Sarah E. Kerr

Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases.

Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions

Cholangiocarcinoma is a highly lethal cancer of the biliary tract. The intrahepatic subtype of cholangiocarcinoma is increasing in incidence globally. Despite technologic advancements over the past decade, little is known about the somatic changes that occur in these tumors. The goal of this study was to determine the frequency of common oncogenes in resected cholangiocarcinoma specimens that could provide potential therapeutic targets for patients diagnosed with cholangiocarcinoma. Formalin-fixed, paraffin-embedded tissue blocks from 94 resected cholangiocarcinomas were used to extract DNA from areas comprising more than 20% tumor. Specimens were evaluated using the Sequenom MassARRAY OncoCarta Mutation Profiler Panel (San Diego, CA). This matrix-assisted laser desorption/ionization-time of flight mass spectrometry single genotyping panel evaluates 19 oncogenes for 238 somatic mutations. Twenty-five mutations were identified in 23 of the 94 cholangiocarcinomas within the following oncogenes: KRAS (n = 12), PIK3CA (n = 5), MET (n = 4), EGFR (n = 1), BRAF (n = 2), and NRAS (n = 1). Mutations were identified in 7 (26%) of 27 extrahepatic cholangiocarcinomas and 16 (24%) of 67 intrahepatic cholangiocarcinomas. When combined with IDH1/2 testing, 40 (43%) of the 94 cholangiocarcinomas had a detectable mutation. MassARRAY technology can be used to detect mutations in a wide variety of oncogenes using paraffin-embedded tissue. Clinical testing for somatic mutations may drive personalized therapy selection for cholangiocarcinomas in the future. The variety of mutations detected suggests that a multiplexed mutation detection approach may be necessary for managing patients with biliary tract malignancy.

Molecular characterization of endometrial cancer: a correlative study assessing microsatellite instability, MLH1 hypermethylation, DNA mismatch repair protein expression, and PTEN, PIK3CA, KRAS, and BRAF mutation analysis.

Endometrial cancer is associated with numeric and structural chromosomal abnormalities, microsatellite instability (MSI), and alterations that activate oncogenes and inactivate tumor suppressor genes. The aim of this study was to characterize a set of endometrial cancers using multiple molecular genetic and immunohistochemical techniques. Ninety-six cases were examined for genomic alterations by MSI, MLH1 promoter hypermethylation, p53 and mismatch repair protein expression (MLH1, MSH2, MSH6, PMS2), and PTEN, PIK3CA, KRAS, and BRAF mutation analysis. At least 1 alteration was identified in 48 of 87 (55%) specimens tested for PTEN, making it the most common abnormality in this study. A PIK3CA alteration was observed in 16 (17%) specimens. Twenty-nine of 94 (31%) MSI tested tumors exhibited an MSI-H phenotype. Of the 29 MSI-H cases, 24 (83%) were positive for methylation of the MLH1 promoter region. Twenty-three (82%) of the 28 MSI-H cases with immunohistochemistry results showed loss of expression of MLH1/PMS2 (n=19), MSH2/MSH6 (n=2), or MSH6 only (n=2). Of the 19 MSI-H cases with loss of MLH1/PMS2 on immunohistochemistry, 18 were positive, and 1 was equivocal for MLH1 promoter hypermethylation. Twelve of 94 cases (13%) analyzed for KRAS mutations were found to have a mutation. No BRAF V600E mutations were indentified. This study provides a comprehensive molecular genetic analysis of commonly analyzed targets in a large cohort of endometrial cancers.

Lung cancer adrenal gland metastasis: Optimal fine‐needle aspirate and touch preparation smear cellularity characteristics for successful theranostic next‐generation sequencing

Multigene molecular testing to guide personalized therapy for oncology patients is of increasing clinical relevance. Molecular testing of fine‐needle aspiration samples is underused, but when acquired with minimally invasive techniques could become the standard of care to obtain theranostic specimens. The aims of the current study were to identify key cytology specimen selection criteria suitable for next‐generation sequencing (NGS) and to determine the prevalence and spectrum of pathogenic alterations in a cohort of patients with AJCC stage IV lung cancer.

