RB1 Alterations Research Articles

Evolutionary trajectories of small cell lung cancer under therapy

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1–3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Clinical and Genomic Predictors of Adverse Events in Newly Diagnosed Glioblastoma.

Adverse clinical events cause significant morbidity in patients with GBM (GBM). We examined whether genomic alterations were associated with AE (AE) in patients with GBM. We identified adults with histologically confirmed IDH-wild-type GBM with targeted next-generation sequencing (OncoPanel) at Dana Farber Cancer Institute from 2013 to 2019. Seizure at presentation, lymphopenia, thromboembolic events, pseudoprogression, and early progression (within 6 months of diagnosis) were identified as AE. The biologic function of genetic variants was categorized as loss-of-function (LoF), no change in function, or gain-of-function (GoF) using a somatic tumor mutation knowledge base (OncoKB) and consensus protein function predictions. Associations between functional genomic alterations and AE were examined using univariate logistic regressions and multivariable regressions adjusted for additional clinical predictors. Our study included 470 patients diagnosed with GBM who met the study criteria. We focused on 105 genes that had sequencing data available for ≥ 90% of the patients and were altered in ≥10% of the cohort. Following false-discovery rate (FDR) correction and multivariable adjustment, the TP53, RB1, IGF1R, and DIS3 LoF alterations were associated with lower odds of seizures, while EGFR, SMARCA4, GNA11, BRD4, and TCF3 GoF and SETD2 LoF alterations were associated with higher odds of seizures. For all other AE of interest, no significant associations were found with genomic alterations following FDR correction. Genomic biomarkers based on functional variant analysis of a routine clinical panel may help identify AE in GBM, particularly seizures. Identifying these risk factors could improve the management of patients through better supportive care and consideration of prophylactic therapies.

Characterizing the Genomic Landscape of the Micropapillary Subtype of Urothelial Carcinoma of the Bladder Harboring Activating Extracellular Mutations of ERBB2

The micropapillary subtype of urothelial carcinoma (MPUC) of the bladder is a very aggressive histological variant of urothelial bladder cancer (UBC). A high frequency of MPUC contains activating mutations in the extracellular domain (ECD) of ERBB2. We sought to further characterize ERBB2 ECD-mutated MPUC to identify additional genomic alterations that have been associated with tumor progression and therapeutic response. In total, 5,485 cases of archived formalin-fixed, paraffin-embedded UBC underwent comprehensive genomic profiling to identify ERBB2 ECD-mutated MPUC and evaluate the frequencies of genomic co-alterations. We identified 219 cases of UBC with ERBB2 ECD mutations (74% S310F and 26% S310Y), of which 63 (28.8%) were MPUC. Genomic analysis revealed that TERT, TP53, and ARID1A were the most common co-altered genes in ERBB2-mutant MPUC (82.5%, 58.7%, and 39.7%, respectively) and did not differ from ERBB2-mutant non-MPUC (86.5%, 51.9%, and 35.3%). The main differences between ERBB2 ECD-mutated MPUC compared with non-MPUC were KMT2D, RB1, and MTAP alterations. KMT2D and RB1 are tumor-suppressor genes. KMT2D frequency was significantly decreased in ERBB2 ECD-mutated MPUC (6.3%) in contrast to non-MPUC (27.6%; P < .001). RB1 mutations were more frequent in ERBB2 ECD-mutated MPUC (33.3%) than in non-MPUC (17.3%; P = .012). Finally, MTAP loss, an emerging biomarker for new synthetic lethality-based anticancer drugs, was less frequent in ERBB2 ECD-mutated MPUC (11.1%) than in non-MPUC (26.9%; P = .018). Characterizing the genomic landscape of MPUC may not only improve our fundamental knowledge about this aggressive morphological variant of UBC but also has the potential to identify possible prognostic and predictive biomarkers that may drive tumor progression and dictate treatment response to therapeutic approaches.

Real-World comprehensive genomic profiling data for diagnostic clarity in pulmonary Large-Cell neuroendocrine carcinoma

BackgroundPulmonary large-cell neuroendocrine carcinoma (LCNEC) is an uncommon subtype of lung cancer believed to represent a spectrum of tumors sharing characteristics of both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Other groups have proposed genomic LCNEC subtypes, including small cell-like, non-small cell-like, and carcinoid-like subtypes. The primary goal of this study was to better define the NSCLC-like subtype with comprehensive genomic profiling (CGP). MethodsAn institutional database was queried to identify tissue specimens (TBx, N = 1,426) and liquid biopsies (LBx, N = 39) submitted for CGP during routine clinical care (8/2014 – 7/2023) with a disease ontology of LCNEC. TBx were profiled with FoundationOne® (F1) or F1CDx, using hybrid-capture technology to detect genomic alterations (GAs). Results1,426 LCNEC samples were genomically profiled. The presence of RB1 and TP53 genomic alterations (GAs) were used to define a SCLC-like subtype (n = 557). A carcinoid-like group was defined by the presence of MEN1 mutation in the absence of TP53 GAs (n = 25). The remaining 844 samples were compared to the SCLC-like group and GAs enriched relative to the SCLC-like samples with a false discovery rate (FDR) < 0.0001 were used to define a NSCLC-like group. These NSCLC-like subtype-defining GAs included SMARCA4, KRAS, FGF3/4/19, STK11, CDKN2A/B, MTAP, and CCND1. Under this schema, 530 samples were classified as NSCLC-like and 314 remained unclassified. ConclusionsLarge-scale CGP can better characterize biologically distinct molecular subtypes in LCNEC. Further studies to define how these molecular subtypes may help inform treatment decisions in this complex and challenging malignancy are warranted.

