Copy Number Gain Research Articles

Abstract 90: Exploring the role of common circulating tumor cell-associated genes in estrogen receptor-positive breast cancer recurrence

Abstract Despite having the best survival among the molecular subtypes, estrogen receptor-positive (ER+) breast cancer has a tendency for late recurrence. Circulating tumor cells (CTCs) are responsible for disease recurrence by seeding tumors at secondary sites. We have previously developed a CTC signature of 90 common regions of gene copy number gain and demonstrated that this signature is significantly more widespread among cells in metastases than primary tumors.1,2 The objective of our current work is to establish whether the expression data from a 133 gene panel largely derived from the CTC signature can be used to assess the risk of recurrence (ROR) in early ER+ breast cancer patients to help guide treatment decisions following surgery. Formalin-fixed, paraffin-embedded (FFPE) ER+ breast tumor biopsies and linked normal (control) tissues from 192 patients are being analyzed with a custom NanoString nCounter probe set of our 133 CTC genes. From data gathered to date, several genes were shown to be significantly overexpressed in tumors compared to reference normal tissues. This includes MMP11, MKI67, LILRB4, UBE2T, BIRC5, CDC6, MYBL2, and NUF2, which had a log2 fold-change of at least 3.5 (n = 34, p < 0.01). The correlation of our CTC genes to ROR is being evaluated by comparison to the results of the Oncotype DX Breast Recurrence Score Test, a well-established clinical test for ROR in ER+ breast cancer, for each patient. So far, it has been shown that the intratumor heterogeneity (ITH) of CTC genes as established by DEPTH2, an ITH algorithm which evaluates mRNA levels, is significantly positively corelated to Oncotype DX score (n = 23, r = 0.41, p = 0.0495). Moving forward, we will create our own ROR scoring system based on gene expression and copy number data from the CTC gene panel and validate it by comparison to Oncotype DX score and other available clinical data for each of the 192 patient samples. The goal of our work is to gain a deeper understanding of the molecular mechanisms that drive metastasis and recurrence in ER+ breast cancer by focusing on CTCs which play a pivotal role in driving these events. 1. Kanwar N, Hu P, Bedard P, Clemons M, McCready D, Done SJ. Identification of genomic signatures in circulating tumor cells from breast cancer. International Journal of Cancer. 2015;137(2):332-344. doi:10.1002/ijc.29399 2. Kanwar N, Balde Z, Nair R, et al. Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response. Cancer Research. 2021;81(24):6196-6206. doi:10.1158/0008-5472.can-21-1079‌ Citation Format: Martin Rotbauer, Melanie Dawe, Philippe Bedard, David Cescon, Susan Done. Exploring the role of common circulating tumor cell-associated genes in estrogen receptor-positive breast cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 90.

Abstract 4763: Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response

Abstract Osteosarcoma is a highly aggressive cancer seen mostly in children and young adults which is characterized by aneuploidy and dramatic structural rearrangements associated with large copy number gain and widespread chromosomal loss. Osteosarcoma therapy has not change in over forty years, highlighting the need for deeper molecular understanding of this disease which could in turn identify new therapeutic opportunities. While DNA-level heterogeneity between tumors has been well-described, not much is known regarding epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum including biopsies, resections, and metastases. ATAC-seq analysis revealed the presence of at least two distinct epigenetic cell states, epigenetic cluster 1 (EC1) and epigenetic cluster 2 (EC2). We used the GREAT algorithm to identify enriched pathways for the genes linked to these differential open regions (~2000 unique peaks per EC). EC1 is defined by genes related to mesenchymal cell proliferation and osteoblast differentiation, whereas EC2 is enriched in genes related to extracellular matrix organization and regulation of kinase activity. Accessible chromatin peaks in EC1 showed transcription factor binding motifs related to development including RUNX2/3, MEOX2, HOXA2/5 and DLX; whereas members of the AP1 complex including FOSL1/2, c-FOS, JUN were enriched in open regions in EC2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. We defined a gene-signature of 343 genes by integrating differential open chromatin with differential gene expression of EC1 and EC2. This gene-signature is able to separate gene expression data of patient samples into 2 clusters, suggesting these epigenetic cell states are found in patient samples. We assigned the patient samples to one EC and performed a survival analysis using only primary patient samples. EC1 shows a lower survival probability than EC2. To identify possible therapeutic implications of these EC, we evaluated a drug response dataset generated with ~40 targeted agents for the same panel of PDX-cell lines. Whereas EC1 cell lines were highly sensitive to a selective inhibitor of Aurora B, cell lines in EC2 were inhibited by a MEK inhibitor. PDX-cell lines injected in a subcutaneous model recapitulated the differential drug response in vivo. In summary, we have identified two epigenetically cell states regulated by a state-specific set of transcription factors defining a gene signature that could help to define osteosarcoma subclasses and predict drug response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne Sayles, Maria Pons Ventura, Alex Lee, Vijay Ramani, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4763.