Multisite Validation Study to Determine Performance Characteristics of a 92-Gene Molecular Cancer Classifier

Purpose: Accurate tumor classification is essential for cancer management as patient outcomes improve with use of site- and subtype-specific therapies. Current clinicopathologic evaluation is varied in approach, yet standardized diagnoses are critical for determining therapy. While gene expression–based cancer classifiers may potentially meet this need, imperative to determining their application to patient care is validation in rigorously designed studies. Here, we examined the performance of a 92-gene molecular classifier in a large multi-institution cohort. Experimental Design: Case selection incorporated specimens from more than 50 subtypes, including a range of tumor grades, metastatic and primary tumors, and limited tissue samples. Formalin-fixed, paraffin-embedded tumors passed pathologist-adjudicated review between three institutions. Tumor classification using a 92-gene quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay was conducted on blinded tumor sections from 790 cases and compared with adjudicated diagnoses. Results: The 92-gene assay showed overall sensitivities of 87% for tumor type [95% confidence interval (CI), 84–89] and 82% for subtype (95% CI, 79–85). Analyses of metastatic tumors, high-grade tumors, or cases with limited tissue showed no decrease in comparative performance (P = 0.16, 0.58, and 0.16). High specificity (96%–100%) was showed for ruling in a primary tumor in organs commonly harboring metastases. The assay incorrectly excluded the adjudicated diagnosis in 5% of cases. Conclusions: The 92-gene assay showed strong performance for accurate molecular classification of a diverse set of tumor histologies. Results support potential use of the assay as a standardized molecular adjunct to routine clinicopathologic evaluation for tumor classification and primary site diagnosis. Clin Cancer Res; 18(14); 3952–60. ©2012 AACR.

Tumor budding in colorectal carcinoma: Confirmation of prognostic significance and histologic cutoff in a population-based cohort

Tumor budding in colorectal carcinoma has been associated with poor outcome in multiple studies, but the absence of an established histologic cutoff for “high” tumor budding, heterogeneity in study populations, and varying methods for assessing tumor budding have hindered widespread incorporation of this parameter in clinical reports. We used an established scoring system in a population-based cohort to determine a histologic cutoff for “high” tumor budding and confirm its prognostic significance. We retrieved hematoxylin and eosin–stained sections from 553 incident colorectal carcinoma cases. Each case was previously characterized for select molecular alterations and survival data. Interobserver agreement was assessed between 2 gastrointestinal pathologists and a group of 4 general surgical pathologists. High budding (≥10 tumor buds in a ×20 objective field) was present in 32% of cases, low budding in 46%, and no budding in 22%. High tumor budding was associated with advanced pathologic stage (P<0.001), microsatellite stability (P=0.005), KRAS mutation (P=0.010), and on multivariate analysis with a >2 times risk of cancer-specific death (hazard ratio=2.57 [1.27, 5.19]). After multivariate adjustment, by penalized smoothing splines, we found increasing tumor bud counts from 5 upward to be associated with an increasingly shortened cancer-specific survival. By this method, a tumor bud count of 10 corresponded to approximately 2.5 times risk of cancer-specific death. The interobserver agreement was good with weighted &kgr; of 0.70 for 2 gastrointestinal pathologists over 121 random cases and 0.72 between all 6 pathologists for 20 random cases. Using an established method to assess budding on routine histologic stains, we have shown that a cutoff of 10 for high tumor budding is independently associated with a significantly worse prognosis. The reproducibility data provide support for the routine widespread implementation of tumor budding in clinical reports.

Targeted next generation sequencing of endoscopic ultrasound acquired cytology from ampullary and pancreatic adenocarcinoma has the potential to aid patient stratification for optimal therapy selection

Background & Aims Less than 10% of registered drug intervention trials for pancreatic ductal adenocarcinoma (PDAC) include a biomarker stratification strategy. The ability to identify distinct mutation subsets via endoscopic ultrasound fine needle aspiration (EUS FNA) molecular cytology could greatly aid clinical trial patient stratification and offer predictive markers. We identified chemotherapy treatment naïve ampullary adenocarcinoma and PDAC patients who underwent EUS FNA to assess multigene mutational frequency and diversity with a surgical resection concordance assessment, where available. Methods Following strict cytology smear screening criteria, targeted next generation sequencing (NGS) using a 160 cancer gene panel was performed. Results Complete sequencing was achieved in 29 patients, whereby 83 pathogenic alterations were identified in 21 genes. Cytology genotyping revealed that the majority of mutations were identified in KRAS (93%), TP53 (72%), SMAD4 (31%), and GNAS (10%). There was 100% concordance for the following pathogenic alterations: KRAS, TP53, SMAD4, KMT2D, NOTCH2, MSH2, RB1, SMARCA4, PPP2R1A, PIK3R1, SCL7A8, ATM, and FANCD2. Absolute multigene mutational concordance was 83%. Incremental cytology smear mutations in GRIN2A, GATA3 and KDM6A were identified despite re-examination of raw sequence reads in the corresponding resection specimens. Conclusions EUS FNA cytology genotyping using a 160 cancer gene NGS panel revealed a broad spectrum of pathogenic alterations. The fidelity of cytology genotyping to that of paired surgical resection specimens suggests that EUS FNA represents a suitable surrogate and may complement the conventional stratification criteria in decision making for therapies and may guide future biomarker driven therapeutic development.

