Copy Number Alteration Burden Research Articles

Tumor Copy Number Alteration Burden as a Predictor for Resistance to Immune Checkpoint Blockade across Different Cancer Types.

Immune checkpoint blockade (ICB) benefits only a subset of advanced cancer patients, and predictive biomarkers for immunotherapy response are needed. Recently, copy number alteration (CNA) burden has been proposed to predict ICB resistance. We assessed this finding using the publicly accessible data for 1661 ICB-treated patients whose tumors were profiled by MSK-IMPACT, an approved targeted assay in clinical care. We tested the hypothesis that the continuous increase in CNA burden is associated with poor overall survival following ICB. In addition, we hypothesized that the combinatorial biomarkers of tumor mutational burden (TMB) and CNA burden would better stratify patients for immune status and ICB response. Of the 1661 cases, 79% (n = 1307) were treated with anti PD-1/PD-L1 and the remaining 21% (n = 354) with anti CTLA-4 or the combination of both. In a multivariate analysis, increase in CNA burden was associated with poor overall survival [HR = 1.52, 95% CI (1.01-2.30), p = 0.04]. The combination of biomarkers TMB and CNA burden stratified patients into four clinically distinct subsets among which "LowTMB/HighCNA" showed the worst survival (p < 0.0001). The four patient subsets had unique CNA profiles and enriched pathways, which could predict transcriptional and phenotypic effects related to immune signaling and CD8+ T-cell abundance in the tumor microenvironment. CNA burden was associated with poor overall survival in patients receiving ICB and could improve patient stratification when incorporated with TMB. These findings may guide patient selection for immunotherapy or alternative strategies.

Abstract A038: Evaluating DCIS progression: A comparative analysis of CNA predictive power derived from lpWGS and WES data

Abstract Ductal carcinoma in situ (DCIS) is a very common non-life threatening, pre-invasive form of breast cancer constituting 25% of all new breast cancer diagnoses in the USA, and is normally treated with invasive measures. However, only 20 to 30% of DCIS cases will progress to life-threatening invasive breast cancer in their lifetime. There is a need for patient stratification to decrease treatment burden and focus resources on patients who actually require treatment. To ameliorate this issue, we aim to develop evolutionary biomarkers that predict both DCIS recurrence and its progression to invasive breast cancer. We conducted an observational longitudinal study in which two DCIS samples from each primary tumor were acquired prior to treatment—to account for intratumor heterogeneity—and followed up with (median event-free patient observation time: 106 months) to classify each patient’s outcome into its respective cohort category of [1] no DCIS recurrence, [2] DCIS recurrence, or [3] invasive ductal carcinoma (IDC) recurrence (progression). Each of the three cohort groups comprises approximately 30 patients, for which our primary data includes low-pass whole genome sequencing (lpWGS) and whole exome sequencing (WES) of the two primary DCIS samples, in addition to WES data for normal tissue control, clinical and epidemiological covariates, and time to recurrence. Preliminary estimates of copy number alterations (CNAs) generated using lpWGS data show that CNA burden, representing the proportion of altered genome, has statistically significant predictive capability for DCIS progression (Univariate Cox regression: p=0.014, HR=1.5). CNA divergence, representing the proportion of non-overlapping altered genome, has predictive capabilities of both recurrence and progression (Univariate Cox regression: Recurrence: p=0.021, HR=0.74; Progression: p=0.024, HR=0.67). In this work, our primary objective is to test if CNAs generated utilizing WES data are comparable in their predictive power to those yielded by lpWGS data for the same human DNA samples. It is not expected that the copy number alteration (CNA) estimates will be identical between the two methods, but rather that mutational burden and intratumor heterogeneity estimated using different data sources are strongly correlated and will have similar predictive capabilities. If we successfully confirm that CNAs estimated with both lpWGS and WES data have similar predictive power of DCIS progression, we will then be able to validate our evolutionary biomarkers using a different cohort for which we only possess WES data. This would also expand the applicability of our evolutionary biomarkers to a larger number of pre-existing datasets. We ultimately aim to develop strong evolutionary biomarkers that, when utilized alongside evolutionary therapies, will revolutionize the clinical management of cancer with the potential to improve both patient prognoses and quality-of-life outcomes. Citation Format: Manasa Iyer, Diego Mallo, Carlo C. Maley, Angelo Fortunato, Luis Cisneros, Lorraine M. King, Marc D. Ryser, Joseph Y. Lo, Allison Hall, Jeffrey R. Marks, Shelley Hwang. Evaluating DCIS progression: A comparative analysis of CNA predictive power derived from lpWGS and WES data [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A038.