Abstract 5214: Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing

Abstract Large case-control and familial studies have established clear cancer-specific risk profiles for several key cancer predisposition genes (CPGs). For example, germline pathogenic variants (PVs) in BRCA1/2 (gBRCA) are associated with increased risk for developing breast, ovarian, pancreatic, and prostate cancers. However, the extent to which gBRCA mutations are involved in mediating the tumorigenesis of other cancer types remains challenging to characterize. We hypothesized that integrating orthogonal features such as selection for biallelic inactivation of the PVs and depletion of canonical somatic drivers among the carriers can enrich the signal for identifying novel gene and cancer type associations. We then extend this framework to identify novel CPGs as well as to understand how tumors arise in patients with PVs in oncogenes. To study this, we leveraged the prospective MSK-IMPACT matched tumor-normal sequencing cohort of 49,291 patients across 77 major cancer types. We study 90 well-known CPGs as well as >300 cancer genes not previously associated with cancer predisposition. Overall, 8% (N=3,964) of patients harbored a PV in high or moderate penetrance CPGs. We identified 90 gene and cancer type associations with enrichment for biallelic inactivation (q<0.05), including 19 novel findings among clinically actionable genes. For example, we find enrichment for biallelic inactivation of BRCA1/2 in unexpected lineages such as hepatobiliary, endometrioid, and ampullary cancers. These tumors were also significantly depleted for somatic gain-of-function driver alterations. Hepatobiliary cancers with gBRCA1 mutations were also enriched for somatic loss of NF1. Among carriers of PVs in oncogenes, we observe two possible mechanisms of first somatic hit towards malignant transformation. We find enrichment for copy number gain or copy neutral loss of heterozygosity of the germline PV in thyroid cancers with a PV in RET. We also find that lung cancers with a germline PV in EGFR frequently developed additional somatic point mutations located in cis with the PV. Investigating genes with no prior association with germline predisposition to cancer, we find evidence for KEAP1 and CIC as likely novel CPGs. Lung (n=8) and thyroid (n=4) cancers with deleterious germline variants in KEAP1 were characterized by loss of the wild-type allele, co-occurring somatic STK11 mutations, and depletion of canonical drivers such as EGFR. We also found biallelic loss of CIC in two patients with Neuroblastoma, each carrying a different germline loss-of-function mutation in CIC. Both tumors were also negative for MYCN and ALK defects. Collectively, our findings expand our understanding of cancer predisposition in cancer, shed new insights into how tumors arise in germline carriers, and provide a framework for identifying new CPGs using population scale tumor-normal paired clinical sequencing data. Citation Format: Miika Mehine, Rebecca Caeser, Yelena Kemel, Daniel Muldoon, Sebastià Franch-Expósito, A. Rose Brannon, Aijazuddin Syed, Ozge Ceyhan-Birsoy, Maksym Misyura, Panieh Terraf, David B. Solit, Marc Ladanyi, Kenneth Offit, Zsofia K. Stadler, Diana L. Mandelker, Yonina R. Murciano-Goroff, Charles M. Rudin, Michael F. Berger, Chaitanya Bandlamudi. Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5214.

Abstract 4331: Comprehensive analysis of copy number variation and sensitivity to targeted therapy in renal cell carcinoma using in-house cancer gene panel testing

Abstract Purpose: Immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) are the main drugs used to treat metastatic renal cell carcinoma (mRCC), but there are no effective biomarkers to determine their use. In this study, we aimed to identify factors that correlate with staging of RCC and drug sensitivity to mRCC based on the results of an in-house cancer gene panel test at Fujita Health University. Methods: 133 patients with RCC (70 non-metastatic RCC and 63 mRCC). DNA was analyzed for mutations in 143 cancer genes using the PleSSision Rapid cancer gene panel test. NextSeq2000 was used as the next-generation sequencer. From the curated reports obtained, factors including each gene mutation information and the correlation between copy number (CN) variation (CNV) cluster analysis information, clinicopathological information, and therapeutic response to ICI or TKI was examined. In this study, the therapeutic response was evaluated with RECIST1.1 and complete response and partial response were determined as objective response (OR). The endpoint was progression-free survival 2 (PFS2) defined as the interval between first-line treatment and the second relapse. Result: Mutational analysis showed no difference in the pattern of genetic variation with or without metastasis. CNV analyses showed a marked trend toward a decrease in CN on homologous recombination repair genes in a part of mRCCs. Cluster analysis of CNV information for each gene in response to TKI treatment in mRCC revealed multiple regions of decreased or increased CN. The main regions of increased CN were shown to be those formed by CN gain of oncogenes on chromosomes 7 and 12. Detailed analysis of this hot spot showed that when the cutoff value of sum of CN for KRAS, ERBB3, CDK4, and MDM2. for chromosome 12 set to 9, 8 (72.7%) of 11 patients with CN 9 or higher were in OR group, while 4 (7.7%) of 52 patients with CN less than 9 were in OR group, showing a significant difference between the two groups (P < 0.001). Similarly, when the cutoff value of sum of CN for EGFR, MET, and BRAF for chromosome 7 set to 8, 5 (83.3%) of 6 patients with CN 8 or higher were in OR group, while 7 (12.3%) of 57 patients with CN < 8 were in OR group, showing a significant difference between the two groups (P < 0.001). On the other hand, genetic markers predicting susceptibility to ICI could not be identified. Kaplan-Meier analysis showed that the group of patients with higher CN values for either chromosome 7 or 12 had better PFS2 after TKI treatment than the group of patients with lower CN values for either chromosome 7 or 12 (P < 0.001). Conclusion: Our results from in-house cancer gene panel tests successfully showed an association between RCC progression and cancer-related gene alterations. Furthermore, the results showed the possibility of predicting TKI treatment sensitivity in mRCC by focusing on CN gain of oncogenes in specific chromosomal regions. Citation Format: Akhito Takeuchi, Mayu Takeda, Eiji Sugihara, Yoshinari Muto, Sachio Nohara, Shigeki Tanishima, Kenji Zennami, Kiyoshi Takahara, Tetsuya Tsukamoto, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Comprehensive analysis of copy number variation and sensitivity to targeted therapy in renal cell carcinoma using in-house cancer gene panel testing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4331.