Concordance and Clinical Significance of Genomic Alterations in Progressive Tumor Tissue and Matched Circulating Tumor DNA in Aggressive-Variant Prostate Cancer.

Sequencing of ctDNA is a minimally invasive approach to reveal the genomic alterations of cancer, however, its comparison with sequencing of tumor tissue has not been well documented in real-world patients with aggressive-variant prostate cancer (AVPC). Concordance of genomic alterations was assessed between progressive tumor tissue and matched ctDNA by next-generation sequencing for 63 AVPC patients. Associations of genomic alterations with progression-free survival (PFS) and overall survival (OS) were investigated using Kaplan-Meier and Cox regression analyses. A total of 161 somatic mutations (SMs) and 84 copy number variants (CNVs) were detected in tumors, of which 97 were also found in ctDNA, giving concordance of 39.6% (97/245) across all SMs and CNVs, 49.7% for SMs only and 20.2% for CNVs only. Across all AVPC patients, chemotherapy was associated with significantly longer median PFS (6 vs. 0.75 mo., P=0.001) and OS (11 vs. 8 mo., P < 0.001) than next-generation hormonal therapy (NHT). Among types of chemotherapy, additional platinum-based chemotherapy was associated with significantly longer median PFS and OS than docetaxel only in patients with TP53, RB1 or PTEN alterations, and in those with ctDNA% ≥ 13.5%. The concordance analysis first provides evidence for combining the sequencing of ctDNA and tumor tissue in real-world AVPC patients. Chemotherapy is associated with significantly better survival than NHT, and the benefit of additional platinum-based chemotherapy may depend on the presence of alterations in TP53, RB1 or PTEN and on a sufficiently high proportion of ctDNA in AVPC patients.

Pooled ctDNA analysis of MONALEESA phase III advanced breast cancer trials

The phase III MONALEESA trials tested the efficacy and safety of the cyclin-dependent kinase (CDK)4/6 inhibitor ribociclib with different endocrine therapy partners as first- or second-line treatment of hormone receptor-positive/humanepidermal growth factor receptor 2-negative advanced breast cancer (ABC). Using the largest pooled biomarker dataset of the CDK4/6 inhibitor ribociclib in ABC to date, we identified potential biomarkers of response to ribociclib. Baseline circulating tumour DNA from patients in the MONALEESA trials was assessed using next-generation sequencing. An analysis of correlation between gene alteration status and progression-free survival (PFS) was carried out to identify potential biomarkers of response to ribociclib. Multiple frequently altered genes were identified. Alterations in ERBB2, FAT3, FRS2, MDM2, SFRP1, and ZNF217 were associated with a greater PFS benefit with ribociclib versus placebo. Patients with high tumour mutational burden (TMB) and with ANO1, CDKN2A/2B/2C, and RB1 alterations exhibited decreased sensitivity to ribociclib versus placebo. Although exploratory, these results provide insight into alterations associated with the improved response to ribociclib treatment and may inform treatment sequencing in patients with actionable alterations following progression on CDK4/6 inhibitors. Validation of potential biomarkers identified here and development of prospective trials testing their clinical utility are warranted. NCT01958021, NCT02422615, NCT02278120.

Combined large cell neuroendocrine carcinoma, lung adenocarcinoma, and squamous cell carcinoma: a case report and review of the literature

BackgroundCombined large cell neuroendocrine carcinoma (C-LCNEC) has a poor prognosis and there is no consensus about the treatment regimen for both LCNEC and C-LCNEC patients.Case presentationThe patient was a 47-year-old female who received surgical resection. The postoperative histology and staging of the tumor suggested C-LCNEC with adenocarcinoma and squamous cell carcinoma and T2aN0M0 stage IB. Next-generation sequencing test showed KIF5B/RET fusion mutation without EGFR, ALK, RB1, and TP53 alterations. Adjuvant chemotherapy with 4-cycle docetaxel plus carboplatin was given and brain metastasis occurred after 10 months.ConclusionsC-LCNEC with adenocarcinoma and squamous cell carcinoma is rare and highly aggressive cancer. Surgical resection and adjuvant chemotherapy with SCLC regimen may improve the disease-free survival and overall survival. The accumulation of similar cases will clarify the profile and management of the disease.

Genomic alterations associated with rapid progression of brain metastases.

