shorter (+) gap segments in rDups, closely mirrored that of BRCA1d tumor-specific tandem duplications (median 12 Kbp; FIG.10F). Although the underlying mechanism will require experimental confirmation, we found that several simple extensions to the replication restart model could explain the full spectrum of reciprocal pair alterations, including rDups (FIG.3G), rDelDups (FIG.11A), and rDels (FIG.11B).

[0227] Replication restart (e.g. break induced replication; BIR) mechanisms are known to be prone to template switching and half-crossovers, and have been previously linked to templated insertions in cancer cells26,29–31see FIG.11. A key decision point between template switching and half-crossover in BIR rests on the fate of the displacement loop following strand invasion26,32. Factors that mediate displacement loop disassembly or nascent strand displacement channel BIR toward template switching with shorter (<5 Kbp) replication tracts whereas factors that stabilize the displacement loop are known to favor long-tract synthesis and half-crossover formation33. In our data, rDups and rDelDups with 67 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 short ectopic (E segment) tracts were exclusively in cis, consistent with a template switch (FIGs.3A-3D). Additionally, for BRCA1-deficiency associated cis rDups, the shorter E segment was always copied in between two longer B segment copies (FIG.3A). In contrast, only half-crossover outcomes (trans rDups and rDelDups) were observed alongside longer E (1-100Kbp) segments (FIG.3A-3B). In both outcomes, the rDup B segment length distribution mirrored the size distribution of tandem duplications in BRCA1d tumors (FIG. 3A, FIG.10F, FIG.11).

[0228] Taken together, our observations suggest reciprocal SVs can be found in both cis and trans forms, with topological and tract length characteristics previously associated with half-crossover and templated insertion outcomes of BIR32,33. We propose a provisional model invoking microhomology-mediated BIR (FIG.3G, FIG.11), extended from experimentally validated models established for BRCA1d-associated tandem duplications23, that plausibly accounts for the full spectrum of reciprocal pairs. Example 8: Footprints of single strand annealing (SSA) in BRCA2-deficient tumors

[0229] Given our LR WGS data, we posited that additional scars of HR-deficiency- specific repair pathways could be detected using long molecule mapping. Single-strand annealing (SSA) is a DSB repair pathway that involves the hybridization of approximately homologous (homeologous) repeat sequences flanking a DSB. Experimental model systems of HR-deficiency have long shown that SSA is active in BRCA2d but not BRCA1d cancers34–36. Since SVs occurring in regions of long sequence homology are considered to be unmappable with short-reads, the contribution of SSA to SV patterns in human HR- deficient cancers has not been previously investigated.

[0230] SSA can tolerate as little as 80% sequence identity when annealing similar sequences deep inside resected breakends36, however, previous genome studies of HR deficiency have only analyzed exact microhomology and have not examined inexact sequence identity. To better assess the burden of SSA in LR WGS profiles, we developed an approach (see Methods) to characterize homeology, or ≥ 80% sequence identity near somatic breakends. Specifically, we exhaustively quantified the fraction of matched bases in an optimal pairwise alignment across pairs of sliding 20 bp bins in the vicinity of breakends. We used these results to build raster images of homeology, which we found to harbor horizontal stretches of pixels representing runs of >80% sequence similarity in the human genome reference (FIG.4A). To identify these patterns automatically, we built a simple image analysis algorithm (see Methods) to quantify homeology run length for candidate junctions. Application of this method to 7,098 breakends across 46 LR WGS samples 68 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 revealed a peak of homeology around 50 bp, spanning from 5 to 200 bp (FIG.4B) and distinct from 0. We thus applied a threshold of 10 bp to call homeologous junctions, which yielded 138 homeologous junctions across 46 LR WGS samples.

[0231] We next asked whether homeologous junctions could be detected in short-read WGS. While only 33 of 133 of LR WGS-derived homeologous junctions were found in the matched short-read WGS tumor data, of 136 homeologous junctions detected by short-read WGS, 99 (73%) showed robust LR WGS support upon reinspection (FIG.4A, see Methods). In both short-read and LR WGS, homeologous junctions were supported by high mapping quality reads (>75% with MAPQ=60 (FIG.12A). Additional, comparison of reference sequence in the vicinity of breakends showed similar fractions of unmappable bases between homeologous and non-homeologous junctions (FIG.12B). These results indicate that the sequence divergence between homeologous breakend sequences was sufficient for the joined sequences to be distinguishable using short-reads. We therefore expanded our analysis of homeology to the full short-read WGS BOPP cohort.