Genomic landscape and efficacy of HER2-targeted therapy in patients with HER2-mutant non-small cell lung cancer.

HER2-targeted therapy provides survival benefits to HER2-mutant non-small cell lung cancer (NSCLC). A better understanding of the clinical and genomic characterization of treatment-naïve HER2-positive NSCLC, as well as the efficacy of and resistance to HER2-targeted therapy in HER2-altered NSCLC, could promote further improvement of HER2 targeted therapy. HER2-altered NSCLC patients was retrospectively included and their genomic profiles were performed by next-generation sequencing. The clinical outcomes included overall response rate, disease control rate and progression-free survival. Among 176 treatment-naïve patients with HER2 alterations, 64.8% harbored HER2 mutations with/without HER2 amplification, and 35.2% carried HER2 amplification only. Molecular characterization was correlated with tumor stage that late-stage NSCLC with HER2 oncogenic mutations showed a higher prevalence of TP53 mutations and a higher tumor mutation burden. However, this correlation was not found in patients with HER2 amplification only. Twenty-one patients with HER2 alterations treated with pyrotinib or afatinib were retrospectively enrolled. Pyrotinib yielded a longer median progression-free survival than afatinib (5.9 [95% CI, 3.8-13.0] vs. 4.0 months [95% CI, 1.9-6.3], P = 0.06) in these patients. Analysis of the genomic profiles before and after anti-HER2 targeted therapies identified de novo HER2 copy number gain and G518W mutation, as well as mutations involving DNA damage repair signaling, SWI-SNF complex, and epigenetic regulations as potential resistance mechanisms. HER2-mutant NSCLC had different molecular features from HER2-amplified NSCLC, and its genomic profile was dependent of tumor stage. Pyrotinib had superior therapeutic effects than afatinib in HER2-altered NSCLC, although larger cohorts are warranted to validate it. HER2-dependent and -independent resistance mechanisms to afatinib and pyrotinib were unveiled.

MP04-08 COMPREHENSIVE ANALYSIS OF COPY NUMBER VARIATION AND SENSITIVITY TO TARGETED THERAPY IN RENAL CELL CARCINOMA USING IN-HOUSE CANCER GENE PANEL TESTING

You have accessJournal of UrologyCME1 Apr 2023MP04-08 COMPREHENSIVE ANALYSIS OF COPY NUMBER VARIATION AND SENSITIVITY TO TARGETED THERAPY IN RENAL CELL CARCINOMA USING IN-HOUSE CANCER GENE PANEL TESTING Akihito Takeuchi, Mayu Takeda, Eiji Sugihara, Yoshinari Muto, Sachio Nohara, Shigeki Tanishima, Kenji Zennami, Kiyoshi Takahara, Tetsuya Tsukamoto, Ryoichi Shiroki, Hideyuki Saya, and Makoto Sumitomo Akihito TakeuchiAkihito Takeuchi More articles by this author , Mayu TakedaMayu Takeda More articles by this author , Eiji SugiharaEiji Sugihara More articles by this author , Yoshinari MutoYoshinari Muto More articles by this author , Sachio NoharaSachio Nohara More articles by this author , Shigeki TanishimaShigeki Tanishima More articles by this author , Kenji ZennamiKenji Zennami More articles by this author , Kiyoshi TakaharaKiyoshi Takahara More articles by this author , Tetsuya TsukamotoTetsuya Tsukamoto More articles by this author , Ryoichi ShirokiRyoichi Shiroki More articles by this author , Hideyuki SayaHideyuki Saya More articles by this author , and Makoto SumitomoMakoto Sumitomo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) are the main drugs used to treat metastatic renal cell carcinoma (mRCC), but there are no effective biomarkers to determine their use. In this study, we aimed to identify factors that correlate with staging of RCC and drug sensitivity to mRCC based on the results of an in-house cancer gene panel test at Fujita Health University. METHODS: 133 patients with RCC (70 non-metastatic RCC and 63 mRCC). DNA was analyzed for mutations in 143 cancer genes using the PleSSision Rapid cancer gene panel test. NextSeq2000 was used as the next-generation sequencer. From the curated reports obtained, factors including each gene mutation information and the correlation between copy number (CN) variation (CNV) cluster analysis information, clinicopathological information, and therapeutic response to ICI or TKI was examined. In this study, the therapeutic response was evaluated with RECIST1.1 and complete response and partial response were determined as objective response (OR). The endpoint was progression-free survival 2 (PFS2) defined as the interval between first-line treatment and the second relapse. RESULTS: Mutational analysis showed no difference in the pattern of genetic variation with or without metastasis. CNV analyses showed a marked trend toward a decrease in CN on homologous recombination repair genes in a part of mRCCs. Cluster analysis of CNV information for each gene in response to TKI treatment in mRCC revealed multiple regions of decreased or increased CN. The main regions of increased CN were shown to be those formed by CN gain of oncogenes on chromosomes 7 and 12. Detailed analysis of this hot spot showed that when the cutoff value of sum of CN for KRAS, ERBB3, CDK4, and MDM2 for chromosome 12 set to 9, 8 (72.7%) of 11 patients with CN 9 or higher were in OR group, while 4 (7.7%) of 52 patients with CN less than 9 were in OR group, showing a significant difference between the two groups (P < 0.001). Similarly, when the cutoff value of sum of CN for EGFR, MET, and BRAF for chromosome 7 set to 8, 5 (83.3%) of 6 patients with CN 8 or higher were in OR group, while 7 (12.3%) of 57 patients with CN < 8 were in OR group, showing a significant difference between the two groups (p<0.001). On the other hand, genetic markers predicting susceptibility to ICI could not be identified. Kaplan-Meier analysis showed that the group of patients with higher CN values for either chromosome 7 or 12 had better PFS2 after TKI treatment than the group of patients with lower CN values for either chromosome 7 or 12 (p<0.001). CONCLUSIONS: Our results from in-house cancer gene panel tests successfully showed an association between RCC progression and cancer-related gene alterations. Furthermore, the results showed the possibility of predicting TKI treatment sensitivity in mRCC by focusing on CN gain of oncogenes in specific chromosomal regions. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e36 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihito Takeuchi More articles by this author Mayu Takeda More articles by this author Eiji Sugihara More articles by this author Yoshinari Muto More articles by this author Sachio Nohara More articles by this author Shigeki Tanishima More articles by this author Kenji Zennami More articles by this author Kiyoshi Takahara More articles by this author Tetsuya Tsukamoto More articles by this author Ryoichi Shiroki More articles by this author Hideyuki Saya More articles by this author Makoto Sumitomo More articles by this author Expand All Advertisement PDF downloadLoading ...

