Hypermutated Genotype Research Articles

The Many Uses of p53 Immunohistochemistry in Gynecological Pathology: Proceedings of the ISGyP Companion Society Session at the 2020 USCAP Annual9 Meeting.

Ancillary immunohistochemistry (IHC) has become a reliable adjunct for subclassification of gynecological neoplasms. An important recent development was optimization and validation of p53 IHC, where 3 abnormal IHC patterns (nuclear overexpression, complete absence, cytoplasmic) were shown to predict underlying TP53 mutations with high accuracy in ovarian carcinomas. p53 IHC now helps in distinguishing high-grade serous from low-grade serous carcinomas. Thereafter, the new interpretation of p53 IHC was quickly adapted for other purposes and similar accuracies were shown in endometrial carcinomas, vulvar squamous cell carcinomas, and ovarian mucinous tumors. However, it required further refinement of the p53 IHC interpretation criteria for each tumor site. A proportion of endometrial endometrioid carcinomas shows an ultramutated or hypermutated genotype due to underlying POLE mutations or mismatch repair deficiency sometimes causing subclonal TP53 mutations, and their distribution can be visualized by p53 IHC. Squamous cell carcinomas and ovarian mucinous tumors show a phenomenon called terminal differentiation where basal cells demonstrate an abnormal pattern of p53 IHC but apical cells do not despite an underlying TP53 mutation. High-grade progression of adult granulosa cell tumors due to a subclonal TP53 mutation has been recently described. Another use of p53 IHC is triaging gynecological sarcomas for molecular testing based on the assumption that TP53-mutated gynecological sarcomas do not harbor cancer driving translocations. Therefore, familiarity with interpretation of p53 IHC is becoming increasingly important for the practicing gynecological pathologist. Furthermore, local optimization of the p53 IHC assay using validated protocols including appropriate low expressing control tissues (eg, tonsil) is vital in order to achieve high diagnostic accuracy, especially for abnormal staining patterns such as complete absence or cytoplasmic, and interlaboratory concordance. p53 IHC is a reliable diagnostic adjunct for histotyping and molecular subtyping of ovarian and endometrial carcinomas, and it paves the way for large-scale studies to validate the prognostic value of p53 IHC in several gynecological tumor types. The technical advances, validated interpretation criteria, and its growing versatility in identifying high-risk neoplasms paired with its widespread availability in pathology departments make p53 IHC perhaps the single most useful IHC stain in gynecological pathology.

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

Abstract 2955: A radiomic-based MRI phenotype is uniquely associated with hypermutated genotype in gliomas

Abstract INTRODUCTION Hypermutation is the excessive accumulation of DNA mutation in cancer cells. This specific hypermutated genotype has been reported in low and high grade gliomas, specifically post-temozolomide treatment and is associated with treatment-resistance. Herein, we sought to identify an imaging-based signature for hypermutated gliomas using a radiomics-based approach. MATERIALS AND METHODS In this IRB-approved retrospective study, we analyzed a total of 101 patients with primary gliomas from the University of Texas MD Anderson Cancer Center. Next generation sequencing (NGS) platforms (T200 and Foundation 1) were used to determine the Mutation burden status in post-biopsy (stereotactic/excisional). Patients were dichotomized based on their mutation burden; 77 hypomutated (<30 mutations) and 24 hypermutated (>=30 mutations or <30 with MMR gene or POLE/POLD gene mutations). Radiomic analysis was performed on the conventional MR images (FLAIR and T1 post-contrast) obtained prior to tumor tissue surgical sampling; and a total of 2480 rotation-invariant radiomic features were extracted using: (i) the first-order histogram and (ii) grey level co-occurrence matrix. The Maximum Relevance Minimum Redundancy technique was used to select the most relevant radiomic features. ROC analysis and leave-one-out cross-validation (LOOCV) were used to assess the performance of the Support Vector Machine (SVM) classifier as and AUC, Sensitivity, Specificity, and p-value were obtained. RESULTS We found 100 radiomic features that can discriminate between hypermutated versus hypomutated gliomas, AUC 96.3% (CI: 90.2%-98.9%), Sensitivity 100%, Specificity 95%, p-value=3.769e-6. CONCLUSION Hypermutated gliomas has a unique radiomic quantitative signature that can be used to predict mutation burden regardless of tumor grade or histopathology. Citation Format: Islam Hassan, Aikaterini Kotrotsou, Carlos Kamiya Matsuoka, Kristin D. Alfaro-Munoz, Nabil Elshafeey, Nancy El Shafei, Pascal O. Zinn, John F. de Groot, Rivka R. Colen. A radiomic-based MRI phenotype is uniquely associated with hypermutated genotype in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2955.