[0232] Analysis of 583 tumor / normal pairs (BRCA1d, BRCA2d, or HRP) revealed 1,248 of 49,561 (2.5%) junctions were homeologous, with a distribution that mirrored LR WGS (FIG.4C). Whilst the median homeology length among these junctions was 40bp, we observed tracts as long as 128bp. We next asked which classes of simple or complex variants harbored homeologous junctions. Comparing distributions across genotypes revealed that BRCA2d tumors had a significantly higher burden and fraction of larger (>1 Kbp) homeologous deletions relative to BRCA1d (RR = 3.93, P = 4.2E-4, Wald test on gamma-Poisson regression) and HR-proficient cancers (RR = 3.28, P = 8.37E-6, Wald test on gamma-Poisson regression, FIG.4D). While we also observed homeologous breakends in other SV classes, the burden of these events did not correlate with HR-proficiency status (data not shown). Notably the median size of (~10 Kbp FIG.4E) of homeologous deletions was consistent with the length of end resection known to occur in BRCA2dsupporting the role of SSA as a backup repair pathway in human BRCA2d tumors (FIG. 4F). Example 9: Reciprocal pairs and homeologous deletions improve BRCA1- vs. BRCA2- deficiency classification

[0233] Having identified SV footprints of backup repair pathways specific to BRCA1d and BRCA2d cancers, we next sought to understand whether these features could improve pan-cancer HR-deficiency classification. One motivation for WGS based classifiers of HR deficiency, which include HRDetect and CHORD2,3, is to identify exceptional responders 69 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 to DNA damaging agents including PARP inhibitors. Notably, these previous algorithms have not used reciprocal pairs nor homeologous deletions as features for HR deficiency classification.

[0234] To assess the predictive value of the SV features highlighted in our study, we built a classifier augmenting the six features used by HRDetect (deletions with microhomology, mhDel; COSMIC single base signature 3 and 8, SBS3 and SBS8; rearrangement signatures 3 and 5, RS3 and RS5, loss of heterozygosity, LOH) with the five highlighted by our study (1-100 kb tandem duplications, rDups, rDelDups, and rDels, homeologous deletions). This yielded an 11 feature random forest classifier called B1+2 (Methods). We augmented our short-read-only WGS BOPP cohort with LR WGS breast cancer cases and 2,662 pan-cancer WGS profiles giving a pan-cancer training set of 62 BRCA1d, 64 BRCA2d, and 2,536 HR-proficient cases. Training B1+2 to predict the probability of BRCA1d (B1 score), BRCA2d (B2 score), and HR-proficient labels across these data revealed only marginal improvement over HRDetect in an independent test set of 1,966 BRCA1d, BRCA2d, and HR-proficient pan-cancer WGS profiles from the Hartwig Medical Foundation (HMF, B1+2, AUC = 0.98, AUPRC = 0.87; HRDetect AUC = 0.97, AUPRC = 0.86, FIG.13A). Indeed, as with HRDetect, classification was primarily driven by the fraction of indels with microhomology (FIGs.13B-13C), and scores were largely similar between the two algorithms (FIG.13D).

[0235] Given that HR deficiency is a disorder of DSB repair and altered genome structure, we next asked how well HR deficiency could be predicted solely on the basis of breakpoint-level structural features. Our goal was to understand whether the B1+2 classifier-specific SV features highlighted in our study, taken together, were independent of those features assessed by the SV features in HRDetect (RS3 and RS5). To assess this, we compared the performance of a random forest classifiers trained using only the SV features in HRDetect (RS3 and RS5) and those additionally used in B1+2 (homeologous deletions, reciprocal pairs, 1-100 Kbp simple duplications). In this analysis, we found substantially better performance (P < 2.2E-16, DeLong test) using a classifier augmented with B1+2 SV features (AUC = 0.93, AUPRC = 0.565, pan-cancer HMF) relative to one using only RS3 and RS5 (AUC = 0.73, AUPRC = 0.323, FIG.5A). Though certain B1+2 classifier- specific SV features were important on their own, we found that these were particularly important in combination (FIGs.5B-5C). The performance improvement was mostly 70 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 clearly attributable to the B1+2-specific SV features recognizing BRCA2d tumors (FIG. 13E).

