High-grade neuroendocrine neoplasms (World Health Organization WHO G3) of the pancreas include both well-differentiated neuroendocrine tumor (WD-NET) and poorly differentiated neuroendocrine ...carcinoma (PD-NEC). According to the WHO classification scheme, the diagnosis of this group of tumors is based on both the histopathology of the tumor and the assessment of proliferation fraction. However, the former can be challenging due to the lack of well-defined histologic criteria, and the latter alone (ie, >20 mitoses/10 high-power fields or Ki67>20%) may not sufficiently distinguish WD-NETs from PD-NECs. Given the considerable differences in treatment strategies and clinical outcome, additional practical modalities are required to facilitate the accurate diagnosis of high-grade pancreatic neuroendocrine neoplasms. We examined 33 cases of WHO G3 neuroendocrine neoplasms of the pancreas and attempted to classify them into WD-NET, small cell PD-NEC (PD-NEC-SCC), and large cell PD-NEC (PD-NEC-LCC) or to designate them as "ambiguous" when an uncertain diagnosis was rendered by any of the observers or there was any disagreement in classification among the 3 observers. To simplify the interpretation, both PD-NEC-SCC and PD-NEC-LCC were considered together as PD-NECs in the final analysis. The initial approach was to assess microscopically a single morphologically challenging hematoxylin and eosin section from each case without the knowledge of Ki67 values, performed independently by 3 pathologists to assess the degree of diagnostic concordance, and then evaluate immunohistochemical staining for surrogate biomarkers of known genotypes of WD-NET and PD-NEC, respectively, and, lastly, complete a clinicopathologic review to establish a final definitive classification. Loss of DAXX or ATRX protein expression defined WD-NET, and abnormal p53, Rb, SMAD4 expression signified PD-NEC. When the chosen section displayed an element of WD histopathology, or other tumor sections contained WHO G1/G2 components, or there had been a prior established diagnosis of a primary WD-NET, the final diagnosis was rendered as a WD-NET with high-grade (G3) progression. If a component of conventional adenocarcinoma was present (in slides not seen in the initial review), the diagnosis was established as a combined adenocarcinoma and PD-NEC. All 3 pathologists agreed on the morphologic classification of 33% of the cases (6 WD-NET, 3 PD-NEC-SCC, and 2 PD-NEC-LCC), were conflicted on 2 cases between PD-NEC-SCC and PD-NEC-LCC, and disagreed or were uncertain on the classification for the remaining 20 cases (61%), which were therefore categorized as ambiguous. In the group of cases in which all pathologists agreed on the classification, the 6 WD-NET cases had either loss of DAXX or ATRX or had evidence of a WD-NET based on additional or prior pathology slides. The 7 PD-NEC cases had abnormal expression of p53, Rb, and/or SMAD4 or a coexisting adenocarcinoma. In the ambiguous group (n=20), 14 cases were established as WD-NETs, based upon loss of DAXX or ATRX in 7 cases and additional pathology evidence of high-grade progression from WD-NET in the other 7 cases; 5 cases were established as PD-NEC based upon abnormal expression of p53, Rb, and/or SMAD4; 1 case remained undetermined with normal expression of all markers and no evidence of entity-defining histologic findings in other slides. On the basis of the final pathologic classifications, the disease-specific survival was 75 and 11 months for the WD-NET and PD-NEC groups, respectively. Thus, we conclude that morphologic diagnosis of high-grade pancreatic neuroendocrine neoplasms is challenging, especially when limited pathologic materials are available, and necessitates better defined criteria. The analysis of both additional sections and prior material, along with an immunohistochemical evaluation, can facilitate accurate diagnosis in the majority of cases and guide the appropriate clinical management and prognosis.
Pancreatic neuroendocrine tumors (PanNETs) are a rare but clinically important form of pancreatic neoplasia. To explore the genetic basis of PanNETs, we determined the exomic sequences of 10 ...nonfamilial PanNETs and then screened the most commonly mutated genes in 58 additional PanNETs. The most frequently mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1, which encodes menin, a component of a histone methyltransferase complex, and 43% had mutations in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain-associated protein) and ATRX (α thalassemia/mental retardation syndrome X-linked). Clinically, mutations in the MEN1 and DAXX/ATRX genes were associated with better prognosis. We also found mutations in genes in the mTOR (mammalian target of rapamycin) pathway in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors.
