APC Alterations Research Articles

Genomic profiling of biliary tract carcinomas by their location.

e16175 Background: Historically, biliary tract cancers have had a poor prognosis. Genomic profiling allows identification of alterations in genes with FDA-approved matched therapies either in these cancers ( FGFR2/3 fusions, certain IDH1 missense mutations), in all solid tumors (e.g. BRAF V600E mutations), or in other cancers (e.g. BRCA1/2 loss-of-function mutations) that could inform treatment. Here we examine the frequency of matched therapies in biliary tract carcinomas and the association with tumor location. Methods: All biliary tract carcinoma patient samples receiving the OncoExTra assay between April 2018 and November 2022 were included. The assay uses tumor-normal, whole-exome, whole-transcriptome sequencing to identify genomic alterations. Alterations in genes with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies were recorded. Results: A total of 155 patient samples were processed: 92 intrahepatic CCA, 34 gall bladder carcinomas, 9 extrahepatic CCA, and 20 ampulla of Vater carcinomas. Targetable alterations were found in 153 (98.7%) samples; 31 (20.3%) had FDA-approved matched therapies, including 5 (3.3%) with microsatellite unstable and/or tumor mutational burden-high (eligible for immunotherapy) and 7 (4.6%) homologous recombinational repair deficient. Alterations of several genes showed a significant association with tumor type (Table). Cancers in the ampulla of Vater were enriched for KRAS, APC and ARID2 alterations; TP53 alterations were less frequent in biliary tract/intrahepatic cancers. Among the 15 FGFR1/2/3 alterations, there were 3 amplifications (1 FGFR1, 1 FGFR2, 1 FGFR3), 3 missense FGFR2 mutations, and 9 FGFR2 fusions. Conclusions: Comprehensive genomic profiling of biliary tract cancers identifies numerous targetable alterations. The distribution of altered genes varies, suggesting that appropriate therapies may differ by location, reflecting different lineage types. [Table: see text]

Abstract 1908: Clinical genomic profiling identifies lower frequency of therapeutically actionable alterations and lower prognostic value of APC inactivation in colorectal cancer patients of African ancestry

Abstract Intro. We compared molecular profiles and clinical outcomes in a large cohort of colorectal cancer (CRC) patients treated at a single tertiary center to investigate differences in the frequency of therapeutically targetable alterations and the prognostic value of somatic driver alterations across ancestry groups. Methods. We analyzed targeted DNA sequencing data from 4,441 CRC patients treated at Memorial Sloan Kettering between 2014 and 2022. Tumors were sequenced using MSK-IMPACT, a next-generation tumor-normal sequencing assay containing up to 505 genes. Genetic ancestry was estimated using reference populations from the 1000 Genomes Project, including European (EUR), African (AFR), East Asian (EAS), South Asian (SAS), and Native American (NAM). Patients were assigned specific ancestry labels when the predominant ancestry fraction was >80% and considered to have admixed ancestry (ADM) otherwise. Clinical actionability of individual genomic features was determined using the OncoKB knowledgebase. Results. Our cohort included 3,265 EUR, 263 EAS, 245 AFR, 89 SAS, and 15 NAM patients; 564 ADM patients were excluded from subsequent analyses. The AFR group had shorter overall survival (OS) from time of diagnosis (median 45.7 months vs. 67.1 months, p<0.0001). The fraction of patients who qualify for immunotherapy based on FDA guidelines, including microsatellite unstable (MSI) or highly mutated (TMB>10 mut/Mb), was lower in AFR vs. EUR (13.5% vs. 20.4%, p=0.008) patients. Among microsatellite stable (MSS) and not highly mutated (TMB<10 mut/Mb) patients, AFR patients had lower rates of clinically actionable alterations than EUR patients (5.6% vs 11.2%, p=0.01). This difference was driven by a depletion of actionable BRAF mutations in the AFR group (1.8% vs. 5.0%, p=0.04). Somatic APC alterations were associated with longer OS in MSS EUR patients (median 64.6 months in APC altered vs. 45.6 months in APC wildtype, p<0.0001), EAS patients (median 63.1 vs. 35.0 months, p=0.0015), and SAS patients (median not reached vs. 39.4 months, p=0.012). However, APC alterations exhibited no prognostic value for the AFR patients (median OS 45.0 vs. 45.9 months, p=0.91). Multivariate analyses accounting for sex, age, primary tumor location, and stage at diagnosis showed an association between APC status and OS in EUR patients (HR 0.64, CI 0.52-0.79, p<0.001), but not in AFR patients (HR 0.74, CI 0.31-1.7, p=0.492). Conclusions. AFR patients had fewer clinically actionable alterations than patients from other ancestries. The prognostic value of somatic APC alterations was also lower in AFR patients. Our findings provide novel insights into the genomic basis of racial disparities in CRC and highlight the need of ancestry stratification for the analysis of associations between molecular profiles and clinical outcomes. Citation Format: Henry Walch, Anisha Luthra, Kanika S. Arora, Michele Waters, Samantha Chin, Christopher J. Fong, Doori Rose, Hannah Williams, Jeese J. Smith, Nikolaus Schultz, Michael F. Berger, Karuna Ganesh, Julio Garcia-Aguilar, Rona Yaeger, Walid K. Chatila, Francisco Sanchez-Vega. Clinical genomic profiling identifies lower frequency of therapeutically actionable alterations and lower prognostic value of APC inactivation in colorectal cancer patients of African ancestry [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 1908.