[0236] We next asked whether B1+2 could distinguish between BRCA1- and BRCA2- deficiency, which are distinct biological states each with possibly distinct therapeutic vulnerabilities37. Since HRDetect was not developed to address this task, we compared B1+2 to a random forest classifier trained on 6 HRDetect features (see above). We found that B1+2 substantially outperformed (AUC=0.92, AUPRC = 0.91) this HRDetect-like classifier (AUC=0.8, AUPRC = 0.83) in distinguishing BRCA1d from BRCA2d tumors (P = 0.005, DeLong test; FIG.5D). B1+2 classifier-specific SV features were particularly important for making this distinction in non-BOPP cancers (FIGs.5E-5F). CHORD3is a more recently published algorithm that also distinguishes between BRCA1 and BRCA2 deficiency. We found a similar performance improvement when augmenting CHORD with the five B1+2 specific features (FIG.14A). These features were also the most important for distinguishing BRCA1 vs BRCA2 deficiency in this augmented CHORD classifier (FIGs.14B-14C). Analyzing B1 vs. B2 biases across B1+2 positive cases (B1+B2>0.5) confirmed that prostate and pancreatic cancer HR-deficiency is significantly enriched in the BRCA2d phenotype relative to breast and ovarian cancer, where BRCA1- and BRCA2 deficiency are equallyFIG.5G). Extending this analysis to non-BOPP samples, we found a lower rate (<5%) of HR-deficiency but with increased bias toward BRCA2- deficiency (hepatocellular carcinoma, sarcoma, FIGs.14D-14E).

[0237] An important use of HR-deficiency genomic signatures is to uncover alternate mechanisms of HR pathway inactivation and assess the pathogenicity of VUSs. To assess this, we investigated B1+2 score positivity in cases excluded from our training and test data, namely those with monoallelic alterations and VUS’s in BRCA1 and BRCA2 as well as cases harboring alterations other HR pathway genes (i.e. PALB2, RAD51C, and 36 other genes, Table 1). Indeed, we found a significantly higher prevalence of B1+2 positivity (score > 0.5) in these cases (12-25%) relative to HR-proficient (<1%) (FIG.5I, BRCA1 / 2 monoallelic or VUS: OR 27.3, P < 2.2E-16; non-BRCA1 / 2 HR pathway genes: OR 32.6, P < 2.2E-16, Fisher’s Exact Test), though substantially lower than for biallelic BRCA1d or BRCA2d cases (95%). This included genes with distinct biases for BRCA1d (BARD1, EME1) vs. BRCA2d (PALB2, RAD51C), consistent with their known HR pathway roles (FIG.14F). These results indicate that the B1+2 classifier may help uncover and subclassify new pathogenic alleles responsible for HR-deficiency. 71 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0238] To further assess the relevance of classifier results, we obtained clinical outcomes for four cases with elevated B1+2 scores among 80 WGS cases profiled at Weill Cornell Medicine (see Methods). Adequate clinical follow up was available for three subjects, all with aggressive cancers associated with poor prognosis demonstrating favorable responses to platinum chemotherapies and / or PARP inhibition (FIGs.15-16). This included a B2-high (B2 = 0.912) de novo metastatic neuroendocrine prostate cancer case (FIG.5J) with an atypical (20.7 months) extracranial complete response to first-line platinum doublet (cisplatin / docetaxel) therapy and a second complete response to platinum rechallenge. Overall survival for this case (37 months) and a second pancreatic adenocarcinoma case (WCM1341, 32 months) exceeded expectation (<1 year) for tumors of this histology and stage. A second pancreatic adenocarcinoma (WCM1462, B2 score = 0.31) with two pathogenic germline and somatic alterations in BRCA2 and an excess of homeologous deletions showed a remarkable 59 month recurrence-free survival after platinum adjuvant therapy and limited maintenance rucaparib. While all three cases also showed high HRDetect and CHORD scores, B1+2 provided additional certainty in distinguishing BRCA1d vs BRCA2d (FIG.5J, FIG.15) consistent with our benchmarking data (FIG.14A). These results motivate the use of B1+2 and more broadly WGS-based HR deficiency classifiers in the clinic.