The aim of this study was to investigate associations between genomic alterations in resected brain metastases and rapid local and distant CNS recurrence identified at the time of postoperative adjuvant radiosurgery. This was a retrospective study on patients who underwent resection of intracranial brain metastases. Next-generation sequencing of more than 500 coding genes was performed on brain metastasis specimens. Postoperative and preradiosurgery MR images were compared to identify rapid recurrence. Genomic data were associated with rapid local and distant CNS recurrence of brain metastases using nominal regression analyses. The cohort contained 92 patients with 92 brain metastases. Thirteen (14.1%) patients had a rapid local recurrence, and 64 (69.6%) patients had rapid distant CNS progression by the time of postoperative adjuvant radiosurgery, which occurred in a median time of 25 days (range 3-85 days) from surgery. RB1 and CTNNB1 mutations were seen in 8.7% and 9.8% of the cohort, respectively, and were associated with a significantly higher risk of rapid local recurrence (RB1: OR 13.6, 95% CI 2.0-92.39, p = 0.008; and CTNNB1: OR 11.97, 95% CI 2.25-63.78, p = 0.004) on multivariate analysis. No genes were found to be associated with rapid distant CNS progression. However, the presence of extracranial disease was significantly associated with a higher risk of rapid distant recurrence on multivariate analysis (OR 4.06, 95% CI 1.08-15.34, p = 0.039). Genomic alterations in RB1 or CTNNB1 were associated with a significantly higher risk of rapid recurrence at the resection site. Although no genomic alterations were associated with rapid distant recurrence, having active extracranial disease was a risk factor for new lesions by the time of adjuvant radiotherapy after resection.

Novel Genomic Risk Stratification Model for Primary Gastrointestinal Stromal Tumors (GIST) in the Adjuvant Therapy Era.

Traditional risk stratification schemes in gastrointestinal stromal tumors (GIST) were defined in the pre-imatinib era and rely solely on clinicopathologic metrics. We hypothesize that genomic-based risk stratification is prognostically relevant in the current era of tyrosine kinase inhibitor (TKI) therapeutics. Comprehensive mutational and copy-number profiling using MSK-IMPACT was performed. We integrated clinicopathologic and genomic parameters and utilized an elastic-net penalized Cox proportional hazards machine learning model for outcome risk stratification. A 3-tier genomic risk stratification model for recurrence-free survival (RFS) in 152 primary localized gastric and 80 small bowel GISTs was proposed. Gastric GISTs were classified as high risk if chr1p deletion or SDHB loss was present, and intermediate risk if chr14q deletion was present or KIT exon 11 mutation was absent. Small bowel GISTs were classified as high risk if MAX/MGA/MYC, CDKN2A, or RB1 alterations were present, and intermediate risk if chr1p deletion or chr5q amplification was present. Compared with conventional risk stratification, genomic risk stratification both upgrades and downgrades, suggesting that conventional risk stratification may underestimate or overtreat some high-risk and low-risk patients, respectively. Longitudinal sequencing detected most KIT-independent genomic alterations at baseline. Subanalysis in 26 SDH-deficient GISTs revealed that presence of TP53 mutations or chr1q amplifications portends worse RFS and disease-free survival. We developed a novel, next-generation genomic risk stratification model for primary gastric and small bowel GISTs, complementing traditional clinicopathologic models. Future independent validation of our model in external cohorts is essential.

Expanding the Clinicopathologic and Molecular Spectrum of Lipoblastoma-Like Tumor in a Series of 28 Cases

Lipoblastoma-like tumor (LLT) is a rare adipocytic neoplasm with a predilection for the vulva. Since 2002, <30 cases have been reported, characterizing it as an indolent tumor that may sometimes recur locally. Diagnosis can be challenging due to its rarity and morphologic overlap with other adipocytic tumors. Thus far, there are no specific molecular or immunohistochemical features to aid in the diagnosis of LLT. Recent case reports have described LLT arising at other sites, including the spermatic cord and gluteal region, suggesting wider anatomical distribution. We present a large series of LLT to further characterize its clinicopathologic and molecular features. Twenty-eight cases of LLT were retrieved from departmental and consult archives (including 8 from a prior series). The cohort comprised 28 patients (8 males, 20 females) with a median age of 28 years (range: 1-80 years). There were 17 primary LLT of the vulva. Other anatomical sites included the scrotum (n = 3), spermatic cord (n = 2), inguinal region (n = 2), limbs (n = 2), pelvis (n = 1), and retroperitoneum (n = 1). Median tumor size was 6.0 cm (range: 1.8-30.0 cm). The tumors had a lobulated architecture and were typically composed of adipocytes, lipoblasts, and spindle cells in a myxoid stroma with prominent thin-walled vessels. Using immunohistochemistry, a subset showed loss of Rb expression (12/23 of samples). Follow-up in 15 patients (median: 56 months) revealed 8 patients with local recurrence and 1 patient with metastases to the lung/pleura and breasts. Targeted DNA sequencing revealed a simple genomic profile with limited copy number alterations and low mutational burden. No alterations in RB1 were identified. The metastatic LLT showed concurrent pathogenic PIK3CA and MTOR activating mutations, both in the primary and in the lung/pleural metastasis; the latter also harbored TERT promoter mutation. One tumor had a pathogenic TSC1 mutation, and one tumor showed 2-copy deletion of CDKN2A, CDKN2B, and MTAP. No biologically significant variants were identified in 8 tumors. No gene fusions were identified by RNA sequencing in 4 tumors successfully sequenced. This study expands the clinicopathologic spectrum of LLT, highlighting its wider anatomical distribution and potential for occasional metastasis. Molecularly, we identified activating mutations in the PI3K-MTOR signaling pathway in 2 tumors, which may contribute to exceptional aggressive behavior.