The Impact of Germline Alterations in Appendiceal Adenocarcinoma.

More than 10% of assessed patients with appendiceal adenocarcinoma have a pathogenic (P) or likely pathogenic (LP) germline variant, including genes implicated in heritable gastrointestinal cancer syndromes, such as Lynch syndrome. We defined the clinical and molecular impact of heritable alterations in appendiceal adenocarcinoma to evaluate the need for dedicated appendiceal screening and prevention strategies in patients with LP/P germline variants. We performed an integrated germline and somatic molecular analysis for patients with confirmed appendiceal adenocarcinoma. Patients underwent paired tumor-normal sequencing for up to 90 hereditary cancer risk genes and 505 genes for somatic mutation profiling. We defined the cooccurrence of LP/P germline variants and second-hit pathogenic somatic alterations. The associations between germline variants and patient clinicopathologic features were also evaluated. Twenty-five of 237 patients (10.5%) carried pathogenic or likely pathogenic germline variants in cancer susceptibility genes. Clinicopathologic characteristics and appendiceal adenocarcinoma-specific survival were similar in patients with or without germline variants. Most (92%, N = 23/25) patients with germline variants demonstrated no second-hit somatic alterations, including loss of heterozygosity. Two patients with a germline APC I1307K low-penetrance founder variant exhibited secondary somatic pathogenic alterations in APC. However, only one patient tumor exhibited APC-mediated WNT signaling dysregulation: a plausible consequence of multiple somatic APC mutations with no germline variant contribution. Four patients had germline variants in PMS2 or MSH2 associated with Lynch syndrome, yet their cancers were microsatellite-stable. Germline variants are likely incidental without a contributory driver role in appendiceal adenocarcinoma. Appendiceal adenocarcinoma screening in patients with germline variants is not clearly merited.

Locoregional treatment for colorectal liver metastases aiming for precision medicine.

In patients with colorectal liver metastases (CLM), surgery is potentially curative. The use of novel surgical techniques and complementary percutaneous ablation allows for curative-intent treatment even in marginally resectable cases. Resection is used as part of a multidisciplinary approach, which for nearly all patients will include perioperative chemotherapy. Small CLM can be treated with parenchymal-sparing hepatectomy (PSH) and/or ablation. For small CLM, PSH results in better survival and higher rates of resectability of recurrent CLM than non-PSH. For patients with extensive bilateral distribution of CLM, two-stage hepatectomy or fast-track two-stage hepatectomy is effective. Our increasing knowledge of genetic alterations allows us to use them as prognostic factors alongside traditional risk factors (e.g. tumor diameter and tumor number) to select patients with CLM for resection and guide surveillance after resection. Alteration in RAS family genes (hereafter referred to as "RAS alteration") is an important negative prognostic factor, as are alterations in the TP53, SMAD4, FBXW7, and BRAF genes. However, APC alteration appears to improve prognosis. RAS alteration, increased number and diameter of CLM, and primary lymph node metastasis are well-known risk factors for recurrence after CLM resection. In patients free of recurrence 2 y after CLM resection, only RAS alteration is associated with recurrence. Thus, surveillance intensity can be stratified by RAS alteration status after 2 y. Novel diagnostic instruments and tools, such as circulating tumor DNA, may lead to further evolution of patient selection, prognostication, and treatment algorithms for CLM.

RNA Sequencing Identifies Novel NRG1 Fusions in Solid Tumors that Lack Co-Occurring Oncogenic Drivers

NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n=7), breast (n=2), colorectal (n=3), esophageal (n=2), ovarian (n=1), pancreatic (n=1), and unknown primary (n=1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n=8) and previously unreported (n=11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

Sinonasal Myxoma: A Distinct Entity or a Myxoid Variant of Desmoid Fibromatosis?

Sinonasal myxoma (SNM) is a rare benign mesenchymal tumor that arises in the sinonasal cavity or maxilla and almost exclusively affects young children. Currently, it is considered a specific entity, but its molecular characteristics have not been reported. Lesions diagnosed as SNM and odontogenic myxoma/fibromyxoma were identified from the participating institutions, and the clinicopathologic features were recorded. Immunohistochemistry for β-catenin was performed in all cases with available tissue. Next-generation sequencing was performed in all cases with SNM. Five patients with SNM were identified, including 3 boys and 2 girls with an age range of 20-36 months (mean: 26 months). The tumors were well defined, centered in the maxillary sinus, surrounded by a rim of woven bone, and composed of a moderately cellular proliferation of spindle cells oriented in intersecting fascicles in a variably myxocollagenous stroma that contained extravasated erythrocytes. Histologically, the tumors resembled myxoid desmoid fibromatosis. Three tested cases showed nuclear expression of β-catenin. In 3 tumors, next-generation sequencing revealed intragenic deletions of APC exons 5-6, 9 and 15, or 16, respectively, with concurrent loss of the other wild-type copy of APC predicted to result in biallelic inactivation. The deletions were identical to those that occur in desmoid fibromatosis, and copy number analysis raised the possibility that they were germline. In addition, 1 case showed the possible deletion of APC exons 12-14, and another case exhibited a CTNNB1 p. S33C mutation. Ten patients with odontogenic myxoma/fibromyxoma were identified, including 4 women and 6 men (mean age: 42 years). Seven tumors involved the mandible and 3 the maxilla. Histologically, the tumors differed from SNM, and all cases lacked nuclear expression of β-catenin. These findings suggest that SNM represents a myxoid variant of desmoid fibromatosis that often arises in the maxilla. The APC alterations might be germline, and therefore, genetic testing of the affected patients should be considered.