[0239] These results demonstrate that the RP-based screening methods disclosed herein are useful for selecting cancer patients for treatment with PARP inhibitors or platinum agents. Example 10: Tailoring Treatment in BRCA1d vs BRCA2d patients

[0240] Although at the present time, clinical therapies based on DNA repair defects have grouped all DNA repair defects under a single class of tumors, we are starting to realize that matching the therapy to a specific DNA repair defect will optimize the effectiveness of therapy. For example, we know that tumors with an ATM mutation are not optimally sensitized with PARP-inhibitors (Marshall et al., Eur Urol.2019 Oct;76(4):452- 458). The use of genetic screens, where every gene can be depleted individually in cells, has revealed that genes that provide a lethal impact for BRCA2-deficiency (e.g., REV7) can have a survival impact for BRCA1-deficiency. If any of these genetic targets would translate into a druggable target in the future, the genetic distinction between BRCA1- deficiency and BRCA2-deficiency would become important. Therefore, the genomic classifiers of BRCA1d vs BRCA2d could have important diagnostic value in the future. EQUIVALENTS 72 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014

[0241] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0242] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0243] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0244] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. 73 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 REFERENCES 1. Ma, J., Setton, J., Lee, N. Y., Riaz, N. & Powell, S. N. The therapeutic significance of mutational signatures from DNA repair deficiency in cancer. Nat. Commun.9, 3292 (2018). 2. Davies, H. et al. HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures. Nat. Med.23, 517–525 (2017). 3. Nguyen, L., W M Martens, J., Van Hoeck, A. & Cuppen, E. Pan-cancer landscape of homologous recombination deficiency. Nat. Commun.11, 5584 (2020). 4. Setton, J., Reis-Filho, J. S. & Powell, S. N. Homologous recombination deficiency: how genomic signatures are generated. Curr. Opin. Genet. Dev.66, 93–100 (2021). 5. Robson, M. et al. 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Claims

Atty. Dkt. No.115872-3014 CLAIMS 1. A method for selecting a cancer patient for treatment with a PARP inhibitor or a platinum agent comprising: (a) detecting levels of reciprocal rearrangements in a biological sample obtained from the cancer patient that are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of a PARP inhibitor or a platinum agent.

2. A method for prolonging survival of a cancer patient comprising administering to the cancer patient an effective amount of a PARP inhibitor or a platinum agent, wherein levels of reciprocal rearrangements in a biological sample obtained from the cancer patient are elevated relative to a control sample obtained from a healthy subject or a predetermined threshold.

3. The method of claim 1 or 2, wherein the levels of reciprocal rearrangements are detected via next-generation whole-genome sequencing.

4. The method of any one of claims 1-3, wherein the cancer patient is diagnosed with, or is at risk of having a homologous recombination deficiency.

5. The method of any one of claims 1-4, wherein the cancer patient harbors a mutation in one or more driver mutations selected from among BRCA2, BRCA1, PALB2, RAD51C, RAD51B, IDH1, CDK4, RB1, FIGNL1, MDC1, BARD1, and RBBP8.

6. The method of any one of claims 1-5, wherein the cancer patient is suffering from a cancer selected from among breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

7. The method of any one of claims 1-6, wherein the cancer patient exhibits stage I, stage II, stage III or stage IV cancer. 79 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 8. The method of any one of claims 1-7, wherein one or more of the reciprocal rearrangements comprise an orientation that is positive (rDup), negative (rDel), or mixed (rDelDup).

9. The method of claim 8, wherein one or more of the reciprocal rearrangements comprise a negative orientation (rDel), and / or mixed orientation (rDelDup) and the cancer patient harbors a BRCA2 mutation.

