Application of detecting chromosome segment information in prediction of extrahepatic metastasis in patients with primary liver cancer

Abstract

The application belongs to the field of prognosis of extrahepatic metastasis of primary liver cancer, and relates to application of chromosome deletion in prediction of extrahepatic metastasis of primary liver cancer. From the perspective of clonal evolution, it is found that deletion of chromosome segments 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p and 22q is significantly enriched in metastatic foci. The number of the seven deletion variations in the primary foci is calculated as a risk score, and it is found that metastatic cancer has a higher risk score compared with primary liver cancer. It is also found that patients with higher risk scores of primary cancer have a shorter time of extrahepatic metastasis, indicating that the seven deletion variations can be used as a risk prediction factor for extrahepatic metastasis after surgical resection of primary liver cancer. In addition, single factor and multi-factor analysis finds that the seven deletion variations have stable prediction ability for the metastasis risk of patients in different data sets. Therefore, the chromosome copy number variation of the primary tumor is a prediction index for extrahepatic metastasis after surgical resection of primary liver cancer, and is a possible treatment target for preventing extrahepatic metastasis in the future.

Description

Technical Field

[0001] This invention belongs to the field of prognosis of extrahepatic metastasis of primary liver cancer, and relates to the application of detecting chromosomal segment information in predicting extrahepatic metastasis in patients with primary liver cancer. Background Technology

[0002] Somatic copy number variations can be used for early tumor diagnosis and newborn genetic screening. The UroVysion Bladder Cancer Kit is designed to diagnose bladder cancer by detecting aneuploidy of chromosomes 3, 7, and 17 and deletion of 9p21 in urine samples from patients with suspected bladder cancer using fluorescence in situ hybridization (FISH). Copy number variations are the second leading cause of birth defects in newborns, making prenatal screening essential. Currently, non-invasive prenatal screening is only available for trisomy 21, trisomy 18, and trisomy 13.

[0003] However, very few currently available cancer diagnostic kits address copy number variations at the chromosomal arm level. Furthermore, there are no reported technologies using chromosomal segment detection information as a risk predictor in the prediction of extrahepatic metastasis risk in patients with primary liver cancer. Summary of the Invention

[0004] To overcome the above-mentioned technical problems, this invention provides the application of detecting chromosomal segment information in predicting extrahepatic metastasis in patients with primary liver cancer.

[0005] In a first aspect, the present invention provides the application of a detection reagent for detecting missing copy number information of chromosomal segments in the preparation of a product for predicting the risk of extrahepatic metastasis in patients with primary liver cancer, wherein the chromosomal segments are 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q.

[0006] Using the above technical solution, this invention, through the study of the amplification and deletion changes of genes at the small fragment and chromosome arm length level, discovered from the perspective of clonal evolution that deletions of chromosome segments 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q are significantly enriched in metastatic cancers.

[0007] Furthermore, the product is a reagent kit or a testing device.

[0008] Furthermore, the risk prediction includes the following steps:

[0009] Step a) assess the copy number of the chromosomal segment in the tumor tissue of the patient with primary liver cancer; step b) compare the assessment results of step a) with the chromosomal segment in normal tissue; and step c) if copy number variation or deletion of the chromosomal segment occurs, the patient has a higher risk of extrahepatic metastasis after surgical resection of primary liver cancer.

[0010] Secondly, the present invention also provides a detection kit or device for predicting the prognosis of extrahepatic metastasis risk in patients with primary liver cancer, wherein the detection kit or device includes a detection reagent for detecting copy number information of chromosomal segments.

[0011] Furthermore, the chromosome segments are 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q.

[0012] The present invention has the following technical advantages over the prior art:

[0013] 1) This invention, through the study of gene amplification and deletion variations at the small fragment and chromosome arm length levels, reveals from a clonal evolutionary perspective that deletions of chromosome segments 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q are significantly enriched in metastatic cancers. The number of these seven deletion variants in the primary tumor was calculated as a risk score, revealing that metastatic cancer has a higher risk score compared to primary liver cancer. Furthermore, it was found that patients with higher risk scores in primary cancer developed extrahepatic metastasis more quickly, suggesting that these seven deletion variants can serve as risk predictors of extrahepatic metastasis after surgical resection of primary liver cancer.

[0014] 2) This invention also found through univariate and multivariate analyses that seven deletion variants have stable predictive ability for the metastasis risk of patients in different datasets. Therefore, chromosomal copy number variations in the primary tumor are a predictive indicator of extrahepatic metastasis after surgical resection of primary liver cancer and a potential therapeutic target for future prevention of extrahepatic metastasis. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0016] Figure 1Weighted genomic instability index (wGII) for primary (PT) and metastatic (MT) hepatocellular carcinoma samples. (A) wGII in PT of patients in the extrahepatic metastasis group (MET) and non-metastatic recurrence group (NRM) in the discovery cohort. (B) wGII in PT and MT of patients in the extrahepatic metastasis group in the discovery cohort. P-values ​​were obtained from unpaired two-ended Wilcoxon rank-sum tests.