Abstract 165: Establishment and characterization of Vogelgram-mimic colorectal cancer organoids as a laboratory tool

Abstract Colorectal cancer is a deadly disease with a 14% five-year survival rate once it metastasizes. The development of colorectal cancer involves a stepwise accumulation of genetic alterations in APC, KRAS, DCC, and p53 that is often described as a “Vogelgram,” named after the Vogelstein laboratory which laid the groundwork for our understanding of the molecular events leading to colorectal cancer. Organoids are a highly physiologically relevant laboratory tool that can provide valuable information regarding the tumor microenvironment. Here, we describe ex vivo establishment of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl colorectal organoids, which behave normally before tamoxifen treatment but acquire malignant behaviors after tamoxifen-induced loss of APC and p53. Colonic crypts from mice with colon-specific expression of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl were isolated and grown in Matrigel and WENRAS medium following the Organoid Core Facility protocol of the Legorreta Cancer Center. Western blot was used to measure levels of KRAS, KRAS G12D, APC, and p53 after 12 days of tamoxifen treatment and images were taken after 6 days of tamoxifen treatment. The organoid growth medium was collected after 3-4 days of incubation and run on the Luminex 200 platform to measure the levels of 50+ cytokines. Organoids were embedded in OCT and cryosectioned for IF staining with p53, p21, Ki67 and Lgr5 antibodies. IF stained organoid slides were imaged by confocal microscope at 40X magnification. Tamoxifen treatment (12 days) induces overexpression of KRAD G12D protein, reduces expression of APC, and abolishes p53 expression. At day 6, distinct morphological changes indicate malignant transformation. This transformation was accompanied by a trend toward decreased BAFF and CCL20. H&E staining revealed clearly visible intra-organoid structures and florescent labeling of Ki67, Lgr5, and p21 visualized by confocal microscope revealed distinct localization of these proteins within the organoid structures. Here, we describe Vogelgram-mimicking organoids that may be used as a valuable laboratory tool to study the processes involved in the stepwise transformation into colorectal cancer. We plan to use this validated system to evaluate drug sensitivity, cytokine secretion, and immune cell interactions along colorectal cancer progression. We also plan to characterize cell populations with single-cell RNA-seq, establish triangulated cultures, and conduct in vivo studies using this transgenic mouse model. Citation Format: Leiqing Zhang, Lindsey Carlsen, Lanlan Zhou, Liz Hernandez Borrero, Wafik S. El-Deiry. Establishment and characterization of Vogelgram-mimic colorectal cancer organoids as a laboratory tool [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 165.

Abstract P3-05-10: Disease features, genomic profiles and outcomes of younger vs. older Chinese hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients

Abstract Background: Breast cancer in younger women has been characterized as a more aggressive disease with relatively poor outcomes compared to breast cancer in older women. This pattern has been attributed to a variety of clinicopathologic characteristics including the enrichment of aggressive HER2+ and TNBC subtypes in younger patients. However, similar to older breast cancer patients, the majority of young breast cancer patients are HR+/HER2-. Here we have evaluated the clinicopathologic characteristics and genomic profiles of real-world HR+/HER2- MBC patients to examine the determinants of outcome for younger vs. older patients in this subtype. Patients and Methods: This study included 65 Chinese patients presenting with HR+/HER2- MBC at the Peking University Cancer Hospital. Blood samples were collected upon metastatic disease diagnosis before treatment and profiled with the targeted 152-gene PredicineCARETM sequencing panel. Fourteen patients were < 40 years, 19 were 40-50 years, and 32 were > 50 years at the time of primary cancer diagnosis. Kaplan-Meier survival analysis was performed to analyze outcomes including overall survival (OS), disease free survival (DFS) and progression free survival (PFS) in association with clinicopathologic and genomic variables. OS was also evaluated in association with age in separate multivariate models using Cox proportional hazards regression. Results: Significant variation across age groups was observed for several clinicopathological features, including molecular subtype classification (p = 0.045), de novo Stage IV disease (p = 0.011), type of adjuvant endocrine therapy (p = 5.56E-06), resistance to adjuvant endocrine therapy (p = 0.015) and receipt of adjuvant chemotherapy (p = 0.042). Luminal B status was most frequent in patients < 40 years (92.31%), whereas de novo Stage IV disease was more prevalent in patients > 50 years (37.5%). Aromatase inhibitors (AIs) were administered as adjuvant endocrine therapy (ET) to 73.68% of women > 50 years vs. 16.67% and 0% of women aged 40-50 years and < 40 years, respectively. Resistance to adjuvant ET was observed in 23.08% of patients < 40 years but was not observed in the other age groups. Adjuvant chemotherapy was received by a higher proportion of patients in the 40-50 year age group (94.44%) compared to patients < 40 years (69.23%) and patients > 50 years (60%). No differences in somatic gene alteration frequencies were observed across age groups, menopausal status, germline mutation status or tumor grade. However, a higher frequency of FGFR1 alterations was observed in patients classified as Luminal B vs. A (p = 0.048). Comparison of profiles across women who received adjuvant ET revealed a higher prevalence of FGFR1 (p = 0.012), ATM (p = 0.047) and CCND2 (p = 0.04) alterations in patients treated with AIs vs. SERMS. In addition, a higher frequency of APC alterations was observed in patients with high (≥ 20%) vs. low (< 20%) Ki67 index (p = 0.035). At the univariate level OS was significantly associated with age (p = 0.039). OS was shortest in patients > 50 years, intermediate in patients < 40 years, and longest in patients 40-50 years. Across all patients, shorter OS was also associated with de novo Stage IV disease (p = 0.0001), Luminal B subtype (p = 0.008), adjuvant ET with AIs vs SERMS (p = 0.028) and the presence of an FGFR1 (p = 0.028) or CCND2 (p = 0.033) alteration. In multivariate analyses, OS remained significantly longer for patients aged 40-50 years after adjustment for de novo Stage IV disease, Luminal B subtype and FGFR1 status (all p < 0.05). Conclusions: In this group of real-world HR+/HER2- MBC breast cancer patients, younger age was not associated with worse outcomes. While current guidelines recommend treatment decisions based on tumor biology rather than age, young HR+ breast cancer patients are more likely to receive chemotherapy. Our findings support the development of biomarker-driven treatment strategies for these patients. Citation Format: Jinhao Wang, Yaxin Liu, Bin Shao, Hang Dong, Tiantian Zheng, Pan Du, Shidong Jia, Bonnie King, Jing Wang, Xiaoran Liu, Huiping Li. Disease features, genomic profiles and outcomes of younger vs. older Chinese hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients [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 P3-05-10.

The Wnt signaling pathway in gastrointestinal cancers.

807 Background: Activation of the Wnt signaling pathway leads to increases in β-catenin levels, which subsequently increases transcription of various genes, including those involved in cell proliferation. Dysregulation of the Wnt signaling pathway has been observed in numerous gastrointestinal (GI) cancers. Here we examine Wnt pathway activation across all GI cancers by investigating alterations in certain genes of the Wnt pathway, which may identify patients eligible for clinical trials. Methods: The Oncomap ExTra genomic profiling assay utilizes tumor-normal, whole-exome, whole-transcriptome DNA and RNA sequencing to identify somatic alterations in tumors. The assay detects single-nucleotide substitutions, indels, copy number alterations, alternative transcripts, and gene fusions. Alterations in genes that activate Wnt signaling and their frequency across all GI cancers was determined. Results: A total of 1928 patients assayed April 2018 to July 2022 were included. The Wnt signaling pathway was activated in 922 (47.8%) patients. Wnt pathway activation varied substantially across GI cancers, from 81.1% in colorectal cancer (CRC) to 3.4% in anal cancer. APC was the most commonly altered gene in CRC, small bowel and biliary carcinomas. CTNNB1 was predominant in liver (41.2%), MEN1 in neuroendocrine (14.6%), and RNF43 in gastric (9.4%) cancer. Of 921 patients with alterations in the Wnt pathway, 5.2% had a co-mutation in another Wnt pathway-related gene. RSPO2/3 fusions were found in 4 CRC, 1 stomach, 1 biliary, and 1 small bowel cancer and were mutually exclusive with other alterations in the Wnt pathway. Conclusions: Wnt signaling is activated in about half of all GI cancers, and APC alterations are the most frequently observed. Whole transcriptome profiling allowed us to identify RSPO2/3 fusions, which also contribute to activation of the Wnt pathway. Wnt activation appears to be a particularly important in CRC, with 81.1% of CRC having a Wnt pathway alteration. Our results highlight the therapeutic potential of targeting the abnormal Wnt/β-catenin signaling pathway in GI malignancies. [Table: see text]

Clinical and mutational profiles of microsatellite instability (MSI-H) in intrahepatic cholangiocarcinoma (iCCA).