10. The method of claim 8, wherein one or more of the reciprocal rearrangements comprise a positive orientation (rDup), and / or mixed orientation (rDelDup) and the cancer patient harbors a BRCA1 mutation.

11. The method of any one of claims 1-10, wherein the PARP inhibitor is iniparib, olaparib, niraparib, rucaparib, talazoparib, veliparib, AG014699, CEP 9722, MK 4827, BMN-673, E7016, or 3-aminobenzamide.

12. The method of any one of claims 1-10, wherein the platinum agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, pyriplatin, picoplatin, or satraplatin.

13. The method of any one of claims 1-12, wherein the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.

14. The method of any one of claims 1-13, further comprising determining an additional HRD signature in the cancer patient.

15. The method of claim 14, wherein the additional HRD signature comprises one or more of loss of heterozygosity (LOH), large scale state transitions (LST), and telomeric imbalance (tAI).

16. The method of claim 14 or 15, wherein the additional HRD signature comprises one or more of single base substitution signature 3 (SBS3), single base substitution signature 8 (SBS8), ID8 signature, ID6 signature, short tandem duplications (RefSig R3), short deletions (RefSig R5), microhomology-mediated indels, and HRD score.

17. The method of any one of claims 1-16, wherein the cancer patient has not received at least one prior line of anti-cancer therapy. 80 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 18. The method of any one of claims 1-16, wherein the cancer patient has received at least one prior line of anti-cancer therapy.

19. A method for discriminating between BRCA1-deficient and BRCA2-deficient tumors in cancer patients comprising: (a) detecting a plurality of structural variant (SV) features in one or more tumor samples obtained from a plurality of cancer patients, wherein the plurality of SV features comprise homeologous deletions, positive reciprocal rearrangements (rDups), negative reciprocal rearrangements (rDels), and mixed reciprocal rearrangements (rDelDups); (b) for each of the one or more tumor samples (i) identifying tumor samples having elevated levels of negative orientation reciprocal rearrangements (rDels) and / or homeologous deletions relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and / or (ii) identifying tumor samples having elevated levels of positive orientation reciprocal rearrangements (rDups) relative to a control biological sample obtained from a healthy subject or a predetermined threshold; and (c) classifying the tumor samples of (b)(i) as BRCA2-deficient tumors and the tumor samples of (b)(ii) as BRCA1-deficient tumors.

20. The method of claim 19, wherein the tumor samples of (b)(i) or (b)(ii) further comprise elevated levels of mixed orientation reciprocal rearrangements (rDelDups) relative to the control biological sample obtained from a healthy subject or a predetermined threshold.

21. The method of claim 19 or 20, wherein the SV features in the one or more tumor samples are detected using whole genome sequencing.

22. The method of any one of claims 19-21, wherein the plurality of SV features further comprise one or more of 1-100 Kbp simple duplications, rearrangement signature 3 (RS3), rearrangement signature (RS5), deletions with microhomology (mhDel), single base 81 of 83 4867-8530-8103.2Atty. Dkt. No.115872-3014 substitution signature 3 (SBS3), single base substitution signature 8 (SBS8), rearrangement signatures 3 and 5, RS3 and RS5, and loss of heterozygosity (LOH).

23. The method of any one of claims 19-22, wherein the size of the homeologous deletions ranges from 1 kbp to 100 kbp.

24. The method of any one of claims 22-23, wherein the tumor samples of (b)(ii) further comprise elevated levels of 1-100 Kbp simple duplications relative to the control biological sample obtained from a healthy subject or a predetermined threshold.

25. The method of any one of claims 19-23, further comprising identifying the cancer patients from which the tumor samples of (b)(i) were obtained and administering to the cancer patients an effective amount of chemotherapy or immunotherapy.

26. The method of any one of claims 19-24, further comprising identifying the cancer patients from which the tumor samples of (b)(ii) were obtained and administering to the cancer patients an effective amount of chemotherapy, optionally wherein the chemotherapy comprises PARP inhibitors or platinum agents. 82 of 83 4867-8530-8103.2

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