Association between copy number alterations estimated using low-pass whole genome sequencing of formalin-fixed paraffin-embedded prostate tumor tissue and cancer-specific clinical parameters

Copy number alterations (CNAs) are frequently observed in early-stage prostate cancer and are associated with disease recurrence and tumor aggressiveness. Cost-effective assessment of CNAs could enhance clinical utility of CNAs. Here, we combined the cost-effectiveness of low-pass (low coverage) whole genome sequencing (LPWGS) and the routine availability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for assessing CNAs in a cohort of 187 men with early-stage localised prostate cancer. We detected well known CNAs in 8p, 8q, 13q and 16q and recurrent gains of the oncogene MYC and losses of the tumor suppressor genes NKX3-1, PTEN and RB1, indicating assay reliability. The estimated burden of CNAs was significantly associated with Gleason score, pathological T stage, surgical margin status and biochemical recurrence. Further, genomic losses or gains in specific chromosomal arms were significantly associated with worse BCR-free survival. Copy number signatures extracted from the LPWGS data showed potential for risk stratifying patients, where signatures S1 and S2 showed significant association to worse BCR-free survival compared to S3. Our study provides clinical validation of the associations between CNAs and tumor aggressiveness in an independent and representative RP cohort, while demonstrating the feasibility of performing LPWGS of FFPE tumor tissue for cost-effective assessment of CNAs.

Comparison of the somatic genomic landscape between central- and peripheral-type non-small cell lung cancer

ObjectiveLung cancer is classified into central and peripheral types based on the anatomic location. The present study aimed to explore the distinct patterns of genomic alterations between central- and peripheral-type non-small cell lung cancers (NSCLCs) with negative driver genes and identify potential driver genes and biomarkers to improve therapy strategies for NSCLC. MethodsWhole-exome sequencing (WES) was performed with 182 tumor/control pairs of samples from 145 Chinese NSCLC patients without EGFR, ALK, or ROS1 alterations. Significantly mutated genes (SMGs) and somatic copy number alterations (SCNAs) were identified. Subsequently, tumor mutation burden (TMB), weighted genome integrity index (wGII), copy number alteration (CNA) burden, Shannon diversity index (SDI), intratumor heterogeneity (ITH), neoantigen load (NAL), and clonal variations were evaluated in central- and peripheral-type NSCLCs. Furthermore, mutational signature analysis and survival analysis were performed. ResultsTP53 was the most frequently mutated gene in NSCLC and more frequently mutated in central-type NSCLC. Higher wGII, ITH, and SDI were found in central-type lung adenocarcinoma (LUAD) than in peripheral-type LUAD. The NAL of central-type lung squamous cell carcinoma (LUSC) with stage III/IV was significantly higher than that of peripheral-type LUSC. Mutational signature analysis revealed that SBS10b, SBS24, and ID7 were significantly different in central- and peripheral-type NSCLCs. Furthermore, central-type NSCLC was found to evolve at a higher level with fewer clones and more subclones, particularly in central-type LUSC. Survival analysis revealed that TMB, CNA burden, NAL, subclonal driver mutations, and subclonal mutations were negatively related to the overall survival (OS) and the progression-free survival (PFS) of central-type LUAD. ConclusionsCentral-type NSCLC tended to evolve at a higher level and might suggest a favorable response to immunotherapy. Our study also identified several potential driver genes and promising biomarkers for the prognosis and prediction of chemotherapy responses in NSCLC.