607 Background: Microsatellite instability (MSI-H) and increased tumor mutation burden (TMB) have been associated with clinical benefit from immunotherapy. A small subset of patients with intrahepatic cholangiocarcinoma (iCCA) have microsatellite instability (MSI-H). Understanding their clinical outcomes and genomic landscape has been limited. These patients may derive clinical benefit from immunotherapy compared to chemotherapy. Methods: 7565 cases of iCCA underwent comprehensive genomic profiling of 324 genes (FoundationOne CDx). Prevalence of genomic alterations in cases with MSI-H were compared with MSI-low and MSS iCCA using Fischer’s exact test with Bonferonni correction. Clinical and pathological data were collected by retrospective chart review for patients with MSI-H/dMMR iCCA. Results: Among 7565 iCCA cases identified, 137 (1.8%) were MSI-H. Compared to MSS/MSI-low cases, MSS-H patients were more likely to be male and have Lynch syndrome. Median TMB was 21.7 (muts/Mb). MSI-H cases were enriched for genomic alterations (GA) in APC (13.9%), TP53 (59.9%), ARID1A (13.9%), and PRBM1 (37.2%). MSI-H cases had lower frequencies of previously identified, actionable iCCA drivers such as FGFR2 fusions (0% v 9%, p=NS), IDH1 (3.7% v 14.5%, p<0.0001) and IDH2 (0% v 4.1%, p=0.007). Compared to MSS/MSI-low, MSI-H cases had higher GA in PIK3CA (16.8% v 6.6%, p=<0.0001), PTEN (10.9% v 2.8%, p=<0.0001), and BRCA2 (10.2% v 2%, p=<0.0001). MSI-H cases had lower GA in CDKN2A (24.1% v 30.9%, p=0.09) and MTAP loss (3.4% v. 15.5%, p<0.0001). Five patients were identified with dMMR/MSI-H iCCA out of 974 patients at a single institution (0.5%). Median age at diagnosis was 53 years (range 46-70) and 83% were male (n=4). Two patients had resectable disease at time of diagnosis. TMB range was 20-29 (muts/Mb). Patients had 4-6 co-mutations on average with the most common GA being TP53, ARID1A and CDKN2A. One patient had Lynch syndrome with germline alteration of MLH1 gene and one patient had a germline BRCA mutation. One patient was noted to have FGFR2 rearrangement. Gemcitabine/Cisplatin was given to 3 of 6 patients as first-line (1L) therapy, with median progression free survival of 4 months. One hundred percent of patients received immunotherapy (IO) as first or second-line (2L) therapies. Patients receiving IO in the 1L had responses ranging from 4 to 9 months. Three patients have ongoing responses. Median overall survival ranged from 12 months to >18 months. Two patients presented with CNS involvement and did not respond to immunotherapy-based regimens. Conclusions: MSI-H represents a small subset of patients with iCCA. High-TMB noted amongst MSI-H iCCA. Currently, MSI testing is not universally performed and may be necessary to identify and best treat these patients. These findings support the development of personalized immunotherapy treatment strategies for this distinct patient population.

MN1 altered astroblastoma with APC and LRP1B gene mutations: a unique variant in the cervical spine of a pediatric patient.

Astroblastomas (AB) are high-grade neoplasms which typically occur within the cerebral hemisphere. However, given the rarity of this neoplasm and the number of variants, the relevance of this molecular makeup is unknown. We sought to describe the clinical presentation, treatment, and pathological analysis of a novel MN1 (meningioma 1) cervical spinal cord astroblastoma variant presenting in a pediatric patient. A retrospective review of electronic medical records was performed with an emphasis on neuroimaging, perioperative course, and pathological analysis. An 11-month-old male with no significant history presented with two weeks of neck stiffness and cervicalgia. Neurologically, the patient was intact without signs of infection or trauma. Cervical CT was unremarkable. A subsequent MRI demonstrated a heterogeneously enhancing intramedullary lesion extending from the craniocervical junction to T4. The patient was treated with perioperative steroids and underwent C1-C3 laminectomies and C4-T4 laminotomies for tumor resection. Upon completion of the durotomy, an exophytic gray-red tumor was appreciated within the epidural space and gross total resection was achieved (no change on intraoperative neurophysiological monitoring) and confirmed on post-operative imaging. Immunohistochemical analysis was consistent with an astroblastoma with atypical diffuse positivity of CD56, CD99, and nuclear OLIG2. Molecular analysis revealed not only MN1 alterations but also changes in genes encoding APC and LRP1B. Both alterations were not previously documented to be associated with an astroblastoma. Our case represents the first report of an infant with an MN1 astroblastoma with APC and LRP1B gene alterations in the cervical spine. Gross total resection paired with a detailed histopathologic analysis is vital for optimizing adjuvant treatment.

Selective Eradication of Colon Cancer Cells Harboring PI3K and/or MAPK Pathway Mutations in 3D Culture by Combined PI3K/AKT/mTOR Pathway and MEK Inhibition.

Colorectal cancer (CRC) is the second deadliest cancer in the world. Besides APC and p53 alterations, the PI3K/AKT/MTOR and MAPK pathway are most commonly mutated in CRC. So far, no treatment options targeting these pathways are available in routine clinics for CRC patients. We systematically analyzed the response of CRC cells to the combination of small molecular inhibitors targeting the PI3K and MAPK pathways. We used CRC cells in 2D, 3D spheroid, collagen gel cultures and freshly isolated organoids for drug response studies. Readout for drug response was spheroid or organoid growth, spheroid outgrowth, metabolic activity, Western blotting and immunofluorescence. We found profound tumor cell destruction under treatment with a combination of Torin 1 (inhibiting mTOR), MK2206 (targeting AKT) and selumetinib (inhibiting MEK) in 3D but not in 2D. Induction of cell death was due to apoptosis. Western blot analysis revealed efficient drug action. Gedatolisib, a dual PI3K/mTOR inhibitor, could replace Torin1/MK2206 with similar efficiency. The presence of PI3K and/or RAS-RAF-MAPK pathway mutations accounted for treatment responsiveness. Here, we identified a novel, efficient therapy, which induced proliferation stop and tumor cell destruction in vitro based on the genetic background. These preclinical findings show promise to further test this combi-treatment in vivo in mice and to potentially develop a mutation specific targeted therapy for CRC patients.