Tissue- and liquid-biopsy based NGS profiling in advanced non-small-cell lung cancer in a real-world setting: The IMMINENT study.

e21154 Background: NGS-based profiling is the current standard for the molecular characterization of advanced NSCLC (aNSCLC) in clinical practice. Here we report the results of tissue- and liquid-biopsy based NGS, using FoundationOne and FoundationLiquid CDx (FM1/FM1L), in the context of a multicenter national access program in a cohort of aNSCLC patients (pts). Methods: Genomic evaluation was performed on FFPE-tumor blocks or blood samples, using FM1 (324 genes) for tissue samples or FM1L (70 genes) for liquid biopsy. Tumor mutational burden (TMB) and microsatellite instability were also evaluated. The aim of this analysis was to investigate the real-world frequency of molecular alterations in aNSCLC and their correlation with patients’ clinical and pathological characteristics. Results: Overall, 232 aNSCLC pts from 11 different institutions were gathered [median age 63 years, never/former or current smokers 29/66%, Performance Status 0-1 93%, adenocarcinoma/squamous 79/13%, metastasis at diagnosis 70%]. 138 pts (59.5%) underwent FM1 and 94 (40.5%) FM1L analyses. Alterations were found in 170 different genes. Median number of mutated genes per sample was 4 (IQ 3-6) in FM1 cohort and 2 (IQ 1-3) in FM1L cohort. In 13 FM1L samples (5.6%) no genetic alterations were detected. In unselected pts, TP53 (57.8%), KRAS (22%), CDKN2A/B (19%) and STK11 (17.2%) alterations were among the most frequently detected. Percentages of ALK, HER2, RET, MET, BRAF, NTRK and ROS1 alterations were 4.3, 4.3, 3.4, 3.0, 2.6, 1.3, 0.9, respectively. Among EGFR-mutant pts (15.1%), in both FM1 and FM1L cohorts, ex19del (31.3/35.7%), L858R (12.5/14.3%) and T790M (0/14.3%) were the most frequently detected. Among KRAS-mutant pts, G12C was the mutation most frequently detected (37%). Alterations in KEAP1 were significantly associated with STK11, SMARCA4 and KRAS alterations. Significant associations between TP53 and RB1 alterations were also detected. Median TMB was 6 (0-100) and was significantly higher in current/former smokers. Median OS was 19.6 months (IQ 6.9-36.1), and significantly lower in pts harboring ≥3 gene mutations.25 pts (10.8%) received target therapy on the basis of molecular profiling with FM1/FM1L. In this subgroup, EGFR was the most frequently actionable gene alteration detected (9), followed by RET (4), ALK (3), KRAS (2), MET (2), NTRK (2), BRAF (1), BRCA (1) and HER2 (1). Conclusions: This analysis confirms that NGS-based molecular profiling (on either tissue or blood samples) of aNSCLC provides important prognostic and predictive information, possibly increasing treatment opportunities. Further analyses will focus on actionable alterations in potentially clinically relevant genes outside “the usual suspects”. In the awareness of the urgent need for real world data and to implement NGS, ATLAS ( https://biomarkersatlas.com/ ) will certainly be a fundamental tool.

Clinical and genomic landscape of EGFR-mutant lung cancers (LCs) with squamous and adenosquamous differentiation.