Tumor biology, immune infiltration and liver function define seven hepatocellular carcinoma subtypes linked to distinct drivers, survival and drug response

Background & aimsTumor heterogeneity is jointly determined by the components of the tumor ecosystem (TES) including tumor cells, immune cells, stromal cells, and non-cellular components. We aimed to identify subtypes using TES-related genes and determine subtype specific drivers and treatments for hepatocellular carcinoma (HCC). MethodsWe collected 68 genesets depicting tumor biology, immune infiltration, and liver function, totaling 2831 genes, and collected mRNA profiles and clinical data for over 6000 tumors from 65 datasets in the GEO, TCGA, ICGC, and several other databases. We designed a three-step clustering pipeline to identify subtypes. The microenvironment, genomic alteration, and drug response differences were systematically compared among subtypes. ResultsSeven subtypes (TES-1/2/3/4/5/6/7) were revealed in 159 tumors from the CHCC-HBV cohort. We constructed a single sample classifier using paired genes (sscpgsTES). TES subtypes were significantly associated with multiple clinical variables including etiology, and survival in 14 of 17 cohorts and the meta-cohort. TES-1 had the poorest prognosis and highest proliferation level. Both TES-2 and TES-7 were immune-enriched, however, TES-2 had a significantly worse prognosis, and hypoxic and immunosuppressive microenvironment. TES-4 had activated Wnt pathway, driven by CTNNB1 mutation. Good prognosis TES-6 exhibited the best differentiation. TES-5 and TES-3 were considered as novel subclasses by comparing with ten previous subtyping systems. TES-5 tumors had high AFP but good overall survival, and ∼45% of them harbored AXIN1 mutation. TES-3 was immune and stromal desert, may be driven by high copy number alteration burden, and had the poorest response to immune checkpoint inhibitor. TES-1 and TES-2 had significantly lower response to transarterial chemoembolization, but they showed significantly higher sensitivity to compound YM-155. ConclusionsTumor ecosystem subtypes expand existing HCC subtyping systems, have distinct drivers, prognosis, and treatment vulnerabilities.

Integrated exome sequencing and microarray analyses detected genetic defects and underlying pathways of hepatocellular carcinoma

We performed whole exome sequencing (WES) and microarray analysis to detect somatic variants and copy number alterations (CNAs) for underlying mechanisms in a case series of hepatocellular carcinoma (HCC) with paired DNA samples from tumor and adjacent nontumor tissues. Clinicopathologic findings based on Edmondson-Steiner (E-S) grading, Barcelona-Clinic Liver Cancer (BCLC) stages, recurrence, and survival status and their associations with tumor mutation burden (TMB) and CNA burden (CNAB) were evaluated. WES from 36 cases detected variants in the TP53, AXIN1, CTNNB1, and SMARCA4 genes, amplifications of the AKT3, MYC, and TERT genes, and deletions of the CDH1, TP53, IRF2, RB1, RPL5, and PTEN genes. These genetic defects affecting the p53/cell cycle control, PI3K/Ras, and β-catenin pathways were observed in approximately 80% of cases. A germline variant in the ALDH2 gene was detected in 52% of the cases. Significantly higher CNAB in patients with poor prognosis by E-S grade III, BCLC stage C, and recurrence than patients with good prognosis by grade III, stage A, grade III and nonrecurrence was noted. Further analysis on a large case series to correlate genomic profiling with clinicopathologic classifications could provide evidence for diagnostic interpretation, prognostic prediction, and target intervention on involved genes and pathways.

Molecular correlates of invasion pattern in HPV-associated endocervical adenocarcinoma: emergence of two distinct risk-stratified tiers.

The pattern-based (Silva) classification of invasive human papilloma virus (HPV)-associated endocervical adenocarcinomas (HPVA) is an established and reproducible method to predict outcomes for this otherwise stage-dependent group of tumours. Previous studies utilising targeted sequencing have shown a correlation between mutational profiles and an invasive pattern. However, such correlation has not been explored using comprehensive molecular testing. Clinicopathologic data including invasive pattern (Silva groups A, B, and C) was collected for a cohort of invasive HPVA, which previously underwent massive parallel sequencing using a panel covering 447 genes. Pathogenic alterations, molecular signatures, tumour mutational burden (TMB), and copy number alterations (CNA) were correlated with pattern of invasion. Forty five HPVA (11 pattern A, 17 pattern B, and 17 pattern C tumours) were included. Patients with pattern A presented at stage I with no involved lymph nodes or evidence of recurrence (in those with >2 months of follow-up). Patterns B and C patients also mostly presented at stage I with negative lymph nodes, but had a greater frequency of recurrence; 3/17 pattern B and 1/17 pattern C HPVAs harboured lymphovascular space invasion (LVI). An APOBEC mutational signature was detected only in Silva pattern C tumours (5/17), and pathogenic PIK3CA changes were detected only in destructively invasive HPVA (patterns B and C). When cases were grouped as low-risk (pattern A and pattern B without LVI) and high-risk (pattern B with LVI and pattern C), high-risk tumours were enriched in mutations in PIK3CA, ATRX, and ERBB2. There was a statistically significant difference in TMB between low-risk and high-risk pattern tumours (P= 0.006), as well as between Pattern C tumours with and without an APOBEC signature (P= 0.002). CNA burden increased from pattern A to C. Our findings further indicate that key molecular events in HPVA correlate with the morphologic invasive properties of the tumour and their aggressiveness. Pattern B tumours with LVI clustered with pattern C tumours, whereas pattern B tumours without LVI approached pattern A genotypically. Our study provides a biologic foundation for consolidating the Silva system into low-risk (pattern A + B without LVI) and high-risk (pattern B with LVI and pattern C) categories.