Integrated Genomic and Clinicopathologic Approach Distinguishes Pancreatic Grade 3 Neuroendocrine Tumor From Neuroendocrine Carcinoma and Identifies a Subset With Molecular Overlap

Distinguishing grade 3 pancreatic neuroendocrine tumor (G3 PanNET) from neuroendocrine carcinoma (PanNEC) is a known diagnostic challenge, and accurate classification is critical because clinical behavior and therapies differ. Although current recommendations suggest that immunohistochemistry for p53, Rb, ATRX, and DAXX can distinguish most cases, some cases remain difficult to classify using this approach. In this study, we reviewed 47 high-grade neoplasms originally diagnosed as pancreatic neuroendocrine neoplasms. In addition to the currently recommended stains, we performed capture-based sequencing of approximately 500 cancer genes and immunohistochemistry for p16 and trypsin or chymotrypsin. Using an integrated molecular and clinicopathologic approach, 42 (89%) of 47 cases had a clear final diagnosis of either G3 PanNET (n = 17), PanNEC (n = 17), or mixed acinar-NEC (n = 8). The 17 G3 PanNETs demonstrated frequent alterations in MEN1 (71%), DAXX (47%), ATRX (24%), TSC2 (35%), SETD2 (42%), and CDKN2A (41%). Contrary to prior reports, TP53 alterations were also common in G3 PanNETs (35%) but were always mutually exclusive with CDKN2A alterations in this group. The 17 PanNECs demonstrated frequent alterations in TP53 (88%), cell cycle genes RB1 (47%), CCNE1/CCND1 (12%), CDKN2A (29%), and in KRAS (53%) and SMAD4 (41%); TP53 was coaltered with a cell cycle gene in 76% of PanNECs. Diffuse strong p16 staining was observed in 69% of PanNECs in contrast to 0% of G3 PanNETs. The 8 acinar-NECs had recurrent alterations in ATM (25%), APC (25%), and STK11 (25%). Five cases remained difficult to classify, 3 of which exhibited overlapping molecular features with alterations in MEN1 with or without ATRX, and RB1 with or without TP53, making it unclear whether to classify as PanNET or PanNEC. Our data demonstrate that molecular profiling and immunohistochemistry for p16 greatly improve the diagnostic accuracy of high-grade pancreatic neuroendocrine neoplasms and identify a subset of rare cases with overlapping features of both PanNET and PanNEC.

Molecular features and race-associated outcomes of SPOP-mutant metastatic castration-resistant prostate cancer.

Inactivating alterations in SPOP frequently occur in prostate cancer and promote increased dependency on androgen receptor (AR)-mediated oncogenic signaling. The presence of SPOP mutation (SPOP-mutant [SPOP-mut]) may therefore impact therapeutic outcomes with AR-directed therapies and docetaxel in metastatic castration-resistant (mCRPC). This was a retrospective study of mCRPC patients treated at an urban academic hospital (n = 103). Patients underwent tumor DNA sequencing to determine SPOP mutational status (SPOP-mut). Outcomes measured were overall survival (OS) from diagnosis and treatment with second-generation AR signaling inhibitor (ARSI) or docetaxel and time to PSAprogression (prostate-specific antigen-progression-free survival[PSA-PFS]) compared by SPOP status using Kaplan-Meier curves and log-rank test. The univariable and multivariable Cox proportional hazard model evaluated the association of SPOPmutation and outcomes adjusted for clinicopathologic features. SPOP-mut was associated with longer PSA-PFS in mCRPC (median 1.79 vs. 0.84 years; p = 0.06) andmultivariate analysis (hazard ratio [HR] = 0.37;95% confidence interval [CI]: 0.17-0.84;p = 0.02). SPOP-mut demonstrated a higher median PSA decline compared to SPOP wild-type (median decline 100% vs. 92%, p = 0.02). SPOP-mut was not associated with OS from the start of ARSI or docetaxel (median OS not reached vs. 2.0 years)or PSA-PFS on docetaxel (median PSA-PFS 0.4vs. 0.5 years) in mCRPC. The majority of SPOPmutations were identified in African American (AA) patients (69.2%) compared to Caucasian patients (30.8%). Race-associated multivariate analysis revealed no significant differences in OS from the start of ARSI or the start of docetaxeland no differences in ARSI or docetaxel PSA-PFS between AA and Caucasian patients. Molecular profiling demonstrated that AA patients had a higher frequency of SPOPmutations and greater heterogeneity of SPOP variants within the coding sequence. Analysis of concurrent genomic alterations revealed that SPOPmutationsco-occur with APC mutations (p = 0.001) and alterations in the Wnt pathway (p = 0.017). Inactivating mutations in SPOP are associated with better response to ARSI treatment in mCRPC overall. Additional analysis with a larger cohort is needed to evaluate the association of SPOP status and outcomes with docetaxel. Race-associated clinical outcomes and molecular features were observed, suggesting the benefit of biomarker-directed therapy selection for individualized patient subsets in guiding treatment decisions for mCRPC patients.

Alterations in APC, BECN1, and TP53 gene expression levels in colon cancer cells caused by monosodium glutamate.