[0017] Figure 2 In the extrahepatic metastasis group of the discovery cohort, the proportion of occurrences of each local (A) or chromosome arm-level SCNA (B) in both "metastasis-selected" and "non-metastasis-selected" subclones. The right-hand plot shows the enrichment of each local or chromosome arm-level SCNA event in both "metastasis-selected" and "non-metastasis-selected" subclones, presented as log10 p-values. P-values ​​were calculated using a binomial test and corrected using the Benjamini–Hochberg method.

[0018] Figure 3 Met SCNAs are associated with metastatic prognosis in patients. (A) Number of Met SCNAs in MT and PT in the extrahepatic metastasis group of the discovery cohort, and in PT in the non-metastatic relapse group. Data points represent the maximum number of Met SCNAs in PT or MT of patients. P-values ​​are derived from unpaired two-ended Wilcoxon rank-sum tests. (B) Kaplan-Meier curves of metastatic survival based on Met SCNA status (higher vs. lower) in PT of patients in the discovery cohort. (C) Kaplan-Meier curves of progression-free survival based on Met SCNA status in PT of patients in the TCGA LIHC cohort. Patients with metachronous metastases were included in analyses (B) and (C).

[0019] Figure 4 Univariate and multivariate Cox regression analyses were conducted to identify factors related to transfer time in the cohort.

[0020] Figure 5 Met SCNAs as a predictor of risk of extrahepatic metastasis in a validation cohort. (A) wGII in PT of patients in the extrahepatic metastasis group and the non-metastatic relapse group in the validation cohort. P values ​​are derived from unpaired two-ended Wilcoxon rank-sum test. (B) Kaplan-Meier curves of metastatic survival based on Met SCNA status in patients' PT in the validation cohort. Patients with metachronous metastases were included in this analysis. (C) Univariate Cox regression analysis of factors related to metastasis time in the validation cohort. Detailed Implementation

[0021] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustration and explanation only and are not intended to limit the invention as detailed in the claims. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional methods, and the experimental materials used are available from commercial companies unless otherwise specified.

[0022] Example 1

[0023] We collected surgical resection specimens from 109 patients with primary liver cancer as a discovery cohort. Based on follow-up information, 78 patients in the cohort developed extrahepatic metastasis, 31 patients did not develop metastasis or recurrence after surgery, and whole-exome sequencing was performed on the collected tumor samples.

[0024] Sequenza (v2.1.2) and Gistic (v2.0) software were used to infer somatic copy number alterations (SCNAs) in liver cancer specimens. The weighted genomic instability index (wGII) was calculated, such as... Figure 1 As shown, in the cohort, compared with patients without metastasis or recurrence, patients with extrahepatic metastases showed a significantly higher wGII in their primary hepatocellular carcinoma (median 0.41 in the extrahepatic metastasis group and 0.29 in the non-metastasis / recurrence group, p = 0.0067), indicating a higher SCNA burden in hepatocellular carcinoma patients with extrahepatic metastases. Overall genomic instability was significantly increased in extrahepatic metastases of hepatocellular carcinoma (median 0.48 in metastatic samples and 0.41 in primary samples, p = 0.027), suggesting that somatic copy number variation events may play a key role in influencing the metastatic potential of hepatocellular carcinoma.

[0025] Example 2

[0026] Primary tumors typically exhibit high intratumoral heterogeneity, meaning they contain multiple genetically differentiated subclonal cell populations. Usually, only a subset of these subclonal cell populations possess metastatic potential and generate distant metastases. Using multi-regional sampling and phylogenetic reconstruction, we analyzed the progression of individual clones from the primary site to the metastatic site, or from the recurrent site to the metastatic site. Based on the mutational patterns of somatic mutations in the primary lesion and paired metastases, all copy number variations were categorized into three types: selected, unselected, and maintained. Variations present only in metastases (subclonal mutations) but absent in the paired primary or recurrent lesions were not defined as selected, as they may have been acquired after cancer cell colonization at the metastatic site and do not affect metastatic progression. For each focal / Arm level SCNA in the extrahepatic metastasis dataset, we calculated the proportions of selected, unselected, and maintained mutations based on paired primary lesions and primary-derived metastases, recurrent lesions, and recurrent-derived metastases. The proportion of metastatic lesions selected can be calculated by selecting / (selected+unselected) to further screen for variants enriched in metastatic lesions.