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

Prognostic Value of Low-Pass Whole Genome Sequencing of Circulating Tumor DNA in Metastatic Castration-Resistant Prostate Cancer.

Multiple treatments are available for metastatic castration-resistant prostate cancer (mCRPC), including androgen receptor signaling inhibitors (ARSI) enzalutamide and abiraterone, but therapy resistance remains a major clinical obstacle. We examined the clinical utility of low-pass whole-genome sequencing (LPWGS) of circulating tumor DNA (ctDNA) for prognostication in mCRPC. A total of 200 plasma samples from 143 mCRPC patients collected at the start of first-line ARSI treatment (baseline) and at treatment termination (n = 57, matched) were analyzed by LPWGS (median: 0.50X) to access ctDNA% and copy number alteration (CNA) patterns. The best confirmed prostate specific antigen (PSA) response (≥50% decline [PSA50]), PSA progression-free survival (PFS), and overall survival (OS) were used as endpoints. For external validation, we used plasma LPWGS data from an independent cohort of 70 mCRPC patients receiving first-line ARSI. Baseline ctDNA% ranged from ≤3.0% to 73% (median: 6.6%) and CNA burden from 0% to 82% (median: 13.1%) in the discovery cohort. High ctDNA% and high CNA burden at baseline was associated with poor PSA50 response (P = 0.0123/0.0081), poor PFS (P < 0.0001), and poor OS (P < 0.0001). ctDNA% and CNA burden was higher at PSA progression than at baseline in 32.7% and 42.3% of the patients. High ctDNA% and high CNA burden at baseline was also associated with poor PFS and OS (P ≤ 0.0272) in the validation cohort. LPWGS of ctDNA provides clinically relevant information about the tumor genome in mCRPC patients. Using LPWGS data, we show that high ctDNA% and CNA burden at baseline is associated with short PFS and OS in 2 independent cohorts.

Clinicopathologic and Molecular Characteristics of Epstein-Barr Virus–Associated Smooth Muscle Tumor Compared With Those of Leiomyoma and Leiomyosarcoma

Epstein-Barr virus (EBV)–associated smooth muscle tumors (EBV-SMTs) are rare smooth muscle neoplasms exclusively associated with immunosuppression, such as in patients with HIV/AIDS, posttransplant, and congenital immunodeficiency. However, the genomic landscape of EBV-SMTs is poorly understood. Leiomyosarcomas harbor genomic instability and multiple recurrent DNA copy number alterations, whereas leiomyomas lack such changes. Thus, this study aimed to fill this knowledge gap by characterizing copy number alterations in EBV-SMTs and correlating this information with clinicopathologic characteristics. Our study investigated and compared the pathologic characteristics and copy number profiles of 9 EBV-SMTs (from 7 post-transplant and AIDS patients), 6 leiomyomas, and 7 leiomyosarcomas, using chromosomal microarray platforms. Our results showed a lower copy number alteration burden in EBV-SMTs and leiomyoma than in leiomyosarcoma. This contrast in the molecular profile between EBV-SMTs and leiomyosarcoma is concordant with the different clinical behaviors and pathologic characteristics exhibited by these tumors. Despite having an overall copy number alteration profile closer to leiomyoma, recurrent copy number gain of oncogenes, such as RUNX1, CCND2, and ETS2, was found in EBV-SMTs. Epigenetic alterations may play an important role in tumorigenesis as recurrent copy number gains were found in histone deacetylases. A gene enrichment analysis also demonstrated enrichment of genes involved in the host response to viral infection, suggesting that the tumor immune microenvironment may play an important role in EBV-SMT tumorigenesis.