Comprehensive molecular profiling of multi-focal prostate cancer and concomitant lymph node metastasis: Implications for tissue-based prognostic biomarkers.

5061 Background: Current tissue-based prognostic biomarker assays claim that assessment of a single biopsy focus is sufficient to predict disease behavior. We analyzed and compared the genetic profiles of multifocal prostate cancer (PCa) with concordant lymph node metastasis (LNM) to determine if expression-based prognostic tests are robust to multifocality. Methods: This IRB-approved study comprised patients who underwent radical prostatectomy and lymph node dissection that revealed N1 or discordant multifocal (low- and high-grade foci) disease. DNA and RNA were co-isolated from each tumor focus pre-identified on formalin fixed paraffin embedded specimens. High depth, targeted DNA and RNA next generation sequencing was performed to characterize the molecular profile of each sample, using the Oncomine Comprehensive (11 patients) or Comprehensive Cancer (DNA, 3 patients) Panels and a custom targeted RNAseq panel comprising genes for deriving prognostic signatures. Results: A total of 67 primary tumor and 17 LNM foci from 14 patients were analyzed. We observed significant intra- and inter-patient molecular heterogeneity. For example, in patient #1, while all 4 regions of high-grade primary tumor showed TP53 somatic mutations and some copy number alterations (CNAs) with two samples from the LNM, tumor areas near the positive margin showed more complete concordance than intraprostatic regions. Critically, a low-grade primary tumor focus in this case showed no somatic mutation or CNA overlap with the high-grade or LNM samples. In patient #4, all tumor and LNM foci shared a large number of somatic mutations, including a frameshift mutation in PTEN, with no high level CNA, consistent with a hypermutated genotype. By targeted RNAseq, low- and high-grade tumors from the same patient showed distinct expression profiles using genes included in prognostic signatures. Conclusions: Our results challenge the claim that expression-based prognostic tests are robust to multifocality. Further studies are needed to better characterize the biologically dominant lesion in multifocal PCa and hold promise for the development of improved prognostic biomarkers.

First-in-human trial of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab in advanced cancer patients.

The combination of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab overcomes intrinsic and acquired resistance in both EGFR-sensitive and EGFR-resistant preclinical models of colorectal cancer (CRC). Utilizing a standard 3+3 design, a phase I study was designed to determine safety, maximum tolerated dose (MTD), and dose-limiting toxicities (DLTs) of the regorafenib plus cetuximab combination among patients with advanced cancer including CRC. Comprehensive genomic profiling was performed on the exceptional responder. Among the 27 patients enrolled the median age was 54 years. None of 19 patients treated at dose level 1 (cetuximab i.v. 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 80 mg daily) experienced a DLT, and 2 of 5 patients treated at dose level 2 (cetuximab i.v. 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 120 mg daily) experienced a DLT (grade 3 thrombocytopenia [n = 1] and grade 3 intra-abdominal bleed [n = 1]). Most common adverse events were grade 1 or 2 rash (20 patients). Of 24 evaluable patients, 11 (46%) patients had clinical benefit (stable disease > 6 cycles or partial response [PR]) (CRC n = 8, one patient each with head and neck cancer, carcinoma of unknown primary, and glioblastoma). A CRC patient, who progressed on anti-EGFR and regorafenib, achieved a PR (46% decrease per RECIST v1.1) lasting 15 months. Genomic profiling of an exceptional responder with response for over 27 cycles revealed hypermutated genotype with microsatellite instability (MSI). Regorafenib 80 mg daily plus cetuximab 200 mg/m2 loading dose, followed by 150 mg/m2 every week is the MTD/recommended phase II dose. The combination demonstrated early signals of activity in wild-type CRC, including 1 exceptional responder with MSI high. clinicaltrials.gov NCT02095054FUNDING. The University of Texas MD Anderson Cancer Center is supported by the NIH Cancer Center Support Grant CA016672. This work was supported in part by the Cancer Prevention Research Institute of Texas grant RP110584 and National Center for Advancing Translational Sciences grant UL1 TR000371 (Center for Clinical and Translational Sciences).