[0027] Arm-level SCNAs significantly enriched in public datasets (Cancer Genome Atlas Research Network, 2017; Gao et al., 2019) or in metastatic lesions of the extrahepatic metastasis dataset (q <= 0.05) were defined as driving Arm-level SCNAs (N.driver = 34, N.passenger = 46). Focal-level SCNAs significantly enriched in primary lesion samples of the discovery cohort extrahepatic metastasis dataset were also divided into two classes: focal-level SCNAs validated in any of the four public datasets (Wang et al., 2013; Cancer Genome Atlas Research Network, 2017; Gao et al., 2019; Zhou et al., 2019) were defined as driving focal-level SCNAs (N.driver = 22, N.passenger = 13). The background probability, i.e., the probability that a passenger SCNA was selected by a metastatic lesion, was calculated based on all passenger SCNAs in the discovery cohort extrahepatic metastasis dataset. Subsequently, based on a binomial test, the proportion of "selected" events was compared with the proportion of "unselected" events. The background probability of focal level SCNAs was p = 0.4964, and the background probability of Arm level SCNAs was p = 0.4928. Three driving focal level SCNAs (11p15.5, 14q32.33, and 4q35.2) and four driving Arm level SCNAs (14q, 19p, 21p, and 22q) were significantly enriched in metastatic lesions, suggesting that these seven copy number variations may promote the metastatic process of hepatocellular carcinoma. Figure 2 ).

[0028] Example 3

[0029] Previous comparisons of primary and metastatic SCNAs in patients with extrahepatic metastases in a cohort revealed seven SCNA events enriched in metastatic tumors of these patients. Given the complexity and multi-step nature of cancer metastasis, a single metastatically selected SCNA may be insufficient to complete the entire metastatic process. We defined the seven copy number variations significantly enriched in metastatic lesions as Met SCNAs, and calculated the number of these seven Met SCNAs in the primary lesion as a risk score. Compared to the primary lesion, metastatic lesions in patients with extrahepatic metastases had significantly higher risk scores. Figure 3A, p = 0.0038). Consistently, primary lesions with metastatic clinical outcomes showed significantly higher risk scores compared to primary lesions from the non-metastatic / recurrent group (p = 0.0077). In the discovery cohort, the median risk score of all primary lesions was defined as a threshold. If the number of Met SCNAs in the primary lesion was greater than or equal to the threshold, the patient was classified as a higher Met SCNA; otherwise, the patient was classified as a lower Met SCNA. The same method was applied to the TCGA LIHC cohort. Considering the practical clinical application value and reducing predictive bias, patients with both primary tumors and metastases were excluded from the analysis, resulting in a total of 79 patients in the cohort, of whom 48 had extrahepatic metastases and 31 did not (non-metastatic / recurrent patients). In the discovery cohort (p = 0.0001), patients with metastatic metastases in their primary lesions (met SCNA counts equal to or greater than the median Met SCNA counts of primary lesions across all patients in the sample set) exhibited significantly shorter metastasis times. Figure 3 B). In the public dataset TCGA LIHC, patients with higher Met SCNA counts also showed worse progression-free survival (p = 0.027). Figure 3 C). A multivariate analysis of these seven SCNA events and the time to distant metastasis after primary liver cancer resection showed that Met_SCNAs had a statistically significant prognostic impact. Figure 4 The above results suggest that SCNA events in the primary tumor are a predictor of extrahepatic metastasis after surgical resection of primary liver cancer, and may even be a potential therapeutic target for preventing extrahepatic metastasis in the future.

[0030] Application Examples

[0031] To further validate the predictive power of Met-SCNAs for metastatic prognosis, we also collected primary tumor data from 65 patients as a validation cohort. During follow-up, 41 patients developed extrahepatic metastases, while 24 patients neither developed metastases nor experienced intrahepatic recurrence. Consistently, compared with patients without metastases or recurrence, patients with extrahepatic metastases showed significantly higher wGII in their primary hepatocellular carcinoma (median 0.32 in the extrahepatic metastasis group and 0.20 in the non-metastatic / recurrence group, p = 0.018), suggesting a higher SCNA burden in patients with extrahepatic metastases. Figure 5 A). By calculating the Met SCNA count in each sample, it was found that in the validation cohort (p = 0.047), patients with metastatic metastases from primary lesions exhibited significantly shorter metastasis times. Figure 5 B). Furthermore, univariate analysis showed that Met_SCNAs also had a statistically significant effect on patient transfer time in the validation cohort (B). Figure 5 C).

[0032] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. The application of a reagent for detecting chromosomal segment copy number information in the preparation of products for predicting the risk of extrahepatic metastasis in patients with primary liver cancer, characterized in that, The chromosome segments are 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q.

2. The application according to claim 1, characterized in that, The product in question is a reagent kit or testing device.

3. The application according to claim 1, characterized in that, The risk prediction includes the following steps: Step a) Assess the copy number of the chromosomal segment in the tumor tissue of the patient with primary liver cancer; Step b) Compare the evaluation results of step a) with the chromosomal segments described in normal human tissue; and Step c) If a copy number variation or deletion of the chromosomal segment occurs, the patient has a higher risk of extrahepatic metastasis after surgical resection of the primary liver cancer.

4. A diagnostic kit or device for predicting the risk of extrahepatic metastasis in patients with primary liver cancer, characterized in that, The test kit or device includes a test reagent for detecting copy number information of chromosome segments, wherein the chromosome segments are 11p15.5, 14q32.33, 4q35.2, 14q, 19p, 21p, and 22q.

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