Construction and validation of a prognostic model for gastrointestinal stromal tumors based on copy number alterations and clinicopathological characteristics.

The increasing incidence of gastrointestinal stromal tumors (GISTs) has led to the discovery of more novel prognostic markers. We aim to establish an unsupervised prognostic model for the early prediction of the prognosis of future patients with GISTs and to guide clinical treatment. We downloaded the GISTs dataset through the cBioPortal website. We extracted clinical information and pathological information, including the microsatellite instability (MSI) score, fraction genome altered (FGA) score, tumor mutational burden (TMB), and copy number alteration burden (CNAB), of patients with GISTs. For survival analysis, we used univariate Cox regression to analyze the contribution of each factor to prognosis and calculated a hazard ratio (HR) and 95% confidence interval (95% CI). For clustering groupings, we used the t-distributed stochastic neighbor embedding (t-SNE) method for data dimensionality reduction. Subsequently, the k-means method was used for clustering analysis. A total of 395 individuals were included in the study. After dimensionality reduction with t-SNE, all patients were divided into two subgroups. Cluster 1 had worse OS than cluster 2 (HR=3.45, 95% CI, 2.22-5.56, P<0.001). The median MSI score of cluster 1 was 1.09, and the median MSI score of cluster 2 was 0.24, which were significantly different (P<0.001). The FGA score of cluster 1 was 0.28, which was higher than that of cluster 2 (P<0.001). In addition, both the TMB and CNAB of cluster 1 were higher than those of cluster 2, and the P values were less than 0.001. Based on the CNA of GISTs, patients can be divided into high-risk and low-risk groups. The high-risk group had a higher MSI score, FGA score, TMB and CNAB than the low-risk group. In addition, we established a prognostic nomogram based on the CNA and clinicopathological characteristics of patients with GISTs.

Accumulation of copy number alterations and clinical progression across advanced prostate cancer

BackgroundGenomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown.MethodsWe performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression.ResultsThe burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10−6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov–Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037).ConclusionsCopy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes.Trial registrationClinicalTrials.gov NCT00268476, registered on December 22, 2005. EudraCT 2004-000193-31, registered on October 4, 2004.

Prognostic Value of Copy Number Alteration Burden in Early-Stage Breast Cancer and the Construction of an 11-Gene Copy Number Alteration Model.

Simple SummaryBreast cancer is a malignancy that poses a significant threat to women’s health. The enormous disease burden has forced a wide range of researchers to develop more accurate prognostic models. Copy number alterations, which are amplifications or deletions of DNA fragments, often predict a poor prognosis. Instead, copy number alteration burden, i.e., the level of CNA, may have a good predictive value for disease prognosis. In this study, we developed a prognostic model for early breast cancer based on CNAB and simplified it. It performed excellently in two external validation sets.The increasing burden of breast cancer has prompted a wide range of researchers to search for new prognostic markers. Considering that tumor mutation burden (TMB) is low and copy number alteration burden (CNAB) is high in breast cancer, we built a CNAB-based model using a public database and validated it with a Chinese population. We collected formalin-fixed, paraffin-embedded (FFPE) tissue samples from 31 breast cancer patients who were treated between 2010 and 2014 at the National Cancer Center (CICAMS). METABRIC and TCGA data were downloaded via cBioPortal. In total, 2295 patients with early-stage breast cancer were enrolled in the study, including 1427 in the METABRIC cohort, 837 in the TCGA cohort, and 31 in the CICAMS cohort. Based on the ROC curve, we consider 2.2 CNA/MBp as the threshold for the CNAB-high and CNAB-low groupings. In both the TCGA cohort and the CICAMS cohort, CNAB-high had a worse prognosis than CNAB-low. We further simplified this model by establishing a prognostic nomogram for early breast cancer patients by 11 core genes, and this nomogram was highly effective in both the TCGA cohort and the CICAMS cohort. We hope that this model will subsequently help clinicians with prognostic assessments.

Absence of NOTCH1 mutation and presence of CDKN2A deletion predict progression of esophageal lesions.