Colorectal cancer (CRC) is a disease with high incidence worldwide. As of 2018, it is the second leading cause of cancer deaths in the world. In Saudi Arabia, the incidence of this disease has been increasing in the younger population. Both genetic and lifestyle factors may have contributed to its increased incidence and pathogenesis. Monosodium glutamate (MSG) is a food flavor enhancer that can be found in many commercial foods, and it can sometimes be used as a substitute to table salt. MSG has been investigated for its possible genotoxicity, yielding controversial results. In the present study, the effect of MSG on cell viability and its effect on expression of APC, BECN1, and TP53 genes in SW620 and SW480 colon cancer cell lines were studied. TP53 is a tumor suppressor gene that functions in modifying DNA errors and/or inducing apoptosis of damaged cells, and both APC and BECN1 genes are involved in CRC and are of importance in cellular growth and metastasis. Cancer cell viability was analyzed using MTT assay, and the results showed a significant increase in the number of viable cells after 24 h of treatment with MSG with different concentrations (0.5, 1.0, 10, 50, and 100mM). Moreover, gene expression results showed a significant increase in the expression levels of APC and BECN1 under specified conditions in both cell lines; conversely, TP53 showed a significant decrease in expression in SW620 cells. Thus, it can be concluded that MSG possibly confers a pro-proliferative effect on CRC cells.

Clinicopathologic and molecular spectrum of testicular sex cord-stromal tumors not amenable to specific histopathologic subclassification

A subset of testicular sex cord-stromal tumors (SCST), which includes neoplasms with mixed histology, cannot be classified into a specific histologic subtype. This study evaluated the clinicopathologic, immunophenotypic and molecular features of 26 SCST not amenable to specific classification by expert uropathologists. Median age at diagnosis was 43 years and median tumor size was 2.4 cm. Follow-up information was available for 18 (69%) patients, with evidence of an aggressive clinical course in 6 patients (4 alive with disease, 2 dead of disease 3 months and 6 months after orchiectomy). Microscopically, SCST not amenable to specific classification demonstrated monophasic epithelioid (9/26, 35%), monophasic spindle cell (5/26, 19%), and biphasic or mixed histology (12/26, 46%). One or more aggressive histopathologic features were seen in 11 cases. DNA sequencing was successful in 22 tumors. Pathogenic CTNNB1 and APC alterations were seen in 7 (33%) and 2 (10%) cases, respectively, with additional variants (e.g., CDKN2A, RB1, TP53, BRCA2) being identified in individual cases. Combined evaluation of morphology, sequencing data and beta-catenin immunohistochemistry resulted in reclassification of 6 (23%) tumors as Sertoli cell tumor, not otherwise specified. This was supported by comparing the methylation profiles of a subset of these tumors and those of typical Sertoli cell tumors. Additionally, a subset of 5 neoplasms (19%) with spindle cell or biphasic histology and SMA expression was characterized by hyperdiploid genomes with recurrent chromosomal gains and absence of driver mutations, possibly representing a distinct tumor type. The SCST that remained not amenable to specific histologic classification (15/26, 58%) were enriched for aggressive histologic features and malignant clinical behavior. In conclusion, this study demonstrated that a subset of testicular SCST that were originally not amenable to specific classification could be reclassified by combined evaluation of morphology, immunohistochemistry and molecular data.

Abstract IA006: The effect of ancestry and the genomics of colorectal cancer: Implications on incidence and outcomes

Abstract Blacks have a higher incidence of colorectal cancer (CRC) and worse survival rates when compared to Whites. Comprehensive genomic profiling was performed in 46,140 colorectal adenocarcinoma cases. Ancestry-informative markers identified 5,301 patients of African descent (AFR) and 33,770 patients of European descent (EUR). AFR were younger, had fewer MSI-H tumors, and had significantly more frequent alterations in KRAS, APC, and PIK3CA. AFR had increased frequency of KRAS mutations specifically KRAS G12D and KRAS G13. There were no differences in rates of actionable kinase driver alterations (HER2, MET, NTRK, ALK, ROS1, RET). In patients with young onset CRC (<50 years), AFR and EUR had similar frequency of MSI-H and TMB-H tumors, and strikingly different trends in APC mutations by age, as well as differences in MAPK pathway alterations. These findings inform treatment decisions, impact prognosis, and underscore the need for model systems representative of our diverse US population. Further studies are needed to investigate the role of colon polyps and the effect of genomics, ancestry and social determinants of health on CRC incidence and outcomes. Citation Format: Parvathi A. Myer. The effect of ancestry and the genomics of colorectal cancer: Implications on incidence and outcomes [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA006.