9129 Background: Outcomes to first-line therapy with osimertinib in patients (pts) with EGFR-mutant (EGFR+) squamous (sq) or adenosquamous (adenosq) LC have not been well described. Pts also undergo adenocarcinoma to squamous transformation (transformed) after treatment with EGFR tyrosine kinase inhibitor (TKI). The clinical and genomic characteristics of pts with EGFR+ sq and adenosq LC are largely unknown. Methods: We clinically and genomically characterized all pts with EGFR+ LC with baseline sq or adenosq histology and transformed histology at Memorial Sloan Kettering Cancer Center. Next-generation sequencing was performed with the MSK-IMPACT. We compared clinical outcomes (time to discontinuation (TTD), overall survival (OS), and post-progression survival after osimertinib discontinuation (PPS)) among the different cohorts. Results: A total of 51 pts were identified: 14 with baseline sq/adenosq treated with first-line (1L) osimertinib; 8 transformed cases post 1L osimertinib; 13 with baseline sq/adenosq treated with early generation TKI; 3 transformed cases post early generation TKI; the remainder of pts had early-stage EGFR+LC with baseline sq/adenosq. Median age was 63, and 57% were female. Eighteen (35%) were former or current smokers. Among pts with baseline sq/adenosq histology who were treated with 1L osimertinib, median TTD was 11.1 months (95%CI 7.7, not reached (NR)), and median OS was 24.3 months (95%CI 11.1 months, NR). Compared to a non-overlapping time-matched cohort with baseline adenocarcinoma treated with 1L osimertinib (N = 55), TTD was numerically shorter in the sq/adenosq cohort (11 vs. 17 months, HR 1.8, p = 0.076), and median OS was significantly shorter (24 vs. 33 months, HR 2.64, p = 0.03). PPS was shortest in baseline sq/adenosq (5 months, HR 2.94, p = 0.023), but similar in transformed (11 months, HR 0.89, p = 0.8) compared to adeno (11 months). The sq/adenosq cohort had significantly higher proportion of L858R EGFR (48% vs 31% p &lt; 0.001) and atypical EGFR mutations at baseline (17% vs 7.3%, p &lt; 0.001) compared to the adenocarcinoma cohort. PIK3CA mutations were enriched at baseline in the sq/adenosq cohort. In the transformed cases, RB1 alterations were present in 13% at baseline. There was a similar distribution of type of EGFR mutations in the non-overlapping pts in the AACR GENIE EGFR+ LC cohort with sq/adenosq histology (N = 4, 50% L858R, 25% atypical). Conclusions: Pts with EGFR+ sq/adenosq LCs exhibit inferior TTD and OS with 1L osimertinib compared to adenocarcinoma controls. Sq/adenosq LCs appear to be molecularly distinct with enrichment in EGFR L858R and atypical EGFR mutations. Transcriptomic profiling of a subset of samples will be presented. Our findings highlight the need for novel therapeutic strategies among pts in this high-risk subgroup.

TP53 and RB1 alterations characterize poor prognostic subgroups in pediatric acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is a poor prognostic subtype of pediatric leukemia. However, the detailed characteristics of many genetic abnormalities are yet to be established in this disease. Although TP53 and RB1 are established as representative tumor suppressor genes in various cancers, alterations of these two genes, especially RB1, have not been characterized in pediatric AML. We performed next-generation sequencing in 328 pediatric AML patients from the Japanese AML-05 trial to ascertain TP53 and RB1 alterations, and their prognostic implications. We identified seven patients with TP53 alterations (2.1%) and six patients with RB1 alterations (1.8%). These alterations were found in only patients without RUNX1::RUNX1T1, CBFB::MYH11, or KMT2A rearrangements. TP53 and RB1 were frequently co-deleted with their neighboring genes PRPF8 and ELF1, respectively. Patients with TP53 alterations had significantly lower 5-year overall survival (OS; 14.3% vs. 71.4%, p < 0.001) and lower 5-year event-free survival (EFS; 0% vs. 56.3%, p < 0.001); similarly, patients with RB1 had significantly lower 5-year OS (0% vs. 71.8%, p < 0.001) and lower 5-year EFS (0% vs. 56.0%, p < 0.001) when compared to patients without these alterations. In gene expression analyses, oxidative phosphorylation, glycolysis, and protein secretion were upregulated in patients with TP53 and/or RB1 alterations. Additionally, Kaplan-Meier analysis revealed that high expressions of SLC2A5, KCNAB2, and CD300LF were related to poor OS of non-core-binding factor AML patients (p < 0.001, p = 0.001, and p = 0.021, respectively). This study will contribute to the development of risk-stratified therapy and precision medicine in pediatric AML.

Abstract 3891: Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer among American men, which has traditionally been treated through hormone therapy. However, after hormone therapy many patients with PCa still develop a more aggressive stage of PCa, called metastasis castration resistant prostate cancer (mCRPC). The second- generation antiandrogens, such as enzalutamide or apalutamide, are used to competitively inhibit the androgen receptor (AR) signaling and achieved great clinical success. However, most of the mCRPC patients would develop resistance to the targeted therapy drugs within 6 months to 2 years after initial administration. Consequently, understanding the molecular mechanism of antiandrogen resistance, has become a critical endeavor to provide a greater benefit to patients. mCRPC is characterized by extensive heterogeneity of genomic copy number alterations, which may lead to antiandrogen resistance. TP53 and RB1 alterations have been reported to be dramatically enriched in mCRPC neuroendocrine cancer and we have previously found that inactivation of both TP53 and RB1 confers resistance to antiandrogen through lineage plasticity, where cancer cells can transdifferentiate from a luminal lineage to a mixture of basal and neuroendocrine lineages, which is no longer dependent on AR signaling. Despite these exciting discoveries, only 10% of patients have TP53 and RB1 loss and it is estimated that approximately 40% of patients develop antiandrogen resistance through an undiscovered mechanism.To identify more genomic alterations which confer antiandrogen resistance, we performed an in vivo library screening and identified some of the frequently depleted genes as top candidates mediating antiandrogen response, including the chromatin helicase DNA-binding factor (CHD1). Strikingly, the loss of CHD1 establishes an altered chromatin landscape and enables the activation of heterogenous resistant subclones to emerge, including clones with ectopic NR3C1/GR, POU3F2/BRN2, TBX2, and NR2F1. This work provided an innovative model to explain the dramatically increased tumor heterogeneity in therapy resistant prostate cancer and suggested potential druggable targets to overcome resistance. Building on those exciting results, we are now examining the underlying mechanism how the epigenetic rewiring in resistant tumor clones confer resistance and monitoring the evolution of those heterogenous resistant subclones. Furthermore, we are revealing the collaborative function of those four resistant driver genes in promoting metastasis to various distal organs through multidisplinary approaches including single cell transcriptomics and 3D organoid modeling. The completion of this study will not only add clarity to the mechanism of antiandrogen resistance but may also lead to the development of novel biomarker and therapeutic approaches to overcome resistance. Citation Format: Xiaong li, Su Deng, Julisa Gonzalez, Carla Rodriguez Tirado, Choushi Wang, Nickolas A. Johnson, Lauren Metang, Ping Mu. Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer. [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 3891.