Currently, surveillance for esophageal squamous cell carcinoma (ESCC) runs a risk of underestimation of early lesions which show absence of iodine staining, with no or only mild histologic changes. The development of molecular markers that indicate risk of progression is thus warranted. We performed whole-exome sequencing on biopsies from two sequential endoscopies of a single esophageal lesion and matching blood samples. There were 27 pairs of age-, gender-, pathologic stage-, and sampling interval-matched progressors and non-progressors identified in a prospective community-based ESCC screening trial. Putative molecular progression markers for ESCC were first evaluated by comparing somatic mutation, copy number alteration (CNA), and mutational signature information among progressors and non-progressors. These markers were then validated with another 24 pairs of matched progressors and non-progressors from the same population using gene alteration status identified by target sequencing and quantitative PCR. Progressors had more somatic mutation and CNA burden, as well as apolipoprotein B mRNA editing catalytic polypeptide-like and age-related signature weights compared with non-progressors. A gene score consisting of somatic NOTCH1 mutation and CDKN2A deletion is predictive of risk of progression in lesions which show absence of iodine staining under endoscopy but have no or only mild dysplasia. This gene score was also validated in an external cohort of matched progressors and non-progressors. Absence of NOTCH1 mutation and presence of CDKN2A deletion are markers of progression in squamous lesions of the esophagus. This gene score would be an ideal indicator for assisting the pathologist in the identification of high-risk individuals who could be potentially 'missed' or subject to a risk underestimation by histologic analysis, and might improve the performance of ESCC surveillance. © 2022 The Pathological Society of Great Britain and Ireland.

Whole genome sequencing of HER2+ metastatic breast cancer and CNA comparison between long term survivor and short-term survivor.

e13019 Background: The introduction of anti-HER2 therapies such as trastuzumab for HER2+ metastatic breast cancer (MBC) has led to significant improvements to disease progression. We and others have reported long-term durable complete response to trastuzumab in patients with HER2+ MBC. We hypothesise that genomic copy number alteration (CNA) and tumour mutational burden (TMB) may act as a prognostic measure of predicting response to trastuzumab in long-term survivors (LTS) compared to short-term survivors (STS). Using whole-genome sequencing (WGS) to detect these alterations, we aim to identify the molecular characteristics of primary and metastatic lesions in patients with metastatic HER2+ breast cancer. Methods: WGS was performed on samples from two patients from a HER2+ MBC cohort; a of long-term survivor (PFS 27 months; OS &gt; 60 months) and a short-term survivor (PFS 4 months; OS &lt; 14 months). Primary tumours, adjacent normal tissue and metastases were sequenced at a mean depth of 60X. Reads were aligned on the hg38 reference genome using Burrows-Wheeler Aligner (bwa), CNA were detected using Control-FREEC to evaluate total CNA burden. Single nucleotide variants were detected using GATK Mutect2 to calculate TMB. Results: Analysis of DNA chromosome disruption (fraction of the genome amplified/deleted) revealed a higher overall fraction of disrupted genome in the LTS primary tumour and metastasis compared with the STS (0.061 and 0.064 vs 0.031 and 0.047, respectively). Further delineation of the distribution of CNA burden in all genomes identified chr1 as the most altered in the STS samples whereas chr17 was the most disrupted in the LTS samples. HER2 and CDK12 were amplified in both LTS and STS samples (4 copies in LTS primary tumour and metastasis, 18 and 14 copies in STS primary tumour and metastasis respectively). A high TMB was estimated in both samples but was higher in the STS primary tumour than in the LTS one (161 vs 142, respectively). TMB was increased in LTS metastatic lesions. NRAS mutation was found in LTS primary tumour but was lost in the metastasis. Conclusions: This pilot study highlights the potential for whole-genome sequencing to identify CNA and TMB in HER2+ MBC in patients who display long-term and short-term survival. Further samples from this cohort need to be studied to investigate the relationship between CNA burden and HER2+ MBC patient survival.

