Methods and kits for tumor molecular subtype typing

Abstract

The present invention relates to an in vitro method for identifying a molecular subtype of a tumor in a cancer patient, and a method of classifying a cancer patient for tumor treatment. The invention also relates to a kit for identifying a molecular subtype of a tumor in a cancer patient.

Description

[0001] This application is a divisional application based on the Chinese patent application with application number 201480027494.0 and application date on August 19, 2014. Technical Field

[0002] The present invention relates to an in vitro method for identifying molecular subtypes of tumors in cancer patients and a method for classifying cancer patients for tumor treatment. The present invention also relates to a kit for identifying molecular subtypes of tumors in cancer patients. Background Art

[0003] Tumor prognosis and treatment response prediction are closely related to the molecular subtype of the tumor. The standard method currently used in the world to detect the receptor status of cancers such as breast cancer is immunohistochemistry (IHC) of formalin-fixed and paraffin-embedded (FFPE) biopsies or excised tissues. Currently, endocrine or targeted systemic therapy (i.e., trastuzumab) is mostly based on IHC.

[0004] FFPE sample preparation and subsequent immunohistochemical staining with specific antibodies are current techniques that can only be performed in pathology laboratories. From the microscopic examination of FFPE tumor tissues coupled with the interpretation of the staining results, pathologists gain further clinically critical information about tumor biology and tumor spread. Moreover, the interpretation of FFPE tissue examination by pathologists is considered a critical column in clinical decision making. In many countries, pathologists are an integral part of so-called case workshops in breast cancer management decisions. Although IHC can be easily performed in centralized facilities, the personal insights and experience of the examining pathologist are very important in individual case decisions.

[0005] However, several studies have demonstrated significant interobserver and technical variability in up to 40% of immunohistochemical results. Furthermore, immunohistochemistry allows only qualitative, or in some cases semiquantitative statements about the respective receptor status.

[0006] Therefore, reliable, objective, quantitative and reproducible detection systems for molecular subtyping of tumors such as breast tumors are needed, which help to select appropriate tumor treatment regimens (patient classification) and allow prognosis and prediction of treatment success and assessment of distant metastasis risk of patients. In addition, such detection systems should allow for decentralized detection suitable for most cancer patients. SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention relates to an in vitro method for identifying molecular subtypes of a tumor in a cancer patient, the method comprising the following steps:

[0009] (a) determining the expression level of RNA transcripts of human epidermal growth factor receptor 2 (HER2) in a sample of the tumor;

[0010] (b) determining the expression level of RNA transcript of estrogen receptor (ESR1) in the sample of the tumor;

[0011] (c) determining the expression level of RNA transcript of progesterone receptor (PGR) in a sample of said tumor; and

[0012] (d) determining the expression level of RNA transcripts of proliferation antigen Ki-67 (Ki67) in a sample of the tumor; and / or

[0013] (e) determining the expression level of RNA transcript of RacGTPase activating protein 1 (RACGAP1) in the tumor sample.

[0014] In one embodiment, determining the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1 comprises determining whether the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1 is below or above a set expression threshold of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1.

[0015] In one embodiment, step (a) is performed before steps (b), (c) and (d) and / or (e).

[0016] In one embodiment, step (d) and / or step (e) is performed after steps (a), (b) and (c).

[0017] In one embodiment, step (a) is performed before step (b), step (b) is performed before step (c), and step (c) is performed before step (d) and / or step (e).

[0018] In one embodiment, the molecular subtype is selected from HER2-positive, triple negative, luminal A, and luminal B.

[0019] In one embodiment, an expression level of RNA transcripts of HER2 that is above a set expression threshold of RNA transcripts of HER2 identifies a molecular subtype of the tumor as HER2-positive.

[0020] In one embodiment,

[0021] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0022] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0023] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0024] - Expression levels of Ki67 RNA transcripts that are lower or higher than a set expression threshold of Ki67 RNA transcripts

[0025] The molecular subtype of the tumor was identified as triple negative.

[0026] In one embodiment, the molecular subtype is luminal A or luminal B.

[0027] In one embodiment,

[0028] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0029] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0030] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0031] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0032] In one embodiment,

[0033] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0034] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0035] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0036] - An expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal A type.

[0037] In one embodiment,

[0038] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0039] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0040] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0041] - Expression levels of Ki67 RNA transcripts that are lower or higher than a set expression threshold of Ki67 RNA transcripts

[0042] The molecular subtype of the tumor was identified as luminal B.

[0043] In one embodiment,

[0044] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0045] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0046] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0047] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0048] In one embodiment,

[0049] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0050] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0051] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0052] - An expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal A type.

[0053] In one embodiment,

[0054] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0055] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0056] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0057] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0058] In one embodiment, the luminal A molecular subtype is associated with a chance of surviving 5 years after treatment without distant recurrence that is at least 11% higher, preferably at least 13% higher, than the chance of surviving 5 years after treatment without distant recurrence associated with the luminal B molecular subtype, and / or the luminal A molecular subtype is associated with a chance of surviving 5 years after treatment that is at least 7% higher, preferably at least 9% higher, than the chance of surviving 5 years after treatment associated with the luminal B molecular subtype.

[0059] In one embodiment, the method comprises step (d), and

[0060] - Expression level of ESR1 RNA transcript that is higher than a set expression threshold of ESR1 RNA transcript

[0061] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 indicates an increased risk of a poor clinical outcome in a cancer patient, in particular an increased risk of distant metastasis.

[0062] In one embodiment, the method comprises step (e), and an expression level of the RNA transcript of RACGAP1 that is higher than a set expression threshold of the RNA transcript of RACGAP1 indicates an increased risk of a poor clinical outcome in the cancer patient.

[0063] In one embodiment, the method comprises steps (d) and (e), and

[0064] - an expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67, and

[0065] - Expression level of RACGAP1 RNA transcript that is higher than a set expression threshold of RACGAP1 RNA transcript

[0066] Indicates an increased risk of adverse clinical outcomes in cancer patients.

[0067] In one embodiment, the method comprises steps (d) and (e), and

[0068] - an expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67, and

[0069] - The expression level of RACGAP1 RNA transcript is higher than the set expression threshold of RACGAP1 RNA transcript

[0070] Indicates a further increased risk of adverse clinical outcomes in cancer patients.

[0071] In one embodiment, the adverse clinical outcome comprises a relative decrease in one or more of survival, recurrence-free survival, and distant recurrence-free survival.

[0072] In one embodiment, the tumor is a solid tumor.

[0073] In one embodiment, the tumor is a breast tumor or is derived from a breast tumor.

[0074] In one embodiment, the cancer is breast cancer.

[0075] In one embodiment, the sample is RNA extracted from a tumor.

[0076] In one embodiment, the expression level of the RNA transcript is determined by reverse transcription (RT) quantitative PCR.

[0077] In one embodiment, the quantitative PCR is fluorescence-based quantitative real-time PCR.

[0078] In one embodiment, the method comprises using an ESR1-specific primer, and / or a HER2-specific primer, and / or a Ki67-specific primer, and / or a PGR-specific primer, and / or a RACGAP1-specific primer, wherein the ESR1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 1 and 2, the HER2-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 4 and 5, the Ki67-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 7 and 8, the PGR-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11, and the RACGAP1-specific primer has a length of 15 to 30 nucleotides and comprises SEQ ID NOs: At least 10 consecutive nucleotides of the sequences of NO: 13 and 14.

[0079] In one embodiment, the method comprises using an ESR1-specific probe, and / or a HER2-specific probe, and / or a Ki67-specific probe, and / or a PGR-specific probe, and / or a RACGAP1-specific probe, wherein the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:3, the HER2-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:6, the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:9, the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:12, and the RACGAP1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:15.

[0080] In one embodiment, the expression level is normalized to the (average) expression level of one or more reference genes in the tumor sample.

[0081] In one embodiment, the one or more reference genes are selected from CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH.

[0082] In other aspects, the present invention relates to a method for classifying cancer patients for tumor treatment, the method comprising, in a first step, identifying molecular subtypes of cancer patient tumors using an in vitro method as described above, and in a second step, selecting a tumor treatment regimen based on the molecular subtypes identified by the in vitro method.

[0083] In one embodiment, the molecular subtype is selected from HER2-positive, triple negative, luminal A, and luminal B.

[0084] In one embodiment,

[0085] - the molecular subtype is HER2-positive, and the tumor treatment regimen comprises administration of an anti-HER2 antibody and a chemotherapeutic agent;

[0086] - the molecular subtype is triple negative, and the tumor treatment regimen includes administration of a chemotherapeutic agent;

[0087] - the molecular subtype is luminal A and the tumor treatment regimen includes endocrine therapy; or

[0088] - the molecular subtype is luminal B, and the tumor treatment regimen comprises endocrine therapy and optionally administration of a chemotherapeutic agent.

[0089] In one embodiment, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a chemotherapeutic agent.

[0090] In one embodiment, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a taxane, preferably docetaxel.

[0091] In one embodiment, the taxane is administered in combination with fluorouracil, epirubicin, and cyclophosphamide (FEC).

[0092] In one embodiment, the tumor is a solid tumor.

[0093] In one embodiment, the tumor is a breast tumor or is derived from a breast tumor.

[0094] In one embodiment, the cancer is breast cancer.

[0095] In other aspects, the present invention relates to a method for treating cancer, comprising, in a first step, classifying a cancer patient for tumor treatment using the in vitro method described above, and in a second step, providing the cancer patient with a selected tumor treatment regimen.

[0096] In other aspects, the present invention relates to a kit for identifying molecular subtypes of a tumor in a cancer patient by a reverse transcription (RT) quantitative PCR method, the kit comprising:

[0097] - at least one pair of HER2-specific primers and at least one HER2-specific probe;

[0098] - at least one pair of ESR1-specific primers and at least one ESR1-specific probe;

[0099] - at least one pair of PGR-specific primers and at least one PGR-specific probe; and

[0100] - at least one pair of Ki67-specific primers and at least one Ki67-specific probe; and / or

[0101] - at least one pair of primers specific for RACGAP1 and at least one probe specific for RACGAP1.

[0102] In one embodiment, the quantitative PCR is fluorescence-based quantitative real-time PCR.

[0103] In one embodiment, detection of the probe is based on amplification-mediated displacement of the probe.

[0104] In one embodiment, the probe is a dual-labeled probe, which comprises a fluorescent reporter group and a fluorescent quencher group.

[0105] In one embodiment, the kit further comprises a reverse transcriptase and a DNA polymerase.

[0106] In one embodiment, the reverse transcriptase and polymerase are provided as an enzyme mix, which allows for one-step reverse transcription (RT) quantitative PCR.

[0107] In one embodiment, the kit further comprises at least one pair of primers specific for a reference gene and at least one probe specific for a reference gene.

[0108] In one embodiment, the reference gene is one or more selected from the following genes: CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH.

[0109] In one embodiment, the kit further comprises at least one control RNA sample.

[0110] In one embodiment, the primers provide an amplicon size of less than 120 bp.

[0111] In one embodiment, the ESR1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 1 and 2, and / or the HER2-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 4 and 5, and / or the Ki67-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 7 and 8, and / or the PGR-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11, and / or the RACGAP1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 13 and 14.

[0112] In one embodiment, the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:3, and / or the HER2-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:6, and / or the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:9, and / or the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:12, and / or the RACGAP1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:15.