Abstract 3553: Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes

Abstract Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye. Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling. Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations. Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes. Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [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 3553.

Abstract 996: Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma

Abstract Uterine leiomyosarcomas are rare, aggressive cancers that represent the most common type of uterine sarcomas. They frequently harbor genetic alterations in TP53, RB1, PTEN, and ATRX. Their benign counterpart, uterine leiomyomas (or fibroids) are very common tumors that may significantly affect women’s quality of life. The majority of leiomyomas harbor genetic alterations affecting either MED12, HMGA2, or FH. A third of leiomyosarcomas have been reported to harbor leiomyoma-associated driver mutations, suggesting that some leiomyomas may have undergone malignant transformation. The leiomyoma/leiomyosarcoma diagnosis is largely based on histopathology. This can be specifically challenging in differentiating leiomyosarcomas from a subset of leiomyomas that display histopathological features resembling malignancy. The aim of this study was to identify molecular biomarkers that can accurately differentiate benign and malignant uterine smooth muscle tumors. 3’RNA sequencing of 48 leiomyosarcomas and 44 leiomyomas revealed that leiomyosarcomas are uniquely characterized by dysregulation of the retinoblastoma pathway. We confirmed upregulation of several members of this pathway, including TOP2A and CDK1, in a publicly available transcriptomic dataset of 49 uterine leiomyosarcomas. The second objective of this study was to explore the gene expression profile of leiomyosarcomas carrying leiomyoma-associated driver mutations. This revealed that unlike leiomyomas, leiomyosarcomas do not form distinct gene expression profiles based on the presence of leiomyoma-associated driver mutations such as MED12. However, leiomyosarcomas with a MED12 mutation showed upregulation of RAD51B and ADAM12, which we have previously highlighted as biomarkers of leiomyomas harboring a MED12 mutation. We identified upregulation of IRS4 and characteristic deletions affecting COL4A5 and COL4A6 in four in-house and three additional uterine leiomyosarcomas from The Cancer Genome Atlas (TCGA) collection. Similar driver alterations have previously been reported in a small subset of leiomyomas but not in leiomyosarcomas. In conclusion, leiomyosarcomas display a gene expression profile characterized by dysregulation of genes related to the retinoblastoma pathway. These include potential biomarkers that could be used in a clinical setting to differentiate leiomyosarcomas from leiomyomas. Citation Format: Sara Khamaiseh, Riitta Koivisto-Korander, Terhi Ahvenainen, Ralf Bützow, Miika Mehine, Pia Vahteristo. Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma [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 996.

Targeting the Molecular and Immunologic Features of Leiomyosarcoma.

Leiomyosarcoma (LMS) is a rare, aggressive mesenchymal tumor with smooth muscle differentiation. LMS is one of the most common histologic subtypes of soft tissue sarcoma; it most frequently occurs in the extremities, retroperitoneum, or uterus. LMS often demonstrates aggressive tumor biology, with a higher risk of developing distant metastatic disease than most sarcoma histologic types. The prognosis is poor, particularly in patients with uterine disease, and there is a need for the development of more effective therapies. Genetically, LMS is karyotypically complex and characterized by a low tumor mutational burden, with frequent alterations in TP53, RB1, PTEN, and DNA damage response pathways that may contribute to resistance against immune-checkpoint blockade monotherapy. The LMS immune microenvironment is highly infiltrated with tumor-associated macrophages and tumor-infiltrating lymphocytes, which may represent promising biomarkers. This review provides an overview of the clinical and pathologic behavior of both soft tissue and uterine LMS and summarizes the genomic and immune characteristics of these tumors and how they may provide opportunities for the development of biomarker-based immune therapies.

Neoplastic Progression in Neuroendocrine Neoplasms of the Pancreas.