Somatic mutation detection and KRAS amplification in testicular germ cell tumors.

Testicular Germ Cell Tumors (TGCT) are the most common cancer among young adult men. The TGCT histopathology is diverse, and the frequency of genomic alterations, along with their prognostic role, remains largely unexplored. Herein, we evaluate the mutation profile of a 15-driver gene panel and copy number variation of KRAS in a large series of TGCT from a single reference cancer center. A cohort of 97 patients with TGCT, diagnosed at the Barretos Cancer Hospital, was evaluated. Real-time PCR was used to assess copy number variation (CNV) of the KRAS gene in 51 cases, and the mutation analysis was performed using the TruSight Tumor 15 (Illumina) panel (TST15) in 65 patients. Univariate analysis was used to compare sample categories in relation to mutational frequencies. Survival analysis was conducted by the Kaplan-Meier method and log-rank test. KRAS copy number gain was a very frequent event (80.4%) in TGCT and presented a worse prognosis compared with the group with no KRAS copy gain (10y-OS, 90% vs. 81.5%, p = 0.048). Among the 65 TGCT cases, different variants were identified in 11 of 15 genes of the panel, and the TP53 gene was the most recurrently mutated driver gene (27.7%). Variants were also detected in genes such as KIT, KRAS, PDGFRA, EGFR, BRAF, RET, NRAS, PIK3CA, MET, and ERBB2, with some of them potentially targetable. Although larger studies incorporating collaborative networks may shed the light on the molecular landscape of TGCT, our findings unveal the potential of actionable variants in clinical management for applying targeted therapies.

TERT Promoter Mutation c.-124C>T Commonly Occurs in Low-Grade Fibromatosis-like Metaplastic Breast Carcinoma.

Low-grade fibromatosis-like metaplastic carcinoma (FLMC) is a very rare subtype of triple-negative metaplastic (spindle cell) breast carcinoma. It is characterized by the proliferation of spindle cells closely resembling fibromatosis, which represents a benign fibroblastic/myofibroblastic breast proliferation. Unlike most triple-negative and basal-like breast cancers, FLMC has a very low potential for metastases, but demonstrates frequent local recurrences. To genetically characterize FLMC. To this end, we analyzed 7 cases by targeted next-generation sequencing for 315 cancer-related genes and performed comparative microarray copy number analysis in 5 of these cases. All cases shared TERT alterations (6 patients with recurrent c.-124C>T TERT promoter mutation and 1 patient with copy number gain encompassing the TERT locus), had oncogenic PIK3CA/PIK3R1 mutations (activation of the PI3K/AKT/mTOR pathway), and lacked mutations in TP53. TERT was overexpressed in all FLMCs. CDKN2A/B loss or mutation was observed in 4 of 7 cases (57%). Furthermore, tumors displayed chromosomal stability, with only few copy number variations and a low tumor mutational burden. We conclude that FLMCs typically show the recurrent TERT promoter mutation c.-124C>T, activation of the PI3K/AKT/mTOR pathway, low genomic instability, and wild-type TP53. In conjunction with previous data of metaplastic (spindle cell) carcinoma with and without fibromatosis-like morphology, FLMC is most likely distinguished by TERT promoter mutation. Thus, our data support the notion of a distinct subgroup within low-grade metaplastic breast cancer with spindle cell morphology and associated TERT mutations.

Clinical, histopathological and molecular risk factors for recurrence of pilocytic astrocytomas: brainstem/spinal location, nestin expression and gain of 7q and 19 are associated with early tumor recurrence.

Pilocytic astrocytomas (PAs) are benign tumors. However, clinically aggressive PAs despite benign histology have been reported, and histological and molecular risk factors for prognosis have not been elucidated. 38 PAs were studied for clinical, histological, and molecular factors, including tumor location, extent of resection, post-operative treatment, glioma-associated molecules (IDH1/2, ATRX, BRAF, FGFR1, PIK3CA, H3F3A, p53, VEGF, Nestin, PD-1/PD-L1), CDKN2A/B deletion, and chromosomal number aberrations, to see if there is any correlation with patient's progression-free survival (PFS). Brainstem/spinal location, extent of resection and post-operative treatment, and VEGF-A, Nestin and PD-L1 expression, copy number gain of chromosome 7q or 19, TP53 mutation were significantly associated with shorter PFS. None of the histological parameters was associated with PFS. Multivariate analyses demonstrated that high Nestin expression, gain of 7q or 19, and extent of removal were independently predictive for early tumor recurrence. The brainstem/spinal PAs appeared distinct from those in the other sites in terms of molecular characteristics. Clinically aggressive PAs despite benign histology exhibited high Nestin expression. Brainstem/spinal location, extent of resection and some molecular factors including Nestin expression and gains of 7q and 19, rather than histological parameters, may be associated with early tumor recurrence in PAs.