[0113] In one embodiment, the tumor is a solid tumor.

[0114] In one embodiment, the tumor is a breast tumor or is derived from a breast tumor.

[0115] In one embodiment, the cancer is breast cancer.

[0116] In another aspect, the present invention relates to the use of a kit as described above for identifying molecular subtypes of a tumor in a cancer patient.

[0117] In another aspect, the present invention relates to the use of the kit as described above in assessing the risk of distant metastasis in a cancer patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1Summary The methods of the invention have possible applications as a backup to conventional methods of subtype classification (eg, IHC) and as a complement to conventional methods of patient classification.

[0119] Fig. 2 shows a partition test evaluating the prognosis and predictive value of the expression levels of HER2, ESR1, PGR, Ki67 and RACGAP1mRNA for the 5-year survival rate of breast cancer patients. First, the available data from 855 tumors were classified to identify HER2-positive tumors (≥threshold 38) by HER2mRNA expression levels. HER2-negative tumors were further classified by ESR1mRNA expression levels. A: ESR1-positive tumors (≥threshold 34) were further classified by Ki67mRNA expression levels (threshold 31.7) followed by PGR mRNA expression levels (threshold 30.2). B: Alternatively, ESR1-positive tumors (≥threshold 34) were further classified by PGR mRNA expression levels (threshold 30.2) followed by Ki67mRNA expression levels (threshold 31.7). Kaplan-Meier analysis of these data is Figure 3 and Figure 4 C: Ki67-positive and Ki67-negative tumors were classified by RACGAP1 mRNA expression level (threshold 34.2). In order to increase the number of patients for further analysis of RACGAP1, data for PGR are not given in the figure. Further consideration of PGR did not change the overall results for RACGAP1. The data (see Table 3) show that RACGAP1 mRNA expression levels below or above the set threshold are associated with particularly significant differences in 5-year survival rate.

[0120] Figure 3 Kaplan Meier analysis of the survival of breast cancer patients with HER2-positive (HER2), luminal A (LumA), luminal B (LumB) or triple negative (TNT) tumors is shown, wherein the molecular subtype of the tumor is identified according to the present invention based on the mRNA expression levels of HER2, ESR1, PGR and Ki67. The luminal A subtype as determined by the inventors is accompanied by an overall survival rate of 97% after 5 years (compared to 87% for luminal B tumors and HER2-positive tumors and 84% for triple negative tumors).

[0121] Figure 4Kaplan Meier analysis of distant metastasis-free survival ("DMFS"; distant recurrence X years after surgery) of breast cancer patients with HER2-positive (HER2), luminal A (LumA), luminal B (LumB), or triple-negative (TNT) tumors is shown, wherein the molecular subtypes of the tumors are identified according to the present invention based on the mRNA expression levels of HER2, ESR1, PGR, and Ki67. The luminal A subtype as determined by the inventors is associated with a DMFS rate of 92% after 5 years (compared to 78% for luminal B tumors, HER2-positive tumors, and triple-negative tumors).

[0122] Figure 5 The multivariate Cox regression analysis of DMFS comparing molecular subtyping by immunohistochemistry (Sotiriou et al. (2009), NEngl J Med, 360 (8): 790-800) with molecular subtyping based on mRNA expression levels of HER2, ESR1, PGR and Ki67 according to the present invention is shown. This analysis clearly shows the superiority of the method of the present invention, because when the results obtained by the method of the present invention are incorporated into the Cox proportional hazard model, the significance of immunohistochemical subtyping is reduced.

[0123] Figures 6 to 9 Shown are 40-ΔΔCT values ​​for markers HER2, ESR1, PGR, and Ki67 determined by RT-qPCR for patient samples 1-6 and batches 1-3.

[0124] Fig.10 Kaplan Meier analysis of overall survival of patients with luminal B tumors is shown. A: When determined by RT-qPCR, patients with luminal B cancers survived significantly longer with docetaxel-FEC than with vinorelbine-FEC (97% vs. 89%, hazard ratio [HR] 0.241; CI: 0.090-0.642, respectively). B: When tumor subtype was determined by IHC, no benefit of docetaxel could be shown for luminal B patients (95% vs. 92%, HR 0.617; CI 0.235-1.623).

[0125] Fig.11Kaplan Meier analysis of overall survival of patients with luminal B tumors adjusted for tumor histology type by Cox regression specified by SAP is shown. A: The docetaxel benefit predicted by the RT-qPCR kit remains significant (97% vs. 88%, HR 0.232; CI 0.087-0.624). B: Subtyping by IHC does not predict docetaxel benefit (96% vs. 92%, HR 0.510; CI 0.184-1.414).

[0126] Fig.12 Kaplan Meier analysis of distant metastasis-free survival (DMFS) for patients with luminal B tumors is shown. A: When determined by RT-qPCR, luminal B patients treated with docetaxel-FEC had a higher chance of remaining free of distant metastasis compared to vinorelbine-FEC (89% vs. 78%, HR 0.471; CI 0.263-0.843, respectively). B: When luminal B tumors were determined by IHC, no survival differences were observed between the different treatment regimens (87% vs. 86%, HR 0.938 CI 0.474-1.856).

[0127] Fig.13 Kaplan Meier analysis of distant metastasis-free survival of patients with luminal B-type tumors adjusted for number of metastatic lymph nodes, tumor size and histological type by Cox regression specified by SAP is shown. A: The effect was still significant (90% vs. 78%, HR 0.409; CI 0.219-0.764) by using the subtyping assay of the present invention. B: In contrast, there was no effect when tumor subtyping was performed by IHC (89% vs. 85%, HR 0.674; CI 0.307-1.481).

[0128] Fig.14 Kaplan Meier analysis of overall survival of patients with HER2-positive tumors is shown. A: When determined by RT-qPCR, HER2-positive patients treated with docetaxel did not have better overall survival than vinorelbine (87% vs. 91%, HR 1.320; CI 0.556-3.132). B: Tumors typed by IHC showed similar results (86% vs. 89%, HR 1.175; CI 0.518-2.663).

[0129] Fig.15Kaplan Meier analysis of distant metastasis-free survival of HER2-positive tumors is shown. A: When determined by the method of the present invention, HER2-positive patients treated with docetaxel had no difference in distant metastasis-free survival compared to vinorelbine (80% vs. 81%, HR 1.070; CI 0.551-2.076). B: Similarly, when determined by IHC, HER2-positive patients treated differently showed no survival difference (78% vs. 77%, HR 0.975; CI 0.516-1.843).

[0130] Fig.16 Kaplan Meier analysis of overall survival of luminal A tumors is shown. A: When determined by RT-qPCR, luminal A patients tended to have lower overall survival when treated with docetaxel compared to vinorelbine (95% vs. 98%, HR 2.471; CI 0.477-12.809). B: In contrast, when determined by IHC, luminal A patients showed a weak but non-significant trend toward longer overall survival when treated with docetaxel compared to vinorelbine (97% vs. 93% HR 0.443; CI 0.114-1.716).

[0131] Fig.17 Kaplan Meier analysis of distant metastasis-free survival of patients with luminal A tumors is shown. A: When determined by RT-qPCR, there was no difference in distant metastasis-free survival for luminal A patients after treatment with docetaxel or vinorelbine (92% vs. 90%, HR 0.826; CI 0.336-2.033) B: IHC-subtype patients treated with docetaxel showed a weak but non-significant trend toward longer distant metastasis-free survival compared to vinorelbine (93% vs. 88%, HR 0.553; CI 0.221-1.386).

[0132] Fig.18 Kaplan Meier analysis of overall survival of patients with TNBC tumors is shown. A: When determined by RT-qPCR, patients with TNBC showed no difference in overall survival after treatment with docetaxel or vinorelbine (84% vs. 83%, HR 0.949; CI 0.338-2.665). B: When determined by IHC, TNBC patients showed a weak but non-significant trend toward longer overall survival (88% vs. 80%, HR 0.552; CI 0.207-1.472).

[0133] Fig.19Kaplan Meier analysis of distant metastasis-free survival of TNBC tumor patients is shown. A: There was no significant difference in distant metastasis-free survival for RT-qPCR-determined TNBC patients with different treatments (74% vs. 78%, HR 1.211; CI 0.523-2.802). B: Compared with vinorelbine, IHC-determined TNBC patients showed a weak but non-significant trend toward longer distant metastasis-free survival when treated with docetaxel (82% vs. 72%, HR 0.615; CI 0.284-1.333).

[0134] Fig. 20 The results of the analysis of Ki67 are shown. A shows a scatter plot of continuous Ki67 estimates by IHC (as shown by % positive cells on the y-axis) and RT-qPCR (as shown by 40-ΔΔCT on the x-axis). The lines show the predetermined thresholds for the statistical analysis plan (horizontal: IHC 20%; vertical: RT-qPCR 34.8 40-ΔΔCT). B summarizes the agreement and disagreement between RT-qPCR and IHC classification (positive [pos] compared to negative [neg]). C shows a Kappa statistic that reveals a high significant correlation (p<0.0001) between the methods, but moderate agreement. D shows the positive and negative percentage agreement (PPA and NPA, respectively) when testing Ki67 by RT-qPCR compared to IHC.

[0135] Fig.21 Kaplan Meier analysis of distant metastasis-free survival of patients with estrogen receptor-positive tumors and discordant Ki67 results between RT-qPCR and IHC are shown.

[0136] From the following detailed description, especially together with the attached Figure 1 Other objects, advantages and features of the invention will become apparent when considered together. DETAILED DESCRIPTION OF THE INVENTION

[0138] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art.

[0139] Hereinafter, elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to form other embodiments. The examples and preferred embodiments of different descriptions should not be construed as limiting the present invention to only the embodiments clearly described. This description should be construed as supporting and including embodiments that combine the embodiments clearly described with disclosed and / or preferred elements of any number. In addition, unless otherwise indicated in the context, any arrangement and combination of all described elements in the application should be considered to be disclosed by the specification sheet of the application.

[0140] Preferably, the terms used herein are in accordance with "A multilingual glossary of biotechnological terms: (IUPAC recommendations)", HGW Leuenberger, B. Nagel, and H. The definitions are as described in Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0141] Unless otherwise indicated, the practice of the present invention will adopt the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2 nd Conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques are explained in "Invitrogen (1996) and "Recombinant DNA Technology (1996)". Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0142] Unless the context requires otherwise, throughout this specification and the appended claims, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of the stated member, integer or step or group of members, integers or steps, but not the exclusion of any other member, integer or step or group of members, integers or steps, although in some embodiments, such other members, integers or steps or groups of members, integers or steps may be excluded, i.e., the subject matter is present in the inclusion of the stated member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) should be understood to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Any and all examples of use provided herein, or exemplary language (e.g., "such as"), are only intended to better illustrate the present invention and do not impose limitations on the scope of the invention otherwise claimed. The language in this specification should not be interpreted as indicating any unclaimed elements that are essential to the implementation of the present invention.

[0143] Several documents are described in the context of the entire specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions for use, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as admitting that the present invention has no rights due to prior inventions prior to such disclosure.