Pancreatic neuroendocrine neoplasms (PanNENs) represent a heterogeneous group of epithelial tumors of the pancreas showing neuroendocrine differentiation. These neoplasms are classified into well-differentiated pancreatic neuroendocrine tumors (PanNETs), which include G1, G2, and G3 tumors, and poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs), which are G3 by definition. This classification mirrors clinical, histologic, and behavioral differences and is also supported by robust molecular evidence. To summarize and discuss the state of the art regarding neoplastic progression of PanNENs. A better comprehension of the mechanisms underpinning neoplastic evolution and progression of these neoplasms may open new horizons for expanding biologic knowledge and ultimately for addressing new therapeutic strategies for patients with PanNENs. Literature review of published studies and the authors' own work. PanNETs can be seen as a unique category, where G1-G2 tumors may progress to G3 tumors mainly driven by DAXX/ATRX mutations and alternative lengthening of telomeres. Conversely, PanNECs display totally different histomolecular features more closely related to pancreatic ductal adenocarcinoma, including TP53 and Rb alterations. They seem to derive from a nonneuroendocrine cell of origin. Even the study of PanNEN precursor lesions corroborates the rationale of considering PanNETs and PanNECs as separate and distinct entities. Improving the knowledge regarding this dichotomous distinction, which guides tumor evolution and progression, will represent a critical basis for PanNEN precision oncology.

Abstract P5-02-14: Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials

Abstract Background: Genetic alterations that contribute to resistance to therapy may be acquired during treatment (tx) for hormone receptor−positive/human epidermal growth factor receptor−negative (HR+/HER2−) advanced breast cancer (ABC). A previous pooled analysis of circulating tumor DNA (ctDNA) in MONALEESA (ML)-2, -3, and -7 identified potential predictive biomarkers for response and resistance to ribociclib (RIB) at baseline (BL). Here, we describe an analysis of paired BL and end of treatment (EOT) samples from ML-2, -3, and -7 to identify acquired mechanisms that may impact resistance to RIB + endocrine therapy (ET) vs placebo (PBO) + ET. Methods: ML-2 (NCT01958021), ML-3 (NCT02422615), and ML-7 (NCT02278120) evaluated efficacy and safety of RIB + ET vs PBO + ET in pre- and postmenopausal patients (pts) with HR+/HER2− ABC treated in first-line (1L) and second-line (2L) settings. Plasma samples were collected at cycle 1 day 1 (C1D1; prior to first therapy exposure) and at EOT (± 28 days of recorded progression). ctDNA was sequenced using a targeted next-generation sequencing panel of ≈550 genes. Genes with an alteration frequency of &amp;gt;5% at EOT, regardless of their frequency at BL, were included. Tumor mutational burden (TMB) was assessed by tx arm; a TMB cutoff of 10 mutations/MB was used to categorize pts as TMB high vs low. To assess differences in the presence of alterations, a McNemar test was performed on paired samples and adjusted (adj) for multiple testing using the false discovery rate (FDR). A Bayesian mixed effects model was used to account for ctDNA fraction and trial and to test for tx-specific resistance by including a tx × visit interaction term. Results: A total of 905 paired samples from ML-2, -3, and -7 were included in this analysis, 441 and 464 samples from pts treated with RIB + ET and PBO + ET, respectively. Overall, 17 genes had an alteration frequency of &amp;gt;5% at EOT. The ctDNA fraction was higher at EOT vs C1D1 in both the RIB (P=.037) and PBO (P=.033) arms. The frequency of alterations in RB1 (10.4% vs 2.0%), ATM (11.3% vs 8.4%), FAT1 (4.8% vs 3.0%), and FAT3 (5.0% vs 2.5%) was higher at EOT vs C1D1 in the RIB arm (FDR-adj P&amp;lt;.10). Alterations in ESR1 were also higher at EOT vs C1D1 in both the RIB (26.3% vs 9.1%) and PBO arms (28.9% vs 5.4%) (FDR-adj P&amp;lt;.0001). Conversely, alterations in GATA3 were higher at EOT in the PBO arm (FDR-adj P=.11). These results were consistent after adjusting for ctDNA fraction. The most common ESR1 mutations were D538G, Y537S/N/C/D, E380Q, and L536H/P/R. Tx × visit interaction effects were observed for RB1 in the RIB arm and GATA3 in the PBO arm, suggesting tx-specific resistance. A tx × visit interaction for ESR1 was also observed, suggesting a larger relative increase in ESR1 mutations with PBO vs RIB. The percentage of pts with high TMB (&amp;gt;10) at EOT increased from 1.1% to 5.7% in the RIB arm and from 1.7% to 3% in the PBO arm. After accounting for ctDNA fraction and trial, a larger numerical increase in TMB was observed for RIB (odds ratio [OR], 9.0; 95% CI, 2.9-32.7) vs PBO (OR, 2.1; 95% CI, 0.7-6.5); however, the model did not support a differential tx effect. Conclusions: This comprehensive analysis of pooled samples from ML-2, -3, and -7 identified acquired gene alterations in pts with HR+/HER2− ABC treated with 1L or 2L RIB + ET or PBO + ET. The frequency of several genes known to contribute to resistance (ESR1, RB1, ATM, FAT1, and FAT3) was higher at EOT vs C1D1 in pts treated with RIB + ET, while ESR1 and GATA3 alterations were higher at EOT vs C1D1 in pts treated with PBO + ET. This paired dataset of BL and EOT samples from pts with HR+/HER2− ABC treated with a CDK4/6 inhibitor and ET is the largest to date and could be used to validate and confirm acquired resistance mechanisms with low alteration frequency. Citation Format: Fabrice Andre, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Yoon-Sim Yap, Debu Tripathy, Yen-Shen Lu, Dennis Slamon, Stephen K. Chia, Mukta Joshi, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Carlos Arteaga. Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials [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 P5-02-14.