PIK3CA copy-number gain and inhibitors of the PI3K/AKT/mTOR pathway in triple-negative breast cancer.

As wider insights are gained on the molecular landscape of triple-negative breast cancer (TNBC), novel targeted therapeutic strategies might become an option in this setting as well. Activating mutations of PIK3CA represent the second most common alteration in TNBC after the TP53 mutation, with a prevalence of ∼10%-15%. Considering the well-established predictive role of PIK3CA mutations for response to agents targeting the PI3K/AKT/mTOR pathway, several clinical trials are currently evaluating these drugs in patients with advanced TNBC. However, much less is known regarding the actionability of PIK3CA copy-number gains, which represent a thoroughly common molecular alteration in TNBC, with a prevalence estimated at 6%-20%, and are listed as "likely gain-of-function" alterations in the OncoKB database. In the present paper, we describe two clinical cases in which patients harboring PIK3CA-amplified TNBC received a targeted treatment with the mTOR-inhibitor everolimus and the PI3K-inhibitor alpelisib, respectively, with evidence of disease response on 18F-FDG positron-emission tomography (PET) imaging. Hence, we discuss the evidence presently available regarding a possible predictive value of PIK3CA amplification for response to targeted treatment strategies, suggesting that this molecular alteration might represent an intriguing biomarker in this sense. Considering that few of the currently active clinical trials assessing agents targeting the PI3K/AKT/mTOR pathway in TNBC select patients based on tumor molecular characterization, and none of these based on PIK3CA copy-number status, we urge for the introduction of PIK3CA amplification as a criterion for patient selection in future clinical trials in this setting.

Abstract PD9-09: Molecular differences between younger versus older estrogen receptor positive/human epidermal growth factor receptor-2 negative breast cancers

Abstract Background: The RxPONDER and TAILORx trials demonstrated benefit from adjuvant chemotherapy in patients &amp;lt; 50 years with node-positive breast cancer and Recurrence Score (RS) 0-25, and with node-negative disease and RS 16-25, respectively. Neither trial showed benefit in older women with RS &amp;lt; 26. It is unclear what explains the interaction between age and adjuvant chemotherapy benefit. Methods: We analyzed transcriptomic and genomic data from n=4,507 ER+/HER2- breast cancers to compare differences in estrogen receptor (ER), proliferation, and immune-related gene expressions, and somatic mutation patterns and mutation burden between younger (&amp;lt; 50 years of age) and older (&amp;gt;55 years) patients. We restricted our analysis to patients in the lower 80% range of in silico RS distribution to mimic the RxPONDER and TAILORx populations. Results: Five data sets were analyzed independently to assess consistency of results (TCGA n=530; microarray cohort A n=865; Cohort B n=609, METABRIC n=867, SCAN-B n=1636). Older patients had significantly higher somatic mutation burden and more frequent copy number gain in ESR1, LATS1, ARID1B, SGK1, and MYB genes (odds ratio [OR] &amp;gt; 8.5, FDR&amp;lt; 0.05), but lower frequency of GATA3 mutations (12% versus 26%, P&amp;lt; 0.0001). Younger patients had higher rate of ESR1 copy number loss (OR: 0.45, FDR: 0.03). There was no difference in proliferation-related gene expression. ESR1 mRNA expression was significantly lower in younger women in all cohorts (P &amp;lt; 0.001). A regression model of ESR1 mRNA expression using age and ER IHC positivity indicated that lower ER expression in younger patients is primarily driven by lower ESR1 mRNA per cancer cell and not by fewer ER positive cells. We also assessed four gene signatures associated with endocrine therapy sensitivity including a 4-gene ERS, a 7-gene ERS-Lum, a 106-gene ERS-Pos signature, and a 59-gene ERS-Neg signature associated with endocrine resistance. In the TCGA and METABRIC cohorts, the ERS, ERS-Lum, and ERS-Pos signatures were all lower (FDR&amp;lt; 0.03) while the ERS-Neg signature was higher (FDR&amp;lt; 0.001) in younger patients. Similarly, in both microarray cohorts, and in the SCAN-B-cohort, the ERS-Pos signature was lower and the ERS-Neg signature was higher in younger patients (FDR&amp;lt; 0.002). Next, we assessed 4 different immune cell signatures that have been associated with response to chemotherapy. In the TCGA, B-cell, T-cell, Mast-cell, and TIS signatures were significantly higher (FDR&amp;lt;.05). In the microarray Cohort-A, B cells and mast cells were significantly higher, and the T cell and TIS signatures showed a trend for higher expression. In Cohort-B, T cells, B cells, TIS, and dendritic cells signatures were significantly higher in younger patients. Significantly higher expression of immune gene signatures in younger patients were also seen in the METABRIC and SCAN-B data sets. The ER-related and immune-related gene signatures showed negative correlation and joint analysis defined three subpopulations in younger women: (i) immune-high/ER-low, (ii) immune-intermediate/ER-intermediate and (iii) immune-low/ER-intermediate, whereas in older women the dominant pattern was immune-low/ER-high. Conclusion: ESR1 mRNA and ER-associated gene expression is lower in ER positive cancers of younger compared to older patients, while immune infiltration is higher. The cytotoxic and endocrine effects of adjuvant chemotherapy could both contribute to the survival benefit seen in younger patients, but the relative contributions of these effects may vary by ER and immune phenotype. We hypothesize that in immune-high/ER-low cancers, the cytotoxic effect of chemotherapy may drive the benefit, whereas in immune-low/ER-intermediate cancers chemotherapy induced ovarian suppression may play a more important role. Citation Format: Tao Qing, Thomas Karn, Mariya Rozenblit, Julia Foldi, Michal Marczyk, Naing Lin Shan, Kim Blenman, uwe Holtrich, Kevin Kalinsky, Funda Meric-Bernstam, Lajos Pusztai. Molecular differences between younger versus older estrogen receptor positive/human epidermal growth factor receptor-2 negative breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-09.