1102P Genomic alterations correlated with the expression of PD-L1 based on the next-generation sequencing in Chinese non-small cell lung cancer

Blockade of programmed cell death protein-1 (PD1)/programmed cell death ligand-1 (PD-L1) interaction is one of important immunotherapies in lung cancers. Current research also focused on the relationship between genomic alterations and the expression of PD-L1. Therefore, we analyzed the molecular characteristics and its correlation with the expression of PD-L1, which may help to establish a reasonable tumor classification strategy to improve immunotherapy. Tissue specimens were obtained from 391 cases suffered from lung cancer. Frequencies of gene alterations were detected and analyzed using next-generation sequencing(152 or 603 genes). All specimens were also performed for PD-L1 (22C3) by immunohistochemistry (IHC) with PD-L1 tumor proportion score (TPS) reported for further analysis. In 391 lung cancer samples, 113 (28.9%) cases had comutations in both TP53 and EGFR, 110 (28.1%) cases had alterations in EGFR only, 94 (24.0%) cases had alterations in TP53 only, and 74(18.9%) cases were wild-type of TP53 and EGFR. Two major alterated genes were TP53 (57.0%) and EGFR (52.9%), both of whose frequencies were significantly higher than the other tested genes (P < 0.05). Groups with alterations in EGFR, APC (3.3%) or RBM10 (4.1%) were significantly associated with the decreased expression of PD-L1 (P < 0.05), while alterations in TP53 were significantly associated with the increased expression of PD-L1 (P < 0.05). Comparative analysis revealed that alterations (single or compound mutation) occured in either EGFR or TP53 affect the PD-L1 expression in this Chinese cohort. Although alterations in either EGFR or TP53 has independent and synergistic effects on the PD-L1 expression, there is limited evidence for those alterations serving as predictive biomarkers for the PD-L1 status. Furthermore, whether patients with both high PD-L1 expression and alterations in EGFR or TP53 can benefit from immunotherapies is still in question, and this subgroup is still worthy of clinical molecular recognition by further study.

Serial Analysis of Gene Mutations and Gene Expression during First-Line Chemotherapy against Metastatic Colorectal Cancer: Identification of Potentially Actionable Targets within the Multicenter Prospective Biomarker Study REVEAL.

Simple SummaryThe emergence of resistant cells remains a major obstacle for chemotherapy treatment of metastatic colorectal cancers. Improvement of the therapeutic response requires a thorough understanding of the mechanisms of resistance as well as informative biomarkers. In the REVEAL study, we have systematically compared the mutational patterns and expression profiles of primary tumor specimens before and after first-line chemotherapy treatment in the metastatic situation. In addition, we analyzed liquid biopsies pre, during, and after treatment. Alterations in gene expression appeared as the major drivers of chemotherapy resistance. We identified a gene expression signature differentiating primary tumors and metastases and validated this signature in two independent patient cohorts. Moreover, we evaluated the expression of two signature genes, SFRP2 and SPP1, as prognostic and potentially druggable biomarkers.Most metastatic colorectal cancer (mCRC) patients succumb to refractory disease due to secondary chemotherapy resistance. To elucidate the molecular changes associated with secondary resistance, we recruited 64 patients with mCRC and hepatic metastases before standard first-line chemotherapy between 2014 and 2018. We subjected DNA from primary tumor specimens (P), hepatic metastasis specimens after treatment (M), and liquid biopsies (L) taken prior to (pre), during (intra), and after (post) treatment to next generation sequencing. We performed Nanostring expression analysis in P and M specimens. Comparative bioinformatics and statistical analysis revealed typical mutational patterns with frequent alterations in TP53, APC, and KRAS in P specimens (n = 48). P and pre-L (n = 42), as well as matched P and M (n = 30), displayed a similar mutation spectrum. In contrast, gene expression profiles classified P (n = 31) and M (n = 23), distinguishable by up-regulation of immune/cytokine receptor and autophagy programs. Switching of consensus molecular subtypes from P to M occurred in 58.3% of cases. M signature genes SFRP2 and SPP1 associated with inferior survival, as validated in an independent cohort. Molecular changes during first-line treatment were detectable by expression profiling rather than by mutational tumor and liquid biopsy analyses. SFRP2 and SPP1 may serve as biomarkers and/or actionable targets.

Somatic targeted mutation profiling of colorectal cancer precursor lesions

BackgroundMost colorectal cancers (CRC) arise from precursor lesions. This study aimed to characterize the mutation profile of colorectal cancer precursor lesions in a Brazilian population.MethodsIn total, 90 formalin-fixed paraffin-embedded colorectal precursor lesions, including 67 adenomas, 7 sessile serrated lesions, and 16 hyperplastic polyps, were analyzed by next-generation sequencing using a panel of 50 oncogenes and tumor suppressor genes. The genetic ancestry of the patients was estimated.ResultsSomatic driver mutations were identified in 66.7% of cases, including alterations in APC (32.2%), TP53 (20.0%), KRAS (18.9%), BRAF (13.3%) and EGFR (7.8%). Adenomas displayed a higher number of mutations, mainly in APC, compared to serrated polyps (73.1% vs. 47.8%, p = 0.026). Advanced adenomas had a significantly higher frequency of mutation in KRAS and a high overall mutation rate than early adenomas (92.9% vs. 59%, p = 0.006). A high degree of ancestry admixture was observed in the population studied, with a predominance of European components (mean of 73%) followed by African (mean of 11.3%). No association between genetic ancestry and type of lesions was found. The mutation profile of Brazilian colorectal precursor lesions exhibits alteration in APC, KRAS, TP53, and BRAF at different frequencies according to lesion type.ConclusionsThese results bestow the knowledge of CRC's biologic history and support the potential of these biomarkers for precursor lesions detection in CRC screening of the Brazilian population.