[0144] In one aspect, the present invention relates to an in vitro method for identifying molecular subtypes of a tumor in a cancer patient, the method comprising the following steps:

[0145] (a) determining the expression level of RNA transcripts of human epidermal growth factor receptor 2 (HER2) in a sample of the tumor;

[0146] (b) determining the expression level of RNA transcript of estrogen receptor (ESR1) in the sample of the tumor;

[0147] (c) determining the expression level of RNA transcript of progesterone receptor (PGR) in a sample of said tumor; and

[0148] (d) determining the expression level of RNA transcripts of proliferation antigen Ki-67 (Ki67) in a sample of the tumor; and / or

[0149] (e) determining the expression level of RNA transcript of RacGTPase activating protein 1 (RACGAP1) in the tumor sample.

[0150] In one embodiment, the method does not include determining the expression level of one or more additional non-reference genes, in particular the expression level of their RNA transcripts. In other words, the expression level of genes other than HER2, ESR1, PGR and Ki67 and / or RACGAP1 and one or more reference genes, in particular the expression level of their RNA transcripts, is not determined.

[0151] In one embodiment, the method does not include any other diagnostic steps, such as histological grading, or determining lymph node status.

[0152] The term "tumor" as used herein refers to all tumor cell growth and proliferation (whether malignant or benign), as well as all precancerous and cancerous cells and tissues. In one embodiment of the invention, the tumor is a solid tumor. In one embodiment, the tumor is a breast tumor or is derived from a breast tumor (e.g., by metastasis).

[0153] As used herein, "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion and / or migration. The term "cancer" of the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, colorectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and the metastasis of the above cancers. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or the metastasis of the above cancer types or tumors. The term cancer of the present invention also includes cancer metastasis. In one embodiment, the cancer is breast cancer.

[0154] The term "breast cancer" refers to a type of cancer that originates in breast tissue, most commonly in the lining of the milk ducts or in the lobules that supply the ducts with milk. Cancers that originate in the ducts are called ductal carcinomas, while those that originate in the lobules are called lobular carcinomas. Occasionally, breast cancer occurs as a metastatic disease. Common sites of metastasis include bones, liver, lungs, and brain. Breast cancer occurs in humans and other mammals. Although the vast majority of human cases occur in women, male breast cancer may also occur. Treatment of breast cancer may include surgery, drugs (hormonal therapy and chemotherapy), radiotherapy, and / or immunotherapy / targeted therapy.

[0155] The term "patient" as used herein refers to any organism such as a vertebrate, in particular any mammal, including humans and other mammals, for example, animals such as rodents, rabbits or monkeys. The rodent may be a mouse, rat, hamster, guinea pig or chinchilla. Preferably, the patient is a human.

[0156] According to the present invention, the term "RNA transcript" includes and preferably relates to "mRNA", which refers to "messenger RNA", and to a "transcript" encoding a peptide or protein. mRNA generally includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro.

[0157] The gene HER2 (also known as ERBB2; location: 17q12, annotation: chromosome: 17; NC_000017.10) encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. Amplification and / or overexpression of this gene has been reported in many cancers, including breast and ovarian tumors. In the NCBI database, two mRNA variants of HER2 are listed, which encode two protein versions. The protein and mRNA sequences can be found in the accession numbers NM_001005862.1 (receptor tyrosine protein kinase erbB-2 isoform b) and NM_004448.2 (receptor tyrosine protein kinase erbB-2 isoform, a precursor). HER2 gene amplification occurs in approximately 10-20% of primary breast cancers.

[0158] The gene ESR1 (position: 6q25, annotation: chromosome 6, NC_000006.11) encodes the estrogen receptor (ER), a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding and transcriptional activation. Estrogen receptors are known to be involved in pathological processes, including breast cancer, endometrial cancer and osteoporosis. There are 4 known ESR1 mRNA variants, in which the transcript variants differ in the 5'UTR and / or use different promoters, but each variant encodes the same protein. 70-80% of all breast cancers are ER positive.

[0159] The gene PGR (also known as PR; location: 11q22-q23, annotation: chromosome: 11; NC_000011.9) encodes the progesterone receptor. Steroid hormones such as progesterone and their receptors are involved in the regulation of eukaryotic gene expression and affect cell proliferation and differentiation of target tissues. This gene uses two different promoters and translation start sites in the first exon, resulting in two mRNA isoforms, A and B. The two isoforms are identical except for the presence of an additional 165 amino acids at the N-terminus of isoform B. 40% of breast tumors are PGR positive.

[0160] The gene Ki-67 (Ki67; location: 10q26.2, annotation: chromosome: 10; NC_000010.10) encodes a nuclear protein that is associated with and may be required for cell proliferation. Two mRNA variants have been described. A related pseudogene is present on chromosome 10. Approximately 25% of breast tumors are Ki67 positive.

[0161] The gene RACGAP1 (position: 12q13.12, annotation: chromosome: 12; NC_000012.11) encodes the RacGTPase activating protein 1. Three splice variants have been described, all encoding the same protein. RACGAP1 is a component of the central spindle protein (spindlin) complex and plays a key role in regulating growth-related processes and differentiation.

[0162] As used herein, the term "expression level" refers to the expression of a specific gene (i.e., HER2, ESR1, PGR, Ki67, or RACGAP1), thereby producing transcripts and / or proteins. According to the present invention, expression levels are determined at RNA transcript levels, particularly mRNA levels (transcription levels), for example, by measuring transcribed mRNA (e.g., by northern blot), by reverse transcription (RT) quantitative PCR, or by direct staining mRNA (e.g., by in situ hybridization).

[0163] In one embodiment, the term "tumor sample" refers to a tumor tissue sample (e.g., a biopsy or excised tissue of a tumor) isolated from a cancer patient. In a preferred embodiment, the tumor tissue sample is a frozen section of a tumor tissue sample, or a chemically fixed tumor tissue sample. In a more preferred embodiment, the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) tumor tissue sample. In one embodiment, the tumor sample is (total) RNA extracted from a tumor tissue sample. In a particularly preferred embodiment, the tumor sample is (total) RNA extracted from a FFPE tumor tissue sample. Those skilled in the art are able to implement RNA extraction protocols. For example, a high-purity RNA paraffin kit (Roche, Basel, Switzerland) or, preferably, an XTRAKT RNA extraction kit XL (Stratifyer Molecular Pathology, Cologne, Germany) can be used to extract total RNA from 5-10 μm curl of FFPE tumor tissue. The sample material to be used / detected can also be stored in a refrigerator, and the method of the present invention can be implemented at a suitable time point after each sample material is thawed. The sample may be obtained from the cancer patient before starting therapeutic therapy, during therapeutic therapy, and / or after therapeutic therapy, ie, before, during or after administration of the cancer treatment.

[0164] As used herein, the term "molecular subtype of a tumor" refers to a tumor subtype characterized by a different molecular profile (e.g., gene expression profile). In one embodiment, the molecular subtype is selected from HER2-positive, triple negative (also called "basal-like"), luminal A, and luminal B. The term "basal-like" refers to the fact that such tumors have some similarities in gene expression with basal epithelial cells. The term "luminal" derives from the similarity in gene expression between tumors and luminal epithelial cells.

[0165] The molecular subtypes differ significantly in clinical outcomes and treatment responses. In one embodiment, the molecular subtype luminal A as defined herein is associated with a probability of surviving 5 years after treatment without distant recurrence that is at least 11%, preferably at least 13%, higher than the probability of surviving 5 years after treatment without distant recurrence associated with the molecular subtype luminal B, and / or the molecular subtype luminal A is associated with a probability of surviving 5 years after treatment that is at least 7%, preferably at least 9%, higher than the probability of surviving 5 years after treatment associated with the molecular subtype luminal B.

[0166] The term "(therapeutic) therapy" as used herein, particularly when related to cancer treatment, refers to any treatment that can improve health status and / or prolong (increase) the life span of a patient. The treatment can eliminate cancer, reduce the size and number of tumors in a patient, prevent or slow the development of cancer in a patient, inhibit or slow the development of new cancers in a patient, reduce the frequency or severity of symptoms in a patient, and / or reduce recurrence in a patient who currently has or has previously had cancer. In one embodiment, the terms "treatment" and "therapeutic therapy" mean one or more of surgical removal of the primary tumor, chemotherapy, hormone therapy, radiotherapy, and immunotherapy / targeted therapy.

[0167] Adjuvant therapy is treatment given in addition to the primary, main or initial treatment. Surgery and combined treatment regimens for cancer treatment have made the term primarily used to describe adjuvant cancer treatment. Examples of adjuvant therapy are additional treatments (e.g., chemotherapy) that are usually given after surgery (postoperatively) where all detectable disease has been removed, but there is still a statistical risk of recurrence due to occult disease. Neoadjuvant therapy is treatment given before the primary, main or initial treatment (e.g., preoperative chemotherapy).

[0168] According to the present invention, the step of "determining the expression level of RNA transcripts" may include: (i) measuring the expression level of RNA transcripts, and (ii) analyzing the measured expression level of RNA transcripts (e.g., by comparing with a reference expression level, such as a set expression threshold), wherein the order of measuring the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1 is independent of the order of analyzing the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1.

[0169] In one embodiment, determining the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1 includes determining whether the expression level of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1 is lower or higher than a set expression threshold of RNA transcripts of HER2, ESR1, PGR and Ki67 and / or RACGAP1. In the case where the expression level is equal to the set expression threshold, the expression level is considered to belong to the group of expression levels higher than the set expression threshold. Therefore, the phrase "higher than the set expression threshold" as used herein includes expression levels higher than or equal to the set expression threshold. An expression level "higher than the set expression threshold" may also be referred to as "expression positive", while an expression level "lower than the set expression threshold" may also be referred to as "expression negative".

[0170] As used herein, the term "setting an expression threshold of an RNA transcript" may refer to an average threshold value (abbreviated as: cut-off) calculated from multiple samples, wherein the multiple samples are obtained from multiple subjects, in particular subjects suffering from cancer. In order to obtain the threshold value, the multiple subjects may include subjects with tumors of different molecular subtypes, such as subjects with HER2-positive tumors, and / or subjects with triple-negative tumors, and / or subjects with luminal A tumors, and / or subjects with luminal B tumors. The threshold value may represent the amount or concentration of the RNA transcript. In one embodiment, the threshold value is given as a CT (cycle threshold) value (see below). In one embodiment, the (relative) expression level and expression threshold value are expressed as 40-ΔCT or 40-ΔΔCT values ​​(see below).

[0171] The term "subject" as used herein refers to any organism such as a vertebrate, in particular any mammal, including humans and other mammals, for example, animals such as rodents, rabbits or monkeys. The rodent may be a mouse, rat, hamster, guinea pig or chinchilla. Preferably, the subject is a human. In one embodiment, the subject is a subject suffering from or suspected of having a disease, in particular cancer, also referred to herein as a "patient". In order to determine the average threshold, at least two subjects, preferably at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500 or at least 2000 subjects are detected.

[0172] Since various clinical studies have been conducted using the gene markers used in the present invention, consistent studies in a training-testing environment will be sufficient to define and validate clinical cut-offs / thresholds for dichotomizing quantitative results into "expression positive" or "expression negative". Therefore, in one embodiment, the threshold is determined based on one or more prior clinical studies. In addition, additional clinical studies may be conducted to establish and validate the threshold. The threshold may be determined / set by techniques known in the art.