Point mutations of homologs as an adaptive solution to the gene loss

Gene loss is common and influences genome evolution trajectories. Multiple adaptive strategies to compensate for gene loss have been observed, including copy number gain of paralogous genes and mutations in genes of the same pathway. By using the Ubl-specific protease 2 (ULP2) eviction model, we identify compensatory mutations in the homologous gene ULP1 by laboratory evolution and find that these mutations are capable of rescuing defects caused by the loss of ULP2. Furthermore, bioinformatics analysis of genomes of yeast gene knockout library and natural yeast isolate datasets suggests that point mutations of a homologous gene might be an additional mechanism to compensate for gene loss.

PDGF gene expression and p53 alterations contribute to the biology of diffuse astrocytic gliomas

Diffuse, histologically lower grade astrocytomas of adults (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) genes. While wild-type (WT) LGAs often evolve quickly to glioblastoma (GBM), mutant tumors typically follow an indolent course. To find possible effectors of these different behaviors, we compared their respective transcriptomes. Unlike mutant LGAs, platelet-derived growth factor (PDGF) signaling was significantly enriched in WT tumors, and PDGFA was the top overexpressed gene in the pathway. Moreover, methylation of the PDGFA and PDGFD promoters emerged as a possible mechanism for their low expression in mutant tumors. Copy number gain of chromosome 7 co-occurred with high expression of PDGFA in WT cases, and high expression of PDGFA was associated with aneuploidy, extracellular matrix (ECM)-related immunosuppressive features and poor prognosis. We also noted that high PDGFA expression in WT cases occurred irrespective of tumor grade and that multiple mechanisms of p53 pathway inactivation accompanied progression to GBM in PDGFA-overexpressing tumors. Conversely, TP53 point mutations were an early and constant feature of mutant LGAs. Our results suggest that members of the PDGF gene family, in concert with different p53 pathway alterations, underlie LGA behaviors.

Real-world effectiveness of darolutamide in metastatic castration-resistant prostate cancer: Experience from a safety-net hospital serving a racially and ethnically diverse patient population.

150 Background: Androgen deprivation therapy (ADT), CYP17 inhibitors (CYP17Is, e.g., abiraterone) and 2nd generation androgen receptor (AR) antagonists (2GARAs: enzalutamide, apalutamide, darolutamide) are important treatments for advanced prostate cancer (PC). Darolutamide is FDA-approved with ADT in non-metastatic castration-resistant PC (M0-CRPC) and with ADT+docetaxel in metastatic castration-sensitive PC (M1-CSPC). However, Phase III trials evaluating darolutamide excluded patients (pts) previously treated with other 2GARAs or CYP17Is, thus clinical cross-resistance remains to be evaluated. While reported that darolutamide may be active in vitro against AR mutants resistant to enzalutamide, this also has not been examined clinically. We assessed the real-world effectiveness of darolutamide in a racially/ethnically diverse population of M1-CRPC pts with prior exposure to CYP17Is and/or other 2GARAs. Methods: With IRB approval, we abstracted clinical data from advanced PC pts receiving care at Ben Taub Hospital, a safety-net public hospital serving a racially/ethnically diverse patient population. We identified 33 M1-CRPC pts (17 non-Hispanic African American, 14 Hispanic white, 1 non-Hispanic white, 1 Asian) and 4 M0-CRPC pts (2 non-Hispanic African American, 2 Hispanic white) treated with darolutamide. Tissue and longitudinal circulating tumor DNA (ctDNA) next-generation sequencing data (Tempus Labs, Chicago, IL), including of the AR gene, were also included. Progression-free survival (PFS) was estimated with the Kaplan-Meier method. Results: Amongst the 33 M1-CRPC pts, before receiving darolutamide, 21 had progressed on CYP17Is but had not received 2GARAs; 11 had progressed on both classes of agents; and 1 had progressed on 2GARA but had not received CYP17Is. Darolutamide suppressed PSA by at least 50% in 4/33 pts (12%, all previously 2GARA-naïve). PFS at 3, 6 and 12 months was 36%, 10%, and 5%, respectively, for the entire M1-CRPC cohort; 42%, 17%, and 8% for pts with prior resistance to CYP17Is but not to 2GARA; 25%, 0% and 0% for pts with prior resistance to 2GARA; and 31%, 0% and 0% for pts with AR mutations detected before darolutamide treatment, respectively. AR mutations that emerged or increased in ctDNA allele fraction while on darolutamide included L702H, H875Y, H100Q, D891H, T878A, L329W and AR copy number gain, suggesting a possible role in darolutamide resistance. All four M0-CRPC pts (no prior CYP17I or 2GARA exposure) had PSA declines &gt;50% and remain progression-free at 12 months of treatment. Conclusions: Sequential treatment with darolutamide after progression on a CYP17I may benefit some M1-CRPC pts. However, prior resistance to enzalutamide/apalutamide and/or associated AR mutations can significantly decrease the likelihood of benefit from darolutamide.