Abstract P1-13-07: Investigating NF1 Mutations in Circulating Tumor DNA of Patients with Hormone-receptor Positive (HR+) Breast Tumors Resistant to CDK4/6 Inhibition (CDK4/6i): A Retrospective Clinical Analysis

Abstract Background: CDK4/6 inhibitors (CDK4/6i) are standard of care for the management of HR+/HER2- metastatic breast cancer (MBC). Genomic alterations that drive resistance to CDK4/6i are diverse, and while the molecular landscape is heterogeneous, several mechanisms of CDK4/6i resistance converge on the RAS/MAPK and PI3K/AKT/mTOR signaling pathways. NF1 downregulates RAS and dampens cellular proliferation. Laboratory-based models demonstrate that loss of NF1 is associated with resistance to endocrine therapy (ET), and emergence of NF1 mutations (NF1m) are correlated with progressive disease (PD) in circulating tumor DNA (ctDNA). While NF1m may diminish CDK4/6i susceptibility, a clear relationship has not been elucidated. The primary objective of this study was to characterize patient (pt) response to CDK4/6i in NF1m HR+/HER2- MBC. Methods: We identified 47 pts with NF1m via a database with one or more ctDNA samples sequenced at variable time-points as part of routine care for MBC. NF1m were categorized as pathogenic (p)NF1m or variants of uncertain significance (VUS) based on their associated Guardant report. We identified 27 pts with HR+/HER2- MBC and NF1m that received at least 1 line of CDK4/6i in the metastatic setting. Intrinsic resistance was defined as PD &amp;lt; 6 months on a CDK4/6i regimen, and acquired resistance was defined as PD &amp;gt;6 months. Pts with intrinsic resistance or acquired resistance and NF1m detected post-PD were categorized as having a resistance phenotype potentially driven by NF1m. Pts with NF1m detected prior to therapy and &amp;gt;6 months clinical response on a CDK4/6i were categorized as having NF1m tumors sensitive to CDK4/6i. Results: The NF1m cohort (n = 27) had 9 pts with pNF1m, while 18 pts expressed VUS. The median age at MBC diagnosis was 54 years, and 67% had visceral metastasis at ctDNA collection. Pts received a median of 1 prior line (range: 0 - 6) of ET or chemotherapy in the metastatic setting before CDK4/6i. Amongst pts with pathogenic variants (n = 9), we found 3 pts with pNF1m were intrinsically resistant to CDK4/6i. Acquired resistance was seen in 1 pt with pNF1m detected post-PD, and 2 pts had evidence of both acquired and subsequent intrinsic resistance to a later line of CDK4/6i. Overall, 67% (6/9) of pNF1m pts demonstrated a CDK4/6i resistance phenotype; mutant allele fraction (AF) ranged from 0.2% - 29.9%, and the mean maximum allele fraction (MAF) was 6.0%. Pre- and post-treatment samples were available on 3 pts with pNF1m, and 1 of these pts had an AF rise from 2.7% to 12.3% when comparing ctDNA pre- and post-CDK4/6i. ctDNA from 4 of 6 resistant tumors harbored other putative drivers including alterations in FGFR, KRAS, PTEN, and RB. We identified 2 counter-examples of pNF1m tumors sensitive to CDK4/6i. These pts expressed relatively low NF1m AF, ranging 0.1% - 0.5% with a mean MAF 0.3%. Another pNF1m pt had intrinsic resistance to initial CDK4/6i but was sensitive to later-line CDK4/6i. In the subgroup of pts with VUS-NF1m (n = 18), a more mixed picture of resistance and sensitivity was seen. 8 pts had intrinsic or acquired resistance, 8 pts had NF1m tumors sensitive to CDK4/6i, and 1 pt had evidence of both; 61% (n = 11) of pts expressed alterations in other resistance mediating genes. 1 pt stopped therapy due to toxicity rather than PD. Conclusions: Our work demonstrates that tumor expression of pNF1m may be associated with CDK4/6i resistance in pts with HR+/HER2- MBC, and allele fraction could be predictive of drug susceptibility. Tumors harboring VUS had varied sensitivity, suggesting that some of these mutations may not be pathogenic, and counter-examples of pNF1m MBC benefiting from CDK4/6i plus ET highlight the complexities in predicting drug response based on single gene alteration. Future effort is warranted to explore the potential impact of NF1 on CDK4/6i resistance, as well as the potential role for therapies targeting the MAPK pathway in this patient population. Citation Format: Maxwell R. Lloyd, Lianne Ryan, Arielle J. Medford, Jennifer C. Keenan, Laura M. Spring, Neelima Vidula, Beverly Moy, Dejan Juric, Leif Ellisen, Aditya Bardia, Seth A. Wander. Investigating NF1 Mutations in Circulating Tumor DNA of Patients with Hormone-receptor Positive (HR+) Breast Tumors Resistant to CDK4/6 Inhibition (CDK4/6i): A Retrospective Clinical Analysis [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 P1-13-07.