[0173] In one embodiment, the 1:1 ratio of the 1:1 ratio of the 1:1 ratio is 1:1 based on the clinical-pathological parameters such as IHC-ISH, and / or overall survival (OS), disease-free survival (DFS) and distant metastasis-free survival (DMFS), and disease-specific survival (DSS) in the training population by a hierarchical test (e.g., SAS Software 9.0.0) to determine / set thresholds (e.g., HE10-97, Pentheroudakis et al. (2009), Breat Cancer Res Treat, 116:131–143) and validated in independent clinical trial populations, such as FFPE tissue samples from the FinHER study (Joensuu et al. (2006), N Engl J Med, 354:809-820).

[0174] In one embodiment, the 40-ΔCT value is calculated as follows: 40-[CT of the respective biomarker of the patient sample (i.e., HER2, ESR1, PGR and Ki67 and / or RACGAP1) - CT of the reference gene (e.g., CALM2) of the patient sample] (= calculation method 1). If more than one reference gene is used, the 40-ΔCT value is calculated as follows: 40-(CT of the respective biomarker of the patient sample - average CT of the selected reference gene of the patient sample) (= calculation method 2). Alternatively, a 40-ΔΔCT value may be used, wherein the 40-ΔΔCT may be calculated as follows: ΔΔCT = 40-[(CT biomarker of the patient sample - CT biomarker of the reference sample) - (CT reference gene of the patient sample - CT reference gene of the reference sample)] (= calculation method 3); for example, 40-ΔΔCT = 40-[(CT Ki67 patient sample - CT Ki67 reference sample) - (CT CALM2 of the patient sample - CT CALM2 of the reference sample)]. In one embodiment, CALM2 is used as a reference gene.

[0175] In an exemplary embodiment, the average threshold value is given as a 40-ΔCT value according to calculation method 2, wherein the average threshold value for HER2 is a 40-ΔCT value of 38, the average threshold value for ESR1 is a 40-ΔCT value of 34, the average threshold value for PGR is a 40-ΔCT value of 30.2, the average threshold value for Ki67 is a 40-ΔCT value of 31.7, and the average threshold value for RACGAP1 is a 40-ΔCT value of 34.2.

[0176] In another embodiment, the relative expression level of the biomarker is given as a 40-ΔΔCT value, which is calculated as follows: 40-[(CT biomarker of patient sample-CT reference gene of patient sample)-(CT biomarker of control sample-CT reference gene of control sample)] (= calculation method 4); for example, 40-ΔΔCT=40-[(CT Ki67 patient sample-CT average CombRef patient sample)-(CT Ki67 control sample-CT average CombRef control sample)]. In one embodiment, CT is the median CT. The CT of the reference gene can be the CT of a single reference gene, or the average CT of two or more reference genes (referred to as average CombRef). Preferably, the same control sample (also referred to as calibrator) is used in all analyses and produces the same RT-qPCR or qPCR results. In one embodiment, the control sample is an equimolar mixture of cell line RNA, in vitro transcribed artificial RNA, or DNA oligonucleotides, which represents a biomarker mRNA or cDNA with a constant ratio or a biomarker amplicon or a portion of a biomarker amplicon. In one embodiment, CALM2 and B2M are used as reference genes, and a positive control (eg, in vitro transcribed artificial RNA) is used as a control sample (calibrator).

[0177] In an exemplary embodiment, the average threshold value is given as a 40-ΔΔCT value according to calculation method 4, wherein on a Versant kPCR instrument AD module (Siemens), the average threshold value for HER2 is a 40-ΔΔCT value of 40.90, the average threshold value for ESR1 is a 40-ΔΔCT value of 38.20, the average threshold value for PGR is a 40-ΔΔCT value of 34.90, and the average threshold value for Ki67 is a 40-ΔΔCT value of 34.80.

[0178] In another exemplary embodiment, the average threshold is given by a 40-ΔΔCT value according to calculation method 4, where On the 480 instrument II (Roche), the mean threshold for HER2 was a 40-ΔΔCT value of 41.1, for ESR1 was a 40-ΔΔCT value of 38.00, for PGR was a 40-ΔΔCT value of 35.50, and for Ki67 was a 40-ΔΔCT value of 35.50.

[0179] In one embodiment, steps (a), (b), (c) and (d) and / or (e) are performed in a random order. In one embodiment, step (a) is performed before steps (b), (c) and (d) and / or (e). In one embodiment, step (d) and / or step (e) are performed after steps (a), (b) and (c). In one embodiment, step (a) is performed before step (b), step (b) is performed before step (c), and step (c) is performed before step (d) and / or step (e).

[0180] In one embodiment, an expression level of RNA transcripts of HER2 that is above a set expression threshold of RNA transcripts of HER2 identifies a molecular subtype of the tumor as HER2-positive.

[0181] In one embodiment,

[0182] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0183] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0184] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0185] - Expression levels of Ki67 RNA transcripts that are lower or higher than a set expression threshold of Ki67 RNA transcripts

[0186] The molecular subtype of the tumor was identified as triple negative.

[0187] In one embodiment, the molecular subtype is luminal A or luminal B.

[0188] In one embodiment,

[0189] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0190] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0191] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0192] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0193] In one embodiment,

[0194] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0195] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0196] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0197] - An expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal A type.

[0198] In one embodiment,

[0199] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0200] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0201] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0202] - Expression levels of Ki67 RNA transcripts that are lower or higher than a set expression threshold of Ki67 RNA transcripts

[0203] The molecular subtype of the tumor was identified as luminal B.

[0204] In one embodiment,

[0205] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0206] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0207] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0208] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0209] In one embodiment,

[0210] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0211] - an expression level of the RNA transcript of ESR1 that is lower than a set expression threshold of the RNA transcript of ESR1;

[0212] - an expression level of the RNA transcript of the PGR that is higher than a set expression threshold of the RNA transcript of the PGR; and

[0213] - An expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal A type.

[0214] In one embodiment,

[0215] - an expression level of HER2 RNA transcripts that is lower than a set expression threshold of HER2 RNA transcripts;

[0216] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1;

[0217] - an expression level of the RNA transcript of PGR that is lower than a set expression threshold of the RNA transcript of PGR; and

[0218] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 identifies the molecular subtype of the tumor as luminal B type.

[0219] In one embodiment, the method comprises step (d), and

[0220] - an expression level of the RNA transcript of ESR1 that is higher than a set expression threshold of the RNA transcript of ESR1, and

[0221] - An expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67 indicates an increased risk of a poor clinical outcome in a cancer patient, in particular an increased risk of distant metastasis.

[0222] In one embodiment, the method includes step (e), and an expression level of RNA transcripts of RACGAP1 that is higher than a set expression threshold of RNA transcripts of RACGAP1 indicates an increased risk of a poor clinical outcome in the cancer patient (compared to the risk in a cancer patient having a tumor having an expression level of RNA transcripts of RACGAP1 that is lower than the set expression threshold of RNA transcripts of RACGAP1).

[0223] Determination of the expression levels of RNA transcripts of Ki67 and RACGAP1 provides more accurate information about the clinical outcome of a cancer patient, wherein, generally, an increased expression level of RNA transcripts of Ki67 or RACGAP1 indicates an increased risk of a poor clinical outcome in a cancer patient (compared to the risk of a cancer patient having a tumor having an expression level of RNA transcripts of Ki67 or RACGAP1 that is lower than a set expression threshold of RNA transcripts of Ki67 or RACGAP1).

[0224] In one embodiment, the method comprises steps (d) and (e), and

[0225] - an expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67, and

[0226] - Expression level of RACGAP1 RNA transcript that is higher than a set expression threshold of RACGAP1 RNA transcript

[0227] Indicates an increased risk of adverse clinical outcomes in cancer patients.

[0228] In one embodiment, the method comprises steps (d) and (e), and

[0229] - an expression level of the RNA transcript of Ki67 that is higher than a set expression threshold of the RNA transcript of Ki67, and

[0230] - Expression level of RACGAP1 RNA transcript that is higher than a set expression threshold of RACGAP1 RNA transcript

[0231] Indicating a further increased risk of a poor clinical outcome in a cancer patient (compared to an increased risk in a cancer patient having a tumor having an expression level of RNA transcripts of Ki67 that is lower than a set expression threshold of RNA transcripts of Ki67, and an expression level of RNA transcripts of RACGAP1 that is higher than a set expression threshold of RNA transcripts of RACGAP1, wherein preferably, the further increase refers to an increase of at least 5%, more preferably an increase of at least 10%).

[0232] The term "clinical outcome" is defined as the clinical outcome of the disease, particularly after treatment, such as alleviation or improvement of symptoms. In one embodiment, poor clinical outcomes include a relative reduction in one or more of survival, recurrence-free survival, and distant recurrence-free survival. The term "recurrence" with respect to cancer includes the reappearance of tumor cells in the same part and organ of the original disease, the metastasis that may occur even many years after the initial diagnosis and treatment of cancer, or local events such as tumor cell infiltration of local lymph nodes. "Distant recurrence" refers to a situation in which cancer cells have spread (metastasized) beyond local lymph nodes to a distant part of the body (i.e., another organ). Recurrence-free survival is generally defined as the time from randomization to first recurrence, recurrence, secondary cancer, or death.

[0233] The term "metastasis" means that cancer cells spread from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the malignant cells peeling off from the primary tumor, invading the extracellular matrix, infiltrating the endothelial basement membrane to enter the body cavity and blood vessels, and then infiltrating the target organ after being transported by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, because tumor cells or components can retain and develop metastatic potential.

[0234] In one embodiment, "increased risk of adverse clinical outcome" means that the chance of surviving 5 years after treatment is at least 20% lower, preferably at least 25% lower, than the chance of surviving 5 years after treatment in cancer patients with tumors having

[0235] - an expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67, and

[0236] - An expression level of the RNA transcript of RACGAP1 that is lower than a set expression threshold of the RNA transcript of RACGAP1.

[0237] In one embodiment, "further increased risk of adverse clinical outcome" means that the chance of surviving 5 years after treatment is at least 30% lower, preferably at least 35% lower, than the chance of surviving 5 years after treatment in a cancer patient with a tumor having

[0238] - an expression level of the RNA transcript of Ki67 that is lower than a set expression threshold of the RNA transcript of Ki67, and

[0239] - An expression level of the RNA transcript of RACGAP1 that is lower than a set expression threshold of the RNA transcript of RACGAP1.

[0240] In one embodiment, the expression level of the RNA transcript is measured by reverse transcription (RT) quantitative PCR (RT-qPCR). Since RNA cannot be directly amplified in PCR, it must be reverse transcribed into cDNA using enzyme-transcriptase. For this purpose, one-step RT-qPCR can be used, which combines the reverse transcription reaction with the DNA amplification by PCR in the same reaction. In one-step RT-qPCR, the RNA template is mixed in a reaction mixture comprising reverse transcriptase, DNA polymerase, primers and probes, dNTPs, salts and detergents. In the first PCR step, the target RNA is reversely transcribed by reverse transcriptase using a target-specific reverse transcription primer. Subsequently, cDNA is amplified using primers / probes and DNA polymerase.