PD07-12 MOLECULAR PROFILING OF MULTI-FOCAL PROSTATE CANCER AND CONCOMITANT LYMPH NODE METASTASIS: IMPLICATIONS FOR TISSUE-BASED PROGNOSTIC BIOMARKERS

You have accessJournal of UrologyProstate Cancer: Detection & Screening II1 Apr 2017PD07-12 MOLECULAR PROFILING OF MULTI-FOCAL PROSTATE CANCER AND CONCOMITANT LYMPH NODE METASTASIS: IMPLICATIONS FOR TISSUE-BASED PROGNOSTIC BIOMARKERS Simpa Salami, Daniel Hovelson, Romain Mathieu, Jeremy Kaplan, Martin Susani, Christopher Russell, Nathalie Rioux-Leclercq, Shahrokh Shariat, Scott Tomlins, and Ganesh Palapattu Simpa SalamiSimpa Salami More articles by this author , Daniel HovelsonDaniel Hovelson More articles by this author , Romain MathieuRomain Mathieu More articles by this author , Jeremy KaplanJeremy Kaplan More articles by this author , Martin SusaniMartin Susani More articles by this author , Christopher RussellChristopher Russell More articles by this author , Nathalie Rioux-LeclercqNathalie Rioux-Leclercq More articles by this author , Shahrokh ShariatShahrokh Shariat More articles by this author , Scott TomlinsScott Tomlins More articles by this author , and Ganesh PalapattuGanesh Palapattu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.387AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Current tissue-based prognostic biomarker assays claim that genetic assessment of a single focus is sufficient to predict disease behavior. We analyzed and compared the genetic profiles of multifocal prostate cancer (PCa) with concordant lymph node metastasis (LNM) to determine if expression based prognostic tests are robust to multifocality. METHODS This IRB-approved study comprised patients who underwent radical prostatectomy and lymph node dissection that revealed N1 disease or discordant multifocal disease (low- and high-grade foci). DNA and RNA were co-isolated from each tumor focus pre-identified on formalin fixed paraffin embedded specimens. High depth, targeted DNA and RNA next generation sequencing was performed to characterize the genetic and transcriptional signature of each sample, using the Oncomine Comprehensive (11 patients) or Comprehensive Cancer (DNA, 3 patients) Panels and a custom targeted RNAseq panel comprising genes for deriving prognostic signatures. RESULTS A total of 67 primary tumor and 17 LMN foci (with control tissue when available) from 14 patients were analyzed. We observed significant intra- and inter-patient molecular heterogeneity. For example, in patient #1, while all four regions of high-grade primary tumor showed TP53 somatic mutations and some broad copy number alterations (CNAs) with two samples from the LNM, tumor areas near the positive margin showed more complete concordance than intraprostatic regions. Critically, a low-grade primary tumor focus in this case showed no somatic mutation or CNA overlap with the high-grade or LNM samples. In patient #4, all tumor and LNM foci shared a large number of somatic mutations, including a frameshift mutation in PTEN, with no high level CNA identified, consistent with a hypermutated genotype. By targeted RNAseq, low-grade and high-grade tumors from the same patient showed distinct expression profiles using genes included in available prognostic signatures (Figure 1). CONCLUSIONS Our results challenge the claim that expression based prognostic tests are robust to multifocality. Additional molecular studies are needed to better characterize the biologically dominant lesion in multi-focal PCa and hold promise for the development of improved prognostic biomarkers. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e132-e133 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Simpa Salami More articles by this author Daniel Hovelson More articles by this author Romain Mathieu More articles by this author Jeremy Kaplan More articles by this author Martin Susani More articles by this author Christopher Russell More articles by this author Nathalie Rioux-Leclercq More articles by this author Shahrokh Shariat More articles by this author Scott Tomlins More articles by this author Ganesh Palapattu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract C50: Phase I dose escalation study of the oral multi-kinase VEGF inhibitor regorafenib and the anti-EGFR monoclonal antibody cetuximab in patients with advanced solid tumors including colorectal cancer