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.

Proteogenomics decodes the evolution of human ipsilateral breast cancer

Ipsilateral breast tumor recurrence (IBTR) is a clinically important event, where an isolated in-breast recurrence is a potentially curable event but associated with an increased risk of distant metastasis and breast cancer death. It remains unclear if IBTRs are associated with molecular changes that can be explored as a resource for precision medicine strategies. Here, we employed proteogenomics to analyze a cohort of 27 primary breast cancers and their matched IBTRs to define proteogenomic determinants of molecular tumor evolution. Our analyses revealed a relationship between hormonal receptors status and proliferation levels resulting in the gain of somatic mutations and copy number. This in turn re-programmed the transcriptome and proteome towards a highly replicating and genomically unstable IBTRs, possibly enhanced by APOBEC3B. In order to investigate the origins of IBTRs, a second analysis that included primaries with no recurrence pinpointed proliferation and immune infiltration as predictive of IBTR. In conclusion, our study shows that breast tumors evolve into different IBTRs depending on hormonal status and proliferation and that immune cell infiltration and Ki-67 are significantly elevated in primary tumors that develop IBTR. These results can serve as a starting point to explore markers to predict IBTR formation and stratify patients for adjuvant therapy.

Molecular profiling of primary uveal melanoma: results of a Polish cohort.

There is no published data regarding the molecular alterations of Polish patients with primary uveal melanoma. We performed whole exome sequencing of 20 primary uveal melanomas (UMs), 10 metastasizing and 10 non-metastasizing cases to identify significant molecular alterations. We detected mutations and copy number variants in the BAP1 gene in 50% (10 cases) of the cases. GNA11 mutations were detected in 50% (10 cases) including nine p.Q209L and one p.R183C. GNAQ mutations gene were detected in 40% (8 cases) and all were p.Q209P. SF3B1, EIF1AX, PLCB4 , and PALB2 mutations were detected in one case each. Genetic aberrations of FBXW7 were detected in 55% of cases, with copy number loss of 10 and missense mutation in one. Gain or loss of copy number was observed in 60%, 60%, and 10% of cases in MYC, MLH1 , and CDKN2A genes, respectively. BAP1 and GNAQ tumor suppressor genes are more often mutated in UM with metastasis, while GNA11 mutations are more frequently detected in non-metastasizing tumors. MYC copy gain was present twice as frequently (80% versus 40%) in cases with versus those without metastases. BAP1 mutation correlated with worse overall survival; while GNA11 mutation and CDKN2A loss correlated with better and worse progression-free survival, respectively. We have confirmed BAP1 prognostic potential and documented frequent MYC amplification in metastasizing cases. Although GNA11 mutation and CDKN2A loss significantly correlated with progression-free survival in our study, our sample size is small. The prognostic significance of GNAQ/GNA11 mutation and CDKN2A loss would require further investigation.

Pediatric dermatofibrosarcoma protuberans: A clinicopathologic and genetic analysis of 66 cases in the largest institution in Southwest China.

Dermatofibrosarcoma protuberans (DFSP) is an uncommon cutaneous tumor in children. Most published articles are sporadic or small series and lack systematically molecular analyses. The aim of our study is to better understand the clinicopathologic and genetic features of these rare lesions. All patients diagnosed with DFSP aged ≤ 18 years were retrospectively reviewed from January 2006 to May 2022. A total of 66 cases (32 male and 34 female patients) were identified, with ages ranging from 0.3 to 18 years (median, 13 years). Tumor locations predominantly occurred on the trunk (38/66, 57.6%), followed by the extremities (20/66, 30.3%) and head/neck (8/66, 12.1%). Histological findings revealed classic (41/66, 62.1%), myxoid (4/66, 6.1%), pigmented (6/66, 9.1%), plaque-like (3/66, 4.5%), giant cell fibroblastoma (GCF; 6/66, 9.1%), and fibrosarcomatous (6/66, 9.1%) variants of DFSP. Immunochemistry revealed minority tumors (9/66, 13.6%) showing patchy or negative staining for CD34. Fluorescence in situ hybridization (FISH) indicated that 49 of 53 tested cases including all detected biopsy specimens (11/11) contained COL1A1-PDGFB fusion, in which the average copy number gain of COL1A1-PDGFB was 0.68. There were four cases negative for COL1A1-PDGFB rearrangement, one of which was found to harbor a novel COL3A1-PDGFB fusion by next-generation sequencing (NGS). Treatment for 63 patients comprised 40 marginal excisions and 23 wide local excisions (WLEs), including 1 with imatinib therapy. Follow-up information was available on 49 patients with a duration of 12-161 months (median, 60 months). Fourteen patients developed tumor recurrence, all with initial marginal excisions. The others survived with no evidence of disease. This study of pediatric DFSP indicates certain discrepancies in clinicopathologic characteristics between children and adults. The majority of pediatric DFSPs contain COL1A1-PDGFB fusion, the same as their adult counterparts. The COL3A1-PDGFB chimerism might be associated with the special morphology of GCF, which needs further investigation. FISH is valuable in biopsy tissues and cases with atypical CD34 immunostaining, while supplementary NGS could be helpful to identify the cytogenetically cryptic DFSP. Overall, an urgent accurate diagnosis is needed to formulate an optimal therapeutic strategy in the pediatric population.

GLI1 Immunohistochemistry Distinguishes Mesenchymal Neoplasms With GLI1 Alterations From Morphologic Mimics.