[0241] In one embodiment, the quantitative PCR is a fluorescence-based quantitative real-time PCR. The fluorescence-based quantitative real-time PCR comprises the use of a fluorescently labeled probe. Preferably, the fluorescently labeled probe consists of an oligonucleotide labeled with a fluorescent reporter dye and a quencher dye (= dual-labeled probe). Suitable fluorescent reporters and quencher dyes / moieties are known to those skilled in the art and include, but are not limited to, the reporter dye / group 6-FAM. TM , JOE TM , And quencher dyes / groups dabcyl, TAMRA TM , BHQ TM -1. BHQ TM -2 or BHQ TM -3. Amplification of the probe-specific product causes cleavage of the probe (=amplification-mediated probe displacement), resulting in an increase in reporter fluorescence. The increase in fluorescence in the reaction is proportional to the increase in target amplification. The increase in fluorescence can be measured in real time using a LightCycler 480II system (Roche) or a Versant kPCR system (Siemens) or a Mx3005P system (Agilent Technologies) or an equivalent real-time instrument for detecting fluorescence derived from the probe. The analytical output is a CT value for each target. The CT (cycle threshold) value is determined by the number of PCR amplification cycles, after which the fluorescence signal of the probe exceeds a certain background signal, where the CT value is a measure of the amount of target molecules in the sample before PCR amplification. Preferably, suitable software (e.g., Microsoft Excel TM ) or statistical packages (e.g., SAS 9.0.0,GraphPad Prism4,Genedata Expressionist TM) The CT value is further analyzed. The CT value can be converted to the amount of absolute target molecules (e.g., ng / μl or molecules / μl) based on the CT results of a standard curve with known target concentrations. Alternatively, the target amount can be reported as an x-fold reduction or increase (=ΔCT) based on a reference. Compared to a high ΔCT (larger difference), a low ΔCT value (smaller difference) indicates a higher target amount relative to a reference. It is appropriate to recalculate ΔCT by subtracting it from a fixed value (such as the number of PCR cycles, e.g., 40). The result is a value directly related to the target amount (higher value=higher amount) and is expressed as 40-ΔCT value, where one integer refers to doubling the target amount (e.g., a value of 34 indicates that it is twice the amount of a value of 33). Depending on the required repeatability and system accuracy, multiple reference measurements can be embedded or the ΔCT of the calibrator can be used to recalculate / standardize the ΔCT of the sample (1-point calibration; Pfaffl (2001), Nucleic Acid Res., 29 (9): e45). By using different fluorophores for specific probes, it is also possible to perform multiple different target assays in the same reaction. In PCR, different targets in multiple assays are amplified in parallel but detected separately using different fluorescence emissions.

[0242] Preferably, the primers used according to the present invention have a length of 15 to 30 nucleotides, in particular deoxyribonucleotides. In one embodiment, the primers are designed to: (1) be specific for the target mRNA sequence (i.e., HER2, ESR1, PGR and Ki67 and / or RACGAP1), (2) provide an amplicon size of less than 120 bp (preferably less than 100 bp), (3) detect all known splice variants of the encoded protein, (4) exclude known polymorphisms (e.g., single nucleotide polymorphisms, SNPs), (5) be mRNA specific (taking into account exons / introns; preferably no DNA amplification), (6) have no tendency to dimerize, and / or (7) have a melting temperature T of 58° C. to 62° C. m Range (preferably, T m is about 60°C).

[0243] As used herein, the term "nucleotide" includes natural (naturally occurring) nucleotides comprising a nitrogenous base selected from adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U); a sugar selected from ribose, arabinose, xylose and pyranose; and deoxyribose (a combination of base and sugar, commonly referred to as a "nucleoside"); and 1 to 3 phosphate groups, and which can form a phosphodiester internucleosidyl linkage. In addition, as used herein, "nucleotide" refers to nucleotide analogs. As used herein, "nucleotide analogs" shall refer to analogs of A, G, C, T or U (i.e., analogs of nucleotides comprising the bases A, G, C, T or U), which can be recognized by DNA or RNA polymerase (either is applicable) and incorporated into a strand of DNA or RNA (either is applicable). Examples of such nucleotide analogs include, but are not limited to, 5-propynyl pyrimidine (i.e., 5-propynyl-dTTP and 5-propynyl-dCTP), 7-deazapurine (i.e., 7-deaza-dATP and 7-deaza-dGTP), aminoallyl-dNTP, biotin-AA-dNTP, 2-amino-dATP, 5-methyl-dCTP, 5-iodo-dUTP, 5-bromo-dUTP, 5-fluoro-dUTP, N4-methyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and α-thio-dNTP. Also included are labeled analogs, such as fluorescent analogs such as DEAC-propylenediamine (PDA)-ATP, analogs based on morpholino nucleotide analogs and locked nucleic acid (LNA) analogs.

[0244] The term "target mRNA sequence specific" used in conjunction with primers used according to the present invention means the ability of the primer to hybridize (i.e., anneal) with the cDNA of the target mRNA sequence under suitable temperature and solution ionic strength conditions, particularly under PCR conditions. Temperature and solution ionic strength conditions determine the stringency of hybridization. Hybridization requires that the two nucleic acids (i.e., primers and cDNA) contain complementary sequences, although depending on the stringency of hybridization, there may be mismatches between bases. In one embodiment, "suitable temperature and solution ionic strength conditions" refer to a temperature range of 58°C-62°C (preferably a temperature of about 60°C) and solution ionic strength commonly used in PCR reaction mixtures. In one embodiment, as determined by a sequence comparison algorithm known in the art, the sequence of the primer is 80%, preferably 85%, more preferably 90%, even more preferably 95%, 96%, 97%, 98%, 99% or 100% complementary to the corresponding sequence of the cDNA of the target mRNA sequence.

[0245] In one embodiment, the primer hybridizes with the cDNA of the target mRNA sequence under stringent or moderately stringent hybridization conditions. "Stringent hybridization conditions" as defined herein include hybridization at 68°C in 5x SSC / 5xDenhardt's solution / 1.0% SDS, and washing in 0.2x SSC / 0.1% SDS at room temperature, or include equivalents thereof recognized in the art (e.g., such conditions, hybridization at 60°C in 2.5x SSC buffer, followed by a step of washing several times in a low buffer concentration at 37°C, and remaining stable). "Moderately stringent hybridization conditions" as defined herein include washing in 3x SSC at 42°C, or equivalents thereof recognized in the art. Salt concentration and temperature parameters can be changed to achieve optimal levels for identifying primers and target nucleic acids. Guidance regarding such conditions is available in the art, such as Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley and Sons, NY).

[0246] In one embodiment, the method of the present invention comprises using an ESR1-specific primer having a length of 15 to 30 nucleotides and comprising at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 1 and 2, and / or a HER2-specific primer having a length of 15 to 30 nucleotides and comprising at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 4 and 5, and / or a Ki67-specific primer having a length of 15 to 30 nucleotides and comprising at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 7 and 8, and / or a PGR-specific primer having a length of 15 to 30 nucleotides and comprising at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11, and / or a RACGAP1-specific primer having a length of 15 to 30 nucleotides and comprising at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11. At least 10 consecutive nucleotides of the sequences of NO: 13 and 14. In one embodiment, the specific primer comprises at least 15 consecutive nucleotides of the sequences shown above.

[0247] In one embodiment, the method comprises using ESR1-specific primers, and / or HER2-specific primers, and / or Ki67-specific primers, and / or PGR-specific primers, and / or RACGAP1-specific primers, wherein the ESR1-specific primers have the sequences of SEQ ID NOs: 1 and 2, the HER2-specific primers have the sequences of SEQ ID NOs: 4 and 5, the Ki67-specific primers have the sequences of SEQ ID NOs: 7 and 8, the PGR-specific primers have the sequences of SEQ ID NOs: 10 and 11, and the RACGAP1-specific primers have the sequences of SEQ ID NOs: 13 and 14.

[0248] Preferably, the probe used according to the present invention has a length of 20 to 35 nucleotides (particularly deoxyribonucleotides). In one embodiment, the probe is designed to: (1) be specific to the target mRNA sequence (i.e., HER2, ESR1, PGR and Ki67 and / or RACGAP1), (2) not contain known polymorphisms (e.g., single nucleotide polymorphisms, SNPs), and / or (3) have a melting temperature T m , which is lower than the melting temperature T of the corresponding primer m About 5℃ to 8℃ higher.

[0249] The term "target mRNA sequence specific" used in conjunction with the probe used according to the present invention means the ability of the probe to hybridize (i.e., anneal) the cDNA of the target mRNA sequence (amplified) under suitable temperature and solution ionic strength conditions, particularly under PCR conditions. The temperature and solution ionic strength conditions determine the stringency of hybridization. Hybridization requires that the two nucleic acids (i.e., probe and cDNA) contain complementary sequences, although depending on the stringency of hybridization, mismatches may still occur between bases. In one embodiment, "suitable temperature and solution ionic strength conditions" refer to the temperature range of 63°C to 70°C and the solution ionic strength commonly used in the PCR reaction mixture. In one embodiment, as determined by a sequence comparison algorithm known in the art, the sequence of the probe is 80%, preferably 85%, more preferably 90%, even more preferably 95%, 96%, 97%, 98%, 99% or 100% complementary to the corresponding sequence of the cDNA of the target mRNA sequence (amplified).

[0250] In one embodiment, the probe hybridizes to the (amplified) cDNA of the target mRNA-sequence under stringent or moderately stringent hybridization conditions as defined above.

[0251] In one embodiment, the method comprises using an ESR1-specific probe having a length of 20 to 35 nucleotides and comprising at least 15 consecutive nucleotides of the sequence of SEQ ID NO:3, and / or a HER2-specific probe, and / or a Ki67-specific probe, and / or a PGR-specific probe, and / or a RACGAP1-specific probe, wherein the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:6, the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:9, the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:12, and the RACGAP1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:15. In one embodiment, the specific probe comprises at least 20 consecutive nucleotides of the sequence shown above.

[0252] In one embodiment, the method comprises using an ESR1-specific probe, and / or a HER2-specific probe, and / or a Ki67-specific probe, and / or a PGR-specific probe, and / or a RACGAP1-specific probe, wherein the ESR1-specific probe has the sequence shown in SEQ ID NO:3, the HER2-specific probe has the sequence shown in SEQ ID NO:6, the Ki67-specific probe has the sequence shown in SEQ ID NO:9, the PGR-specific probe has the sequence shown in SEQ ID NO:12, and the RACGAP1-specific probe has the sequence shown in SEQ ID NO:15.

[0253] Preferably, the probe as defined above is a dual-labeled probe comprising a fluorescent reporter group and a fluorescent quencher group.

[0254] In one embodiment, the expression level is normalized with the (average) expression level of one or more reference genes in the tumor sample. As used herein, the term "reference gene" means a gene that has a relatively constant expression level based on RNA transcript / mRNA levels in the system being tested, i.e., cancer. Such genes may be referred to as housekeeping genes. In one embodiment, the one or more reference genes are selected from CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH, preferably CALM2 and / or B2M.

[0255] As used herein, CALM2 refers to calmodulin-2, phosphorylase kinase, delta (Ref. Seq. (mRNA): NM_001743), B2M refers to beta-2 microglobulin (Ref. Seq. (mRNA): NM_004048), RPL37A refers to 60S ribosomal protein L37a (Ref. Seq. (mRNA): NM_000998), GUSB refers to beta-glucuronidase (Ref. Seq. (mRNA): NM_000181), HPRT1 refers to hypoxanthine-phosphoribosyl-transferase 1 (Ref. Seq. (mRNA): NM_000194), and GAPDH refers to glyceraldehyde-3-phosphate-dehydrogenase (Ref. Seq. (mRNA): NM_002046).