Abstract Background: The combination of multi-kinase VEGF inhibitor regorafenib and anti-EGFR monoclonal antibody cetuximab overcame intrinsic and acquired resistance in both EGFR-sensitive and EGFR-resistant preclinical models of colorectal cancer (CRC). (Clin Cancer Res; 21(13); 2975-83) Methods: We designed a phase I study to determine the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), and dose-limiting toxicities (DLTs) of the oral multi-kinase inhibitor regorafenib and the anti-EGFR monoclonal antibody cetuximab in patients (pts) with advanced cancer including metastatic colorectal cancer. Tumor responses were assessed using RECIST v1.1. Results: Twenty seven pts were enrolled between May 2014 and August 2015. 22 (81%) pts were evaluable for toxicity and response. 5 heavily pre-treated pts were not evaluable for DLTs because they discontinued the trial before the end of the DLT window, mainly because of disease progression. 21 pts were treated at dose level 1 (cetuximab IV 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 80 mg daily) and 6 pts at dose level 2. MTD was exceeded at dose level 2 (cetuximab IV 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 120 mg daily), with 2 DLTs observed, including G3 thrombocytopenia (n = 1) and G3 thrombocytopenia with intraperitoneal bleeding (n = 1). The most common adverse events observed in all patients across both dose levels tested included G1-2 rash, G1-2 hypomagnesaemia, G1 myalgia, G1 fatigue, G1 nausea/vomiting. In addition other AE's included G2 hand-foot syndrome (n = 5), and G2 hypertension (N = 2). One patient with KRAS wt CRC achieved a partial response (PR) (46% decrease) lasting 15 months who was previously resistant to cetuximab. Another KRAS wt CRC patient with a hyper-mutated genotype [Lynch syndrome (MSI High), both BRCA1 and 2 mutations, FGFR3 mutation] achieved stable disease (SD) for > 10 months. 10 patients (37%) achieved SD>4 months or PR that included renal cell carcinoma (n = 1), EGFR mutant glioblastoma multiforme (n = 1), squamous cell cancer (n = 1) and carcinoma of unknown primary (n = 1). Conclusions: The combination of cetuximab and regorafenib was well tolerated at doses of cetuximab IV 200 mg/m2 followed by 150 mg/m2 weekly, with regorafenib 80 mg daily. Anticancer activity was observed in patients with wild type colorectal cancer. Citation Format: Vivek Subbiah, David S. Hong, Behrang Amini, Sarina Piha-Paul, Joanna Grace Fernandez, Siquing Fu, Apostolia M. Tsimberidou, Aung Naing, Filip Janku, Daniel D. Karp, Michael Overman, Cathy Eng, Scott Kopetz, Funda Meric-Bernstam, Gerald S. Falchook. Phase I dose escalation study of the oral multi-kinase VEGF inhibitor regorafenib and the anti-EGFR monoclonal antibody cetuximab in patients with advanced solid tumors including colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C50.

Precision medicine program for whole-exome sequencing (WES) provides new insight on platinum sensitivity in advanced prostate cancer (PCa).

158 Background: WES has provided insight into the genomic landscape of PCa. The next step for precision medicine requires prospective patient (pt) follow-up and clinical and functional validation of both common and low frequency mutations. We describe a precision medicine WES program and illustrate how this can be used to identify novel biomarkers associated with response. Methods: Metastatic PCa pts were prospectively enrolled. WES of metastatic biopsies or rapid autopsies and normal DNA were sequenced by Illumina HiSeq 2500. Results: Tumor-normal pairs from 71 pts with metastatic PCa (11 hormone naïve, 38 CRPC, 23 NEPC) were sequenced including 3 rapid autopsies, with a biopsy success rate of >95%, avg tumor purity 10-95%, avg coverage 85X. The spectrum of mutations across metastatic PCa and how serial biopsies and rapid autopsies were used to assess heterogeneity and clonal evolution will be presented. WES from a metastatic PCa pt (PM12) with exceptional response to platinum with complete remission of liver and lung metastases at 2 years follow-up, demonstrated a hypermutated genotype and hemizygous deletion of the DNA repair gene FANCA in both his primary and metastatic PCa. A loss of function germline variant was detected within the second allele, and only the mutated allele was expressed in his tumor. Genome editing of FANCA using CRISPR in PCa cell lines resulted in cisplatin hypersensitivity and a significant decrease in FANC complex formation. In patient derived xenograft (PDX) models, PM12’s PDX was significantly more sensitive to cisplatin compared to a control Pca PDX of similar morphology but lacking FANCA deletion. By screening larger cohorts, FANCA loss was detected by FISH in 16% of localized PCa (n= 69), 14% metastatic Pca (n= 29), and not detected in any benign prostate (n=69). Conclusions: Our study provides a proof of principle for developing a precision medicine approach to cancer care with the capability to identify potential biomarkers of response. Our findings suggest that a subset of PCa with FANCA loss may be particularly vulnerable to cytotoxic therapy and provides biologic rationale to help explain the exceptional response of pt PM12 to platinum chemotherapy.

MLH1‐silenced and non‐silenced subgroups of hypermutated colorectal carcinomas have distinct mutational landscapes

Approximately 15% of colorectal carcinomas (CRCs) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumours, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analysed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRCs (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of the hypermutated CRCs exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations, and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRCs lacked MLH1 silencing, contained tumours with the highest mutation rates ('ultramutated' CRCs), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRCs. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRCs with MLH1 silencing had greatly reduced levels of WNT signalling and increased BRAF signalling relative to non-hypermutated CRCs. Our findings suggest that hypermutated CRCs include one subgroup with fundamentally different pathways to malignancy than the majority of CRCs. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.