The genomic landscape of cholangiocarcinoma reveals the disruption of post-transcriptional modifiers

Molecular variation between geographical populations and subtypes indicate potential genomic heterogeneity and novel genomic features within CCA. Here, we analyze exome-sequencing data of 87 perihilar cholangiocarcinoma (pCCA) and 261 intrahepatic cholangiocarcinoma (iCCA) cases from 3 Asian centers (including 43 pCCAs and 24 iCCAs from our center). iCCA tumours demonstrate a higher tumor mutation burden and copy number alteration burden (CNAB) than pCCA tumours, and high CNAB indicates a poorer pCCA prognosis. We identify 12 significantly mutated genes and 5 focal CNA regions, and demonstrate common mutations in post-transcriptional modification-related potential driver genes METTL14 and RBM10 in pCCA tumours. Finally we demonstrate the tumour-suppressive role of METTL14, a major RNA N6-adenosine methyltransferase (m6A), and illustrate that its loss-of-function mutation R298H may act through m6A modification on potential driver gene MACF1. Our results may be valuable for better understanding of how post-transcriptional modification can affect CCA development, and highlight both similarities and differences between pCCA and iCCA.

Tumor copy number alterations were associated with primary endocrine resistance in ER+ breast cancer.

e12567 Background: Several studies has showed alterations in genes were associated with endocrine resistance in breast cancer. Nevertheless, genomic landscape in primary endocrine-resistant breast cancer has not been thoroughly reported. Whether the genomic landscape of primary endocrine-resistant breast cancer is different from that of secondary endocrine-resistant breast cancer is unknown. Methods: We analyzed the genomic landscape of primary tumor of consecutive patients with estrogen-receptor positive (ER+) breast cancer in real-world clinical practice in our center between September, 2019 to December, 2020. According to the duration of endocrine treatment, we classified them into primary endocrine resistance group and secondary endocrine resistance group. Results: A total of 41 tumor samples of which 25 cases in primary endocrine resistance and 16 cases in secondary endocrine resistance were included for analysis. TP53 (65.9%, 27/41), PIK3CA (46.3%, 19/41), MYC (36.6%, 15/41), MCL1 (31.7%, 13/41), CCND1 (31.7%, 13/41) were the most frequently altered genes in all 41 BCs. Copy number amplification of MYC was common in primary endocrine resistant group (48.0%, 12/25), while lower in secondary endocrine resistant group (18.8%, 3/16, P = NS). Copy number alteration (CNA) of CCND1, MCL1, FGF19, ZNF217, ZNF703 and FGFR1 were commonly observed in primary endocrine resistance group. Percentage of CNA in all mutation types was significant increased in primary endocrine resistance group than in secondary resistance group (33.0% vs. 21.0%, P = 0.03). Then we further analyzed and found CNA burden, which was defined as number rather the percentage of the CNA event, was associated with the prognosis in ER+ breast cancer. When defining 6 as the cut-off value of CNA-High, DFS, PFS and OS were practically poorer in patients with CNA-High than those with CNA-Low. Conclusions: Copy number alterations were more commonly seen in primary endocrine-resistant breast cancer and associated with prognosis in ER+ breast cancer. This founding urged to further investigate the prognostic value of CNA burden in predicting the effectiveness of endocrine therapy.

Proteogenomic characterization of 2002 human cancers reveals pan-cancer molecular subtypes and associated pathways

Mass-spectrometry-based proteomic data on human tumors—combined with corresponding multi-omics data—present opportunities for systematic and pan-cancer proteogenomic analyses. Here, we assemble a compendium dataset of proteomics data of 2002 primary tumors from 14 cancer types and 17 studies. Protein expression of genes broadly correlates with corresponding mRNA levels or copy number alterations (CNAs) across tumors, but with notable exceptions. Based on unsupervised clustering, tumors separate into 11 distinct proteome-based subtypes spanning multiple tissue-based cancer types. Two subtypes are enriched for brain tumors, one subtype associating with MYC, Wnt, and Hippo pathways and high CNA burden, and another subtype associating with metabolic pathways and low CNA burden. Somatic alteration of genes in a pathway associates with higher pathway activity as inferred by proteome or transcriptome data. A substantial fraction of cancers shows high MYC pathway activity without MYC copy gain but with mutations in genes with noncanonical roles in MYC. Our proteogenomics survey reveals the interplay between genome and proteome across tumor lineages.

Comparative Analysis of Differentially Mutated Genes in Non-Muscle and Muscle-Invasive Bladder Cancer in the Chinese Population by Whole Exome Sequencing.