[0256] In other aspects, the present invention relates to a method for classifying cancer patients for tumor treatment, the method comprising, in a first step, identifying molecular subtypes of the cancer patient's tumor using the in vitro method defined above, and in a second step, selecting a tumor treatment regimen based on the molecular subtype identified by the in vitro method.

[0257] "Classifying cancer patients for tumor treatment" according to the present invention comprises allocating cancer patients to patient groups with specific molecular tumor subtypes, which subsequently allows physicians to select the most appropriate tumor treatment regimen.

[0258] In one embodiment, the method of classifying cancer patients for tumor treatment does not include any other diagnostic steps, such as histological grading or determining lymph node status, in addition to the step of identifying the molecular subtype of the cancer patient's tumor using the in vitro method defined above.

[0259] In one embodiment, the molecular subtype is selected from HER2-positive, triple negative, luminal A, and luminal B.

[0260] In one embodiment,

[0261] - the molecular subtype is HER2-positive, and the tumor treatment regimen comprises administration of an anti-HER2 antibody and a chemotherapeutic agent;

[0262] - the molecular subtype is triple negative, and the tumor treatment regimen includes administration of a chemotherapeutic agent;

[0263] - the molecular subtype is luminal A and the tumor treatment regimen includes endocrine therapy; or

[0264] - the molecular subtype is luminal B, and the tumor treatment regimen comprises endocrine therapy, and optionally, administration of a chemotherapeutic agent.

[0265] Monoclonal anti-HER2 antibodies including trastuzumab and pertuzumab It can be administered alone or in combination. Trastuzumab is only effective against cancers that overexpress HER2. Other monoclonal antibodies such as ertumaxomab Clinical trials are currently underway. Anti-HER2 antibodies can be further modified to include therapeutic moieties / reagents, such as cytotoxic agents, drugs (e.g., immunosuppressants), chemotherapeutic agents, or radionuclides or radioisotopes. Cytotoxins or cytotoxic agents include any reagent that is harmful to cells and particularly capable of killing cells. Examples include maytansine (mertansine) (DM1), paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (tenoposide), vincristine, vinblastine, colchicine, adriamycin, daunomycin, dihydroxy anthracin dione (dihydroxy anthracin dione), mitoxantrone, mithramycin, actinomycin D, amanitin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs or homologues of the above substances. Other suitable therapeutic agents for forming antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin D (formerly known as actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In preferred embodiments, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A. Other therapeutic moieties include therapeutic moieties that act on mRNA and / or protein synthesis. Several inhibitors of transcription are known. For example, actinomycin D is a transcription inhibitor and DNA damaging agent that inserts into DNA and thereby inhibits the initiation phase of transcription. Flavopiridol targets the elongation phase of transcription. α-amanitin binds directly to RNA polymerase II, which causes inhibition of the initiation and elongation phases. Anti-HER2 antibodies can also be combined with radioactive isotopes such as iodine-131, yttrium-90 or indium-111 to produce cytotoxic radiopharmaceuticals. An alternative to administering anti-HER2 antibodies is to administer small compounds targeting HER2, such as lapatinib or

[0266] Chemotherapeutic agents according to the present invention include cytostatic compounds and cytotoxic compounds. Traditional chemotherapeutic agents work by killing cells that divide rapidly, which is one of the main characteristics of most cancer cells. According to the present invention, the term "chemotherapeutic agent" includes taxane compounds, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines and anthracycline analogs, etoposide, bleomycin, vinorelbine, cyclophosphamide, antimetabolites, antimitogens and alkylating agents, including agents of the combined antibody conjugates disclosed above, and combinations of the above substances. According to the present invention, mentioning chemotherapeutic agents includes any prodrug, such as esters, salts or derivatives of the agent such as conjugates. Examples are conjugates of the agent with a carrier substance, such as paclitaxel bound to a protein, such as paclitaxel bound to albumin. Preferably, the salt of the agent is pharmaceutically acceptable. Chemotherapeutic agents are often given in combination, usually for 3-6 months. One of the most common treatments is cyclophosphamide plus doxorubicin (adriamycin; belonging to the group of anthracyclines and anthracycline analogs), referred to as AC. Sometimes a taxane drug such as docetaxel is added and the regimen is called CAT; taxanes attack the microtubules of cancer cells. Another common therapy that produces equivalent results is cyclophosphamide, methotrexate, which is an antimetabolite, and fluorouracil, which is a nucleoside analog (CMF). Another standard chemotherapy regimen includes fluorouracil, epirubicin, and cyclophosphamide (FEC), which can be supplemented with docetaxel or vinorelbine.

[0267] Endocrine therapy (anti-hormone therapy) by administering drugs such as tamoxifen that block / downregulate estrogen and / or progesterone receptors Fulvestrant Alternatively, use an aromatase inhibitor such as anastrozole or letrozole Blocking estrogen production targets cancers that require estrogen to continue growing. However, aromatase inhibitors are only suitable for postmenopausal patients. This is because the active aromatase in postmenopausal women is different from the prevalent form in premenopausal women, and therefore these agents are ineffective in inhibiting the main aromatase in premenopausal women.

[0268] In one embodiment, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a chemotherapeutic agent.

[0269] In one embodiment, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a taxane, preferably docetaxel.

[0270] In one embodiment, the taxane is administered in combination with fluorouracil, epirubicin, and cyclophosphamide (FEC).

[0271] In other aspects, the present invention relates to a method for treating cancer, the method comprising, in a first step, classifying a cancer patient for tumor treatment using the in vitro method defined above, and in a second step, providing the cancer patient with a selected tumor treatment regimen. The tumor treatment regimen is selected based on the molecular subtype identified by the in vitro method defined above.

[0272] In one embodiment, the method comprises using the quantitative results obtained by the in vitro method as defined above to support or direct decision making for adjuvant / neoadjuvant chemotherapy.

[0273] In yet another aspect, the present invention relates to a kit for identifying molecular subtypes of tumors in cancer patients by a reverse transcription (RT) quantitative PCR method, the kit comprising:

[0274] - at least one pair of HER2-specific primers and at least one HER2-specific probe;

[0275] - at least one pair of ESR1-specific primers and at least one ESR1-specific probe;

[0276] - at least one pair of PGR-specific primers and at least one PGR-specific probe; and

[0277] - at least one pair of Ki67-specific primers and at least one Ki67-specific probe; and / or

[0278] - at least one pair of primers specific for RACGAP1 and at least one probe specific for RACGAP1.

[0279] In one embodiment, the quantitative PCR is fluorescence-based quantitative real-time PCR.

[0280] In one embodiment, detection of the probe is based on amplification-mediated displacement of the probe.

[0281] In one embodiment, the probe is a dual-labeled probe, which comprises a fluorescent reporter group and a fluorescent quencher group.

[0282] In one embodiment, the kit further comprises a reverse transcriptase and a DNA polymerase.

[0283] In one embodiment, the reverse transcriptase and the polymerase are provided in the form of an enzyme mix, which allows for one-step reverse transcription (RT) quantitative PCR.

[0284] In one embodiment, the kit further comprises at least one pair of reference gene-specific primers and at least one reference gene-specific probe. In one embodiment, the reference gene is one or more selected from the following: CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH, preferably CALM2 and / or B2M.

[0285] In one embodiment, the kit further comprises at least one control RNA sample. In one embodiment, the at least one control RNA sample is used as a positive control and / or control sample (calibrator), wherein preferably, the at least one control RNA sample comprises a synthetic mRNA encoding one or more genes selected from HER2, ESR1, PGR, Ki67, RACGAP1 and one or more reference genes (or parts thereof). In one embodiment, the one or more reference genes are selected from CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH, preferably CALM2 and / or B2M.

[0286] In one embodiment, the kit may further comprise DNase and a DNase reaction buffer.

[0287] Preferably, the primers and probes are as further defined above together with the in vitro method of the invention.

[0288] In one embodiment, the primers provide an amplicon size of less than 120 bp, preferably less than 100 bp.

[0289] In one embodiment, the ESR1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 1 and 2, and / or the HER2-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 4 and 5, and / or the Ki67-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 7 and 8, and / or the PGR-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11, and / or the RACGAP1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 13 and 14. In one embodiment, the specific primer comprises at least 15 consecutive nucleotides of the sequences shown above.

[0290] In one embodiment, the ESR1-specific primers have the sequences of SEQ ID NOs: 1 and 2, and / or the HER2-specific primers have the sequences of SEQ ID NOs: 4 and 5, and / or the Ki67-specific primers have the sequences of SEQ ID NOs: 7 and 8, and / or the PGR-specific primers have the sequences of SEQ ID NOs: 10 and 11, and / or the RACGAP1-specific primers have the sequences of SEQ ID NOs: 13 and 14.

[0291] In one embodiment, the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO: 3, and / or the HER2-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO: 6, and / or the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO: 9, and / or the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO: 12, and / or the RACGAP1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO: 15. In one embodiment, the specific probe comprises at least 20 consecutive nucleotides of the sequence shown above.

[0292] In one embodiment, the ESR1-specific probe has a sequence of SEQ ID NO:3, and / or the HER2-specific probe has a sequence of SEQ ID NO:6, and / or the Ki67-specific probe has a sequence of SEQ ID NO:9, and / or the PGR-specific probe has a sequence of SEQ ID NO:12, and / or the RACGAP1-specific probe has a sequence of SEQ ID NO:15.

[0293] Preferably, the probe as defined above is a dual-labeled probe comprising a fluorescent reporter group and a fluorescent quencher group.

[0294] In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is a breast tumor or is derived from a breast tumor (e.g., by metastasis). In one embodiment, the cancer is breast cancer.

[0295] As used herein, the term "kit of parts (abbreviated as: kit)" refers to an article product, which includes one or more containers, and optionally, a data carrier. The one or more containers may be filled with one or more of the above-mentioned components or reagents. The kit may include additional containers, which contain, for example, diluents, buffers and other reagents such as dNTPs. The data carrier may be a non-electronic data carrier, such as a graphic data carrier such as an information booklet, an information table, a bar code or an accession code, or an electronic data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronic data carrier. The accession code allows access to a database, such as an Internet database, a central or decentralized database. The data carrier may include instructions for using the kit in the method of the present invention. The data carrier may include a threshold or reference level of an RNA transcript such as mRNA. In the case where the data carrier includes an accession code that allows access to a database, the threshold or reference level is stored in the database. In addition, the data carrier may include information and instructions on how to implement the method of the present invention.

[0296] In another aspect, the present invention relates to a kit for use as defined above for identifying molecular subtypes of a tumour in a cancer patient.

[0297] In another aspect, the present invention relates to the use of a kit as defined above for assessing the risk of distant metastasis in a cancer patient.

[0298] The present invention overcomes major shortcomings of current standards and state of the art immunohistochemical diagnostic methods as well as newer methods.

[0299] The present invention allows for reliable molecular subtyping of tumors, particularly breast tumors, which will allow physicians to select the most appropriate tumor treatment regimen.

[0300] Thanks to the preferred order of evaluating 4 or 5 markers (preferably, first determining the expression level of HER2 RNA transcripts), there is no misclassification of HER2-positive into luminal A and B subtypes, which is often seen in subtype analysis based on hierarchical cluster analysis or correlation analysis (Perou et al. (2000), Nature, 406: 747-752; TCGA (Cancer Genome Atlas Network) (2012), Nature, 490: 61-70). In addition, there is no false positive and clinical misclassification of HER2-negative tumors, which, according to Perou et al., can occur up to 30%.