Kinase Genotype Analysis of Gastric Gastrointestinal Stromal Tumor Cytology Samples Using Targeted Next-Generation Sequencing

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Matching maternal isodisomy in mucinous carcinomas and associated ovarian teratomas provides evidence of germ cell derivation for some mucinous ovarian tumors

The tissue derivation of mucinous ovarian carcinoma remains a mystery; however, rare tumors are associated with mature teratoma. Two decades ago, studies of chromosomal heteromorphisms and DNA polymorphisms proved that ovarian teratomas arise during female gametogenesis. We sought to exploit the relationship between mucinous carcinoma and associated teratoma to provide molecular evidence for tissue of origin. Seventeen cases of mucinous ovarian carcinoma were studied, 6 of which had associated mature teratoma. DNA was extracted from the mucinous carcinoma, teratoma, and normal dissected tissue from formalin-fixed, paraffin-embedded sections. Twelve polymorphic microsatellite markers were used to allelotype each sample. Alleles from the teratomas and carcinomas were scored as homozygous (1 allele present in the tumor when normal tissue was heterozygous), heterozygous (2 alleles present matching normal tissue), or noninformative (normal tissue was homozygous). Of the 6 carcinoma/teratoma pairs, 2 showed complete matching homozygosity for informative markers (isodisomy), whereas 2 showed matching heterozygosity. One case did not have the corresponding teratoma available for comparison but demonstrated complete homozygosity and was presumed to be isodisomic. The remaining case had a teratoma homozygous for 7 of 10 informative markers, whereas the matching carcinoma was homozygous for only 2 of these markers. Carcinomas without associated teratoma demonstrated variable zygosity. Microsatellite polymorphism analysis demonstrates that mucinous ovarian carcinomas usually clonally match associated teratomas when present and often show evidence of complete isodisomy, indicating that at least some mucinous carcinomas arise from female gametes and thus are of germ cell origin. The zygosity patterns in mucinous carcinomas without teratoma suggest that these tumors may arise through a different mechanism.

A 92-gene cancer classifier predicts the site of origin for neuroendocrine tumors.

A diagnosis of neuroendocrine carcinoma is often morphologically straight-forward; however, the tumor site of origin may remain elusive in a metastatic presentation. Neuroendocrine tumor subtyping has important implications for staging and patient management. In this study, the novel use and performance of a 92-gene molecular cancer classifier for determination of the site of tumor origin are described in a series of 75 neuroendocrine tumors (44 metastatic, 31 primary; gastrointestinal (n=12), pulmonary (n=22), Merkel cell (n=10), pancreatic (n=10), pheochromocytoma (n=10), and medullary thyroid carcinoma (n=11)). Formalin-fixed, paraffin-embedded samples passing multicenter pathologist adjudication were blinded and tested by a 92-gene molecular assay that predicts tumor type/subtype based upon relative quantitative PCR expression measurements for 87 tumor-related and 5 reference genes. The 92-gene assay demonstrated 99% (74/75; 95% confidence interval (CI) 0.93–0.99) accuracy for classification of neuroendocrine carcinomas and correctly subtyped the tumor site of origin in 95% (71/75; 95% CI 0.87–0.98) of cases. Analysis of gene expression subsignatures within the 92-gene assay panel showed 4 genes with promising discriminatory value for tumor typing and 15 genes for tumor subtyping. The 92-gene classifier demonstrated excellent accuracy for classifying and determining the site of origin in tumors with neuroendocrine differentiation. These results show promise for use of this test to aid in classifying neuroendocrine tumors of indeterminate primary site, particularly in the metastatic setting.