Glioma-associated oncogene 1 ( GLI1 ) alterations have been described in pericytoma with t(7;12), gastroblastoma, plexiform fibromyxoma, and an emerging class of GLI1 -rearranged or amplified mesenchymal neoplasms including "nested glomoid neoplasm". The immunophenotype of these tumor types is nonspecific, making some cases difficult to diagnose without sequencing. The utility of GLI1 immunohistochemistry (IHC) in distinguishing nested glomoid neoplasms and pericytomas with t(7;12) from morphologic mimics is unknown. To investigate the diagnostic value of GLI1 IHC, we determined its sensitivity and specificity in a "test cohort" of 23 mesenchymal neoplasms characterized by GLI1 alterations, including 12 nested glomoid neoplasms (7 GLI1 -rearranged, 4 GLI1 amplified, and 1 unknown GLI1 status), 9 pericytomas with t(7;12), 1 gastroblastoma, and 1 malignant epithelioid neoplasm with PTCH1 :: GLI1 fusion. GLI1 IHC was 91.3% sensitive in this cohort; all tumors except 2 pericytomas with t(7;12) expressed GLI1. GLI1 was also expressed in 1 of 8 (12%) plexiform fibromyxomas. Nineteen of 22 GLI1-positive tumors showed nuclear and cytoplasmic staining, while 3 showed nuclear staining only. GLI1 IHC was 98.0% specific; among morphologic mimics [40 well-differentiated neuroendocrine tumors, 10 atypical lung carcinoids, 20 paragangliomas, 20 glomus tumors, 20 solitary fibrous tumors, 10 Ewing sarcomas, 10 alveolar rhabdomyosarcomas (ARMS), 10 BCOR -altered sarcomas, 10 myoepitheliomas, 9 myopericytomas, 9 epithelioid schwannomas, 9 ossifying fibromyxoid tumors, 10 biphasic synovial sarcomas, 10 PEComas, 31 gastrointestinal stromal tumors, 10 inflammatory fibroid polyps, 11 pseudoendocrine sarcomas], 5 of 249 tumors expressed GLI1 (2 well-differentiated neuroendocrine tumors, 1 ARMS, 1 Ewing sarcoma, 1 BCOR -altered sarcoma). GLI1 IHC was also performed on a separate cohort of 13 molecularly characterized mesenchymal neoplasms in which GLI1 copy number gain was identified as a putatively secondary event by DNA sequencing (5 dedifferentiated liposarcoma [DDLPS], 2 adenosarcomas, 2 unclassified uterine sarcomas, 1 leiomyosarcoma, 1 ARMS, 1 intimal sarcoma, 1 osteosarcoma); 2 DDLPS, 1 ARMS, and 1 unclassified uterine sarcoma expressed GLI1. Lastly, because pleomorphic sarcomas sometimes show GLI1 amplification or copy number gain, GLI1 IHC was performed on a separate "pleomorphic sarcoma" cohort: GLI1 was expressed in 1 of 27 DDLPS, 1 of 9 leiomyosarcomas, and 2 of 10 pleomorphic liposarcomas, and it was negative in 23 well-differentiated liposarcomas and 9 unclassified pleomorphic sarcomas. Overall, GLI1 IHC was 91.3% sensitive and 98.0% specific for mesenchymal tumor types with driver GLI1 alterations among morphologic mimics. GLI1 expression was less frequent in other tumor types with GLI1 copy number gain. Given its specificity, in the appropriate morphologic context, GLI1 IHC may be a useful diagnostic adjunct for mesenchymal neoplasms with GLI1 alterations.

An integrative approach toward identification and analysis of therapeutic targets involved in HPV pathogenesis with a focus on carcinomas.

Persistent infection of high-risk HPVs is known to cause diverse carcinomas, mainly cervical, oropharyngeal, penile, etc. However, efficient treatment is still lacking. Identify and analyze potential therapeutic targets involved in HPV oncogenesis and repurposing drug candidates. Integrative analyses were performed on the compendium of 1887 HPV infection-associated or integration-driven disrupted genes cataloged from the Open Targets Platform and HPVbase resource. Potential target genes are prioritized using STRING, Cytoscape, cytoHubba, and MCODE. Gene ontology and KEGG pathway enrichment analysis are performed. Further, TCGA cancer genomic data of CESC and HNSCC is analyzed. Moreover, regulatory networks are also deduced by employing NetworkAnalyst. We have implemented a unique approach for identifying and prioritizing druggable targets and repurposing drug candidates against HPV oncogenesis. Overall, hundred key genes with 44 core targets were prioritized with transcription factors (TFs) and microRNAs (miRNAs) regulators pertinent to HPV pathogenesis. Genomic alteration profiling further substantiated our findings. Among identified druggable targets, TP53, NOTCH1, PIK3CA, EP300, CREBBP, EGFR, ERBB2, PTEN, and FN1 are frequently mutated in CESC and HNSCC. Furthermore, PIK3CA, CCND1, RFC4, KAT5, MYC, PTK2, EGFR, and ERBB2 show significant copy number gain, and FN1, CHEK1, CUL1, EZH2, NRAS, and H2AFX was marked for the substantial copy number loss in both carcinomas. Likewise, under-explored relevant regulators, i.e., TFs (HINFP, ARID3A, NFATC2, NKX3-2, EN1) and miRNAs (has-mir-98-5p, has-mir-24-3p, has-mir-192-5p, has-mir-519d-3p) is also identified. We have identified potential therapeutic targets, transcriptional and post-transcriptional regulators to explicate HPV pathogenesis as well as potential repurposing drug candidates. This study would aid in biomarker and drug discovery against HPV-mediated carcinoma.