[0301] The present invention provides methods and kits for validating / re-evaluating uncertain or contradictory results of immunochemical analysis, particularly in the following groups of breast cancer patients identified by IHC: ESR1 / PGR negative (approximately 30% of all breast cancer patients), ESR1 / PGR weakly positive (approximately 15% of all breast cancer patients), HER2 3+ (tumor biopsy IHC, not validated by resected tumor IHC; approximately 15% of all breast cancer patients), and HER 2+ (approximately 20% of all breast cancer patients).

[0302] The methods and kits of the present invention are useful for supporting or directing decision making for adjuvant / neoadjuvant chemotherapy for luminal breast cancer, since the luminal A and luminal B subtypes can be distinguished by reliable detection of Ki67- and / or RACGAP1 RNA transcripts. This is particularly useful for patients with ESR1 / PGR-positive, grade 2 tumors (approximately 50% of all breast cancer patients).

[0303] The present invention allows for the differentiation of luminal A and luminal B tumors, which have significantly different overall survival and distant metastasis-free survival rates. While luminal B patients have a high risk of continued metastasis, particularly in the first 5 years after treatment (e.g., surgery), the risk for luminal A patients is constant and significantly lower. Thus, the methods and kits also allow for the assessment of a patient's risk of developing distant metastasis.

[0304] By using the method of the present invention, about 77% of luminal tumors in the FinHER study (Joensuu et al. (2006), N Engl J Med, 354: 809-820) were assigned to the low-risk group luminal A, and there was no intermediate risk group. In addition, the classification into luminal A and luminal B reduced the significance of histological grade and nodal status (two parameters that are usually very important for tumor prognosis). Therefore, the uncertainty about the treatment of grade 2 tumors is resolved in a clinically relevant way.

[0305] The methods and kits of the invention also allow prediction of response to systemic therapy thanks to quantitative determination of RNA transcript expression levels of ESR1, HER2, Ki67, and optionally RACGAP1. This is particularly useful for patients with invasive breast cancers ≥ 1 cm with indeterminate IHC (approximately 40% of all breast cancer patients).

[0306] The novelty of the present invention includes not only the estimation based on mRNA of breast cancer biomarkers, but also the algorithmic definition of subtypes. In addition to low Ki67, the mandatory PGR positive defined by luminal A type is also included as the original standard, which was not included in the 2011 St.Gallen clinical guidance when the present invention was submitted. In addition, tumors showing HER2mRNA expression exceeding a predetermined threshold are classified as HER2-positive, regardless of ESR1 / PGR status, which is also derived from the 2011 international guidance, and this guidance is still valid. All these new aspects contribute to the predictive value of the method and kit based on RT-qPCR of the present invention. In fact, these new technologies based on the estimation of biomarkers based on RT-qPCR and the new theory based on the classification algorithm provide more accurate and more meaningful patient subtype typing in the FinHer clinical trial, which ultimately provides a predictive tool for the benefits of docetaxel in the adjuvant therapy environment.

[0307] In summary, the present invention primarily addresses the currently unmet clinical need for reliable, reproducible and quantifiable assessment of prognosis and prediction of treatment success in at least 50% of all breast cancer patients (see also Figure 1 ).

[0308] The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of the present invention. Example

[0309] Example 1: Determination of mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR)

[0310] RNA was isolated from paraffin-embedded formalin-fixed tissue (=FFPE tissue). More specifically, total RNA was extracted from 5-10 μm rolls of FFPE tumor tissue using a high-purity RNA paraffin kit (Roche, Basel, Switzerland) or an XTRAKT RNA extraction kit XL (Stratifyer Molecular Pathology, Cologne, Germany), quantified by Ribogreen RNA quantification (Molecular Probes, Eugene, OR), and quality identified by real-time fluorescence RT-PCR of a reference gene RPL37A fragment. It was found that there were differences between different extraction methods when comparing the quantitative data of target genes in serial sections by different methods. For the purposes of the present invention, the XTRAKT RNA extraction kit XL is preferably used. 2.5 μl of each qualified RNA extract (about 50-100 ng) was determined by qRT-PCR as described below.

[0311] For gene expression analyzed in detail by quantitative RT-PCR method, primers on both sides of the target region and fluorescently labeled probes for intermediate hybridization were used. NCBI primer design tool (www.ncbi.nlm.nih.go) was used to select target-specific primers and probes. RNA-specific primer / probe sequences were used to enable RNA-specific measurements to be performed by locating primer / probe sequences that pass through exon / exon boundaries. In addition, primers / probes that do not bind to sequence regions with known polymorphisms (SNPs) were selected. In the case of multiple isoforms of the same gene, primers for amplifying all related splice variants were selected. The specificity of all primer pairs was checked by conventional PCR reaction. After further optimizing primers / probes, the primers and probes listed in Table 1 obtained the best results. These primers / probes are superior to primers / probes known in the prior art, for example, in terms of specificity and amplification efficiency. In order to standardize the amount of sample RNA, genes CALM2 and B2M were selected as reference genes because they were not differentially regulated in the analyzed samples.

[0312]

[0313]

[0314] Table 1. Primers and probes used.

[0315] Conducted Validation experiments showed that the efficiency of target and control amplification was approximately equal, which is a prerequisite for relative quantification of gene expression by comparing the ΔCT method. In order to perform expression analysis of target genes in biological samples, 4x dual assay-mixtures were prepared by mixing the primers / probes of the two specific assays. In order to detect the CT value separately, the assay probes were modified with different fluorescent probes. Each 4x assay-mixture contained 2 μM of unmodified forward and reverse primers and 1.2 μM of probe. For each reaction, 2.5 μl of total RNA extracted from FFPE sections (see above) was mixed with 2.5 μl of assay-mixture, 2.5 μl of enzyme-mixture and 2.5 μl of water in one well of a 96-well light reaction plate. According to the manufacturer's instructions, the PCR reaction was measured under appropriate conditions (5min.50°C, 20sec.95°C, 15sec.95°C, 1min.60°C; 40 cycles) using a Versant kPCR cycler (Siemens) or Light Cycler 480 (Roche). Experiments with, for example, cell lines, healthy control samples, samples of defined molecular tumor subtypes can be used to standardize experimental conditions before measuring currently unclassified biological sample controls.

[0316] Example 2: Classification of tumors based on mRNA expression levels of HER2, ESR1, PGR, Ki67, and optionally RACGAP1 Subtype classification

[0317] For the evaluation of breast tumors, 855 of 1010 breast cancer patients from the FinHER study (Joensuu et al. (2006), N Engl J Med, 354: 809-820) were used. The average thresholds (given as 40-ΔCT values) were as follows: HER2: 38; ESR1: 34; PGR: 30,2; Ki67: 31,7; and RACGAP1: 34,2. Based on the evaluation of previous studies (Koutras et al. (2008), Brit. J. of Canc., 99: 1775-1785; Pentheroudakis et al. (2009), Breast Cancer Res Treat 2009, 116: 131-143), the thresholds for HER2 and ESR1 were determined using 268 samples and applied to the FinHER study (proportional hazard model validation). CALM2 was used as a reference gene.

[0318] Based on the mRNA expression levels of HER2, ESR1, PGR and Ki67 (negative / low indicates expression levels lower than the set expression threshold; positive / increased indicates expression levels higher than the set expression threshold), the tumor is assigned to the molecular subtypes HER2-positive (HER2), luminal A (LumA), luminal B (LumB) and triple negative (TNT). As shown in Table 2, the tumor molecular subtyping according to the present invention is different from those based on prior art methods, such as immunohistochemistry (Goldhirsch et al. (2011), Annals of Oncology, 22: 1736-1747, St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011) and only analyzing 3 gene markers (Sotiriou et al. (2009), N Engl J Med, 360 (8): 790-800).

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[0320] Table 2. Molecular subtyping of tumors.

[0321] Fig. 2 shows the prognosis and predictive value of the expression level of HER2, ESR1, PGR, Ki67 and RACGAP1mRNA for the 5-year survival rate of breast cancer patients.These data show that the RACGAP1mRNA expression level below or above the set threshold is related to the special significant difference of 5-year survival rate.More particularly, the increased RACGAP1mRNA expression level significantly reduces the survival probability.If the mRNA expression of RACGAP1 increases, and Ki67mRNA expression is low, the survival probability is further reduced (see Table 3).Utilizing the current analysis scheme, these risk patients (which will require different types of follow-up care, such as using specific imaging methods to check) are still undiscovered.

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[0323] Table 3. RACGAP1 / Ki67 mRNA expression and survival probability.

[0324] Using Kaplan Meier analysis, the inventors analyzed the survival of patients with HER2-positive, luminal A, luminal B, and triple-negative tumors, respectively, wherein the molecular subtypes of the tumors were identified according to the present invention, i.e., based on the mRNA expression levels of HER2, ESR1, PGR, and Ki67. Figure 3 As shown in , as identified by the present inventors, the luminal A subtype is associated with a 97% overall survival rate after 5 years (compared to 87% for luminal B and HER2-positive tumors and 84% for triple-negative tumors).

[0325] The inventors also analyzed distant metastasis-free survival ("DMFS"; distant recurrence X years after surgery) of patients with HER2-positive, luminal A, luminal B, and triple-negative tumors, respectively, where the molecular subtypes of the tumors were identified according to the invention. Figure 4 As shown in , as identified by the present inventors, the luminal A subtype is associated with a 92% distant metastasis-free survival after 5 years (compared to 78% for luminal B, HER2-positive and triple-negative tumors).

[0326] In a multivariate analysis of overall survival using the most important histopathological standard parameters (tumor size, nodal status and histological grade), molecular subtyping according to the invention proved to be highly significant, whereas histological grade and nodal status lost their significance.

[0327] Finally, a multivariate Cox regression analysis of DMFS was performed, comparing molecular subtyping by immunohistochemistry (Sotiriou et al. (2009), N Engl J Med, 360(8):790-800) with molecular subtyping by the method according to the present invention based on mRNA expression levels of HER2, ESR1, PGR and Ki67 ( Figure 5 ). This analysis clearly demonstrated the superiority of the method of the present invention, since the immunohistochemical subtyping lost its significance when the results obtained by the method of the present invention were incorporated into the Cox proportional hazards model. Example 3: Measurement by reverse transcription (RT) quantitative PCR (RT-qPCR) mRNA expression levels of biomarkers HER2, ESR1, PGR, Ki67 and determination of molecular subtypes

[0328] RNA was isolated from FFPE tissue. More specifically, total RNA was extracted from 10 μm FFPE breast tumor tissue sections using the XTRAKT RNA extraction kit (Stratifyer Molecular Pathology, Cologne, Germany). The RNA eluate was used directly without determining the concentration. 2.5 μl of RNA from each extract was assayed by RT-qPCR as described below.