Objective: To characterize the spectra of mutations in non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) in the Chinese population to identify any mutational features and find potential therapeutic targets. Materials and methods: We collected fresh bladder tumor samples from NMIBC (n = 9) and MIBC patients (n = 11) along with adjacent normal bladder tissue specimen and peripheral blood sample. Using whole exome sequencing (WES), we analyzed the mutation spectra of those NMIBC and MIBC bladder cancer (BCa) specimen. Results: Our results demonstrated that 95% of BCa patients (19/20) had varying degrees of driver gene mutations, FGFR3 (45%), KMT2D (40%), PIK3CA (35%), ARID1A (20%), EP300 (20%), KDM6A (20%), KMT2C (20%), and STAG2 (20%) were the most frequently mutated genes in BCa patients. NMIBC and MIBC exhibited different genomic alterations. FGFR3 (67%), PIK3CA (56%), and RHOB (44%) were the most frequently mutated genes in NMIBC patients. Of note, RHOB mutation only occurred in NMIBC, whereas mutations of KMT2D (55%), TP53 (36%) and KMT2B (27%) were frequently detected in MIBC, and TP53 and KMT2B mutation only occurred in MIBC. The frequency of mutations in DNA-damage repair (DDR) gene was higher in MIBC than that in NMIBC (91 vs 78%, 6.2 vs 2.4 gene mutations per patient). Copy number alterations (CNAs) occurred at more diverse chromosomal locations in NMIBC, but the CNA burden was higher in MIBC [9.01 (2.07–31.51) vs 4.98 (0.99–9.73) mutations/Mb]., the trend of which was consistent with the tumor mutation burden (TMB) [8.26 (4.63–21.84) vs 5.58 (3.87–9.58) mutations/Mb]. Among the current set of single-base substitution (SBS) signatures including SBS 1, 2, 5, 13, and 40, we identified one differently expressed signature between NMIBC and MIBC patients: SBS13. Conclusions: There were different gene mutational characteristics and signatures between NMIBC and MIBC in the Chinese population. Frequency of DDR, CNA burden and TMB were higher in MIBC. Our analysis revealed that several genes in NMIBC did not overlap with those reported in MIBC, suggesting that a fraction of NMIBC and MIBC likely developed secondary to different precursor lesions.

Genomic and Metabolic Hallmarks of SDH- and FH-deficient Renal Cell Carcinomas

BackgroundSuccinate dehydrogenase-deficient and fumarate hydratase-deficient renal cell carcinomas (SDHRCC and FHRCC) are rare kidney cancers driven by loss of TCA cycle enzymes. ObjectiveTo define and compare the genomic and metabolomic hallmarks of SDHRCC and FHRCC. Design, setting, and participantsWe analyzed SDHRCC and FHRCC tumors with either immunohistochemical evidence of loss of protein expression or genomically confirmed biallelic inactivation of SDHA/B/C/D/AF2 or FH. Outcome measurements and statistical analysisSomatic alterations were identified using clinical pipelines, with allele-specific copy number alterations (CNAs) identified using FACETS. Mass spectrometry–based metabolomic profiling was performed on available SDHRCC and FHRCC tumors. Results and limitationsTumors were analyzed for 42 patients (25 FHRCC, 17 SDHRCC). In the germline analysis, 16/17 SDHRCCs harbored a germline alteration in SDHB, whereas only 17/22 FHRCCs had pathogenic germline FH variants. SDHRCCs had a lower mutation burden (p = 0.02) and CNA burden (p = 0.0002) than FHRCCs. All SDHRCCs presented with deletion of chromosome 1p (overlapping SDHB), whereas FHRCCs demonstrated high but not ubiquitous loss of 1q (FH locus). Both SDHRCCs and FHRCCs exhibited significant idiopathic accumulation of the metabolite guanine. FHRCC tumors had elevated levels of urea cycle metabolites (argininosuccinate, citrulline, and fumarate), whereas SDHRCC tumors had elevation of numerous acylcarnitines. These characteristic metabolic changes allowed identification of a previously unrecognized SDH-deficient RCC. ConclusionsDespite sharing similar genetic etiology, SDHRCC and FHRCC represent distinct molecular entities with unique genetic and metabolic abnormalities. Patient summaryKidney cancers driven by loss of the gene encoding either the succinate dehydrogenase or fumarate hydratase enzyme are rare. We sought to define and compare the genetic and metabolic features of these cancer entities.