[0329] For RT-qPCR, primers flanking the target region and a hydrolysis probe 5'-fluorescently labeled with a 3'-TAMRA or 3'-Dabcyl quencher were used for each target. The primers and probes used are listed in Table 1. In order to correct for different amounts of sample RNA, the genes CALM2 and B2M were used as reference genes for normalizing expression results. RT-qPCR was performed in duplicate in the following combinations: HER2 / ESR1, Ki67 / B2M, and PGR / CALM2. Each of the three 4x assay mixtures contained 2 μM unmodified forward and reverse primers and 1.2 μM probe. For each assay, a master mixture with 2.5 μl of assay mixture, 2.5 μl of 4x enzyme mixture (TaqManFast Virus1-Step MasterMix, Life Technologies), and 2.5 μl of water per reaction was prepared, and 7.5 μl of each master mixture was distributed in triplicate to a kPCR 96-well reaction plate. 2.5 μl of total RNA extracted from FFPE sections (see above), or alternatively, positive (IVT RNA) or negative control (water) were added to each well. Analysis of RNA eluates from 3 patient samples was performed using 3 batches of assay mix and enzyme mix. One-step RT-qPCR reactions were measured using a Versant kPCR cycler (Siemens) according to the manufacturer's instructions with the following thermal profile: 5 min 50°C, 20 sec 95°C, one cycle per reaction, and 15 sec 95°C, 1 min 60°C for 40 cycles.

[0330] Six patient samples along with positive and negative controls were analyzed using three batches of the assay mixture.40-ΔΔCT values ​​were calculated according to the description provided above (Calculation Method 4). Figures 6 to 9 The 40-ΔΔCT values ​​for each marker for each sample and batch are shown. The thresholds (given as 40-ΔΔCT values) used to classify a biomarker as positive or negative were as follows: HER2: 40.90; ESR1: 38.20; PGR: 34.90; Ki67: 34.80. The molecular subtypes were defined as shown in Table 4.

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[0333] Table 4. Molecular subtyping of patient samples.

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[0335] Table 5. 40-ΔΔCT values ​​and isoform calling for assay mixture batch 1.

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[0337] Table 6. 40-ΔΔCT values ​​and isoform estimates for assay mixture batch 2.

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[0339] Table 7. 40-ΔΔCT values ​​and isoform estimates for assay mixture batch 3.

[0340] Only slight differences were observed in the results between different reagent batches. The assay performance was very robust and reproducible for all markers. Tables 5 to 7 show the 40-ΔΔCT values ​​and positive or negative results for each marker and conversion to the respective subtypes defined in Table 4 (see Table 2). The marker results (positive / negative) and subtypes were consistent for all 6 samples measured using 3 different reagent batches.

[0341] Example 4: Comparison of breast cancer subtyping by RT-qPCR and IHC based on clinical results

[0342] Figures 10 to 19 Kaplan-Meier survival curves of different tumor subtypes (luminal B, HER2-positive, luminal A, and triple negative breast cancer [TNBC]) treated with different chemotherapeutic agents (docetaxel or vinorelbine) are shown. The subtypes determined by RT-qPCR according to the present invention are shown in Figure A, while the subtypes determined by IHC are shown in Figure B. The dotted line indicates the 5-year time point for the result calculation.

[0343] Figures 10 to 19 The data shown in the together emphasize the advantages of the new RNA-based breast cancer classification by RT-qPCR over conventional protein-based IHC. The data show a clear advantage of docetaxel treatment for luminal B tumors. In contrast, IHC-based subtyping failed to show a clear advantage in any subtype with some trends in luminal A, luminal B, and triple-negative breast cancer patients. However, because these 3 subtypes include ~80% of all patients, the IHC-based subtyping cannot provide any clinically useful predictive information.

[0344] Importantly, after combined treatment with fluorouracil, epirubicin, and cyclophosphamide (FEC), patients with luminal B tumors identified by RT-qPCR received statistically significant and exclusive benefits from docetaxel compared to vinorelbine. These patients remained metastasis-free and survived significantly longer after treatment with docetaxel, however, their results were significantly worse when receiving vinorelbine. This difference in results between the two chemotherapy regimens was not observed in any other subtype except luminal B. Interestingly, for luminal A tumors, docetaxel seemed to be inferior to vinorelbine. Although this effect did not reach statistical significance, it clearly ruled out the possible beneficial effect of docetaxel in treating luminal A patients, as seen in luminal B patients identified by RT-qPCR.

[0345] On the contrary, when subtyping is performed by IHC patients, the effect of the type of regimen on clinical results is ambiguous due to inaccurate classification. This in turn produces a variety of uncertain trends that cannot be relied upon for clinical decision making. In summary, the present invention undoubtedly identifies a specific group of breast cancer patients who respond favorably to docetaxel rather than vinorelbine, thereby showing the predictive power of luminal B-type tumors in a specific environment. This is a very important characteristic for diagnostic determination, considering that as shown in the FinHER clinical trial, docetaxel is more commonly associated with side effects than vinorelbine, a fact that necessitates a reduction in the scheduled starting dose.

[0346] Therefore, the present invention can be used for appropriate allocation of adjuvant breast cancer treatment. In particular, as shown for the first time in the context of a randomized clinical trial, breast cancer subtyping by RT-qPCR allows limiting docetaxel-containing regimens to patients with luminal B tumors, which are more responsive to docetaxel treatment, and allows consideration of alternative chemotherapy for patients with other tumor subtypes.

[0347] Example 5: Comparison of RT-qPCR and conventional IHC staining based on their sensitivity to Ki67

[0348] A large number of cases were Ki67 negative by IHC (16.58%) but Ki67 positive by RT-qPCR. This demonstrates that the mRNA assay of the present invention has higher sensitivity and robustness than the prior art protein-based assessment. In part, this may be due to various technical limitations of IHC-based methods (e.g., lack of protein protection due to fixation, and / or antigen retrieval issues, analysis time after tissue excision, etc.) or staining interpretation issues (e.g., interpretation of weaker nuclear staining).

[0349] Due to the higher sensitivity of RNA-based Ki67 assessment by RT-qPCR, its agreement with IHC assessment of Ki67 is only moderate (see Fig. 20 In addition, this resulted in a low NPA (53.82%) of the present invention when localized IHC assessment was used as a reference method ( Fig. 20 In contrast, PPA was high, as RT-qPCR identified the majority of positive IHC cases (94.2%). In summary, both methods were significantly correlated (p<0.0001), but only moderately consistent.

[0350] Table 8 is a contingency table showing the association between RT-qPCR-based and IHC-based subtypes in the FinHer study population, where N is the number of observations, % (cell) is the overall frequency of 16 possible subtype combinations, % (column) is the distribution of RT-qPCR subtypes in IHC subtypes, and % (row) is the distribution of IHC subtypes in subtypes determined by the present invention. Setting IHC subtype as the "reference standard", TNBC (85.71%) and HER2-positive (79.43%) tumors have the highest consistency, and luminal A (65.38%) and luminal B (61.22%) tumors have the lowest consistency.

[0351] Example 6: Subtyping based on RT-qPCR and IHC mainly differs in the assessment of luminal B type

[0352] When ESR1, PGR, HER2, and Ki67 were combined for subtyping using the same algorithm, the higher sensitivity of RT-qPCR-based assessment of Ki67 compared with IHC-based assessment resulted in substantial differences in the identification of luminal A and luminal B patients.

[0353] Of the 189 breast cancer patients classified as luminal A by IHC, only 53.97% were also classified as luminal A by RT-qPCR, while 39.68% turned out to be luminal B patients. In contrast, only 6.35% were reclassified as HER2-positive and 0% were triple-negative. In addition, of the 251 patients classified as luminal B by IHC, 71.71% were also classified as luminal B by RT-qPCR, while 19.12% turned out to be luminal A patients, 6.77% were reclassified as HER2-positive, and 2.39% were triple-negative. In contrast, only 61.22% of tumors classified as luminal B by RT-qPCR were also classified as luminal B by conventional IHC.

[0354] These data show that moderate agreement was observed between the two assays for determining luminal B tumors sensitive to docetaxel, which was mainly caused by the limited sensitivity and / or robustness of the semiquantitative assessment of Ki67 by IHC.

[0355] Example 7: High Ki67 by RT-qPCR but low Ki67 by IHC is associated with a higher risk of distant metastasis

[0356] The method of the present invention demonstrated additional discriminatory power when considering a population containing only ER-positive cases determined by IHC. In this case, Ki67 was found to be discordant between IHC-based and RT-qPCR-based assays. Since Ki67 was concordant by RT-qPCR and IHC in 514 of 686 available datasets (74.92%), the number of samples used to demonstrate the superiority of mRNA-based assessment was limited (n=172).

[0357] However, despite the small sample numbers, Ki67 positivity by RT-qPCR indicated an increased risk of distant metastasis in the discordant setting. Fig.21 As shown in Figure A, at 5 years of follow-up, 5% of ER-positive patients with low Ki67 mRNA expression had distant metastasis, while 15% of patients showing high Ki67 mRNA expression had distant metastasis (HR 3.315). This trend was not affected by multivariate analysis ( Fig.21 Figure B in the figure).

[0358] This suggests that the higher sensitivity of the method of the present invention regarding Ki67 assessment provides additional prognostic information compared to conventional IHC staining.

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Claims

1. An in vitro method for identifying molecular subtypes of a tumor in a cancer patient, the method comprising the following steps: (f) determining the expression level of RNA transcript of human epidermal growth factor receptor 2 (HER2) in a sample of the tumor; (g) determining the expression level of RNA transcript of estrogen receptor (ESR1) in the sample of the tumor; (h) determining the expression level of RNA transcript of progesterone receptor (PGR) in a sample of said tumor; and (i) determining the expression level of RNA transcripts of proliferation antigen Ki-67 (Ki67) in a sample of the tumor; and / or (j) determining the expression level of RNA transcript of RacGTPase activating protein 1 (RACGAP1) in the sample of the tumor.

2. The method of claim 1, wherein determining the expression level of RNA transcripts of HER2, ESR1, PGR, Ki67 and / or RACGAP1 comprises determining whether the expression level of RNA transcripts of HER2, ESR1, PGR, Ki67 and / or RACGAP1 is below or above a set expression threshold of RNA transcripts of HER2, ESR1, PGR, Ki67 and / or RACGAP1.

3. The method of any one of claims 1 to 2, wherein the molecular subtype is selected from HER2-positive, triple negative, luminal A, and luminal B.

4. The method of any one of claims 1 to 3, wherein the molecular subtype is luminal A or luminal B.

5. The method of any one of claims 1 to 4, wherein the cancer is breast cancer.

6. A kit for identifying molecular subtypes of tumors in cancer patients by reverse transcription (RT) quantitative PCR, the kit comprising: - at least one pair of HER2-specific primers and at least one HER2-specific probe; - at least one pair of ESR1-specific primers and at least one ESR1-specific probe; - at least one pair of PGR-specific primers and at least one PGR-specific probe; and - at least one pair of Ki67-specific primers and at least one Ki67-specific probe; and / or - at least one pair of primers specific for RACGAP1 and at least one probe specific for RACGAP1.

7. The kit of claim 6, wherein the ESR1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 1 and 2, and / or wherein the HER2-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 4 and 5, and / or wherein the Ki67-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 7 and 8, and / or wherein the PGR-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 10 and 11, and / or wherein the RACGAP1-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequences of SEQ ID NOs: 13 and 14.

8. The kit according to any one of claims 6 to 7, wherein the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:3, and / or wherein the HER2-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:6, and / or wherein the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:9, and / or wherein the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:12, and / or wherein the RACGAP1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 consecutive nucleotides of the sequence of SEQ ID NO:

15.

9. The kit of any one of claims 6 to 8, wherein the cancer is breast cancer.

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