The 2010 World Health Organization (WHO) classification recommends that pancreatic neuroendocrine tumors (PanNETs) be graded on the basis of the mitotic rate and Ki67 index, with grade 2 (G2) PanNETs ...defined as having a mitotic rate of 2 to 20 mitotic figures/10 high-power fields or a Ki67 index of 3% to 20%. Grade 3 (G3) pancreatic neuroendocrine carcinoma (NEC) is defined as having >20 mitotic figures/10 high-power fields or a Ki67 index of >20%. However, some PanNETs show discordance between the mitotic rate and Ki67 index, usually having a Ki67 index in the G3 range but a mitotic rate suggesting G2, prompting us to examine the clinical significance of the Ki67 index in a large series of clinically well-characterized mitotic G2 PanNETs. Mitotic G2 well differentiated PanNETs, surgically resected at our institutions were reviewed. Of those, 19 cases had a Ki67>20% and were selected as the study group of grade-discordant (mitotic count G2/Ki67 index G3) PanNETs. For comparison, 53 grade-concordant (both mitotic count and Ki67 index G2) PanNETs matched for presenting stage with the discordant group as well as 43 morphologically poorly differentiated (either small cell or large cell type) pancreatic NECs were also included. The percentage of Ki67-positive neoplastic cells was quantified by manual counting of at least 500 cells on printed photographic images of "hot spots." The mean Ki67 index for grade-concordant and grade-discordant PanNETs and poorly differentiated NECs were 8.1% (range, 3% to 20%), 40% (range, 24% to 80%), and 70% (range, 40% to 98%), respectively. Overall, patients with grade-discordant PanNETs had significantly longer survival time compared with the patients with poorly differentiated NEC (median survival of 54.1 vs. 11 mo and 5 y survival of 29.1% vs. 16.1%; P=0.002). In addition, the survival time of the patients with grade-discordant PanNETs was shorter than that of the patients with grade-concordant PanNETs (median survival of 67.8 mo and 5 y survival of 62.4%); however, the difference was not statistically significant (P=0.2). Our data support the notion that the mitotic rate and Ki67 index-based grades of PanNETs can be discordant, and when the Ki67 index indicates G3, the clinical outcome is slightly worse. More importantly, we demonstrate that well differentiated PanNETs that are G3 by Ki67 are significantly less aggressive than bona fide poorly differentiated NECs, suggesting that the current WHO G3 category is heterogenous, contains 2 distinct neoplasms, and can be further separated into well differentiated PanNET with an elevated proliferation rate and poorly differentiated NEC.
Since the initial description of pancreatic endocrine physiology and the recognition of islet cell tumors in the 1800s, there have been noteworthy advances in the pathobiology of pancreatic ...neuroendocrine neoplasms (PanNENs), and definition of the important distinction between well-differentiated neuroendocrine tumor (PanNET) and poorly differentiated neuroendocrine carcinoma (PanNEC). The evolving knowledge has resulted in a continuous update in terminology, classification, and grading system for this group of neoplasms. Pancreatic neuroendocrine tumors associated with hereditary conditions have been linked to unique molecular and genetic events, and sporadic PanNETs have specific gene signatures. Based on accumulative experience and knowledge, therapeutic strategies have been defined for this group of neoplasms.
To review the evolution and description of the pathologic-genomic evolution of PanNENs, and to facilitate accurate pathologic interpretation for the corresponding clinical management.
Literature review of published studies and author's own work.
Evolving experience and knowledge have established subtypes of pancreatic neuroendocrine neoplasms, based on their genotype and phenotype. Accurate pathologic interpretation of the specific neoplasm has significant implications for therapy and prognosis.
The Ki67 labeling index is known to correlate with survival in patients with neuroendocrine tumors (NETs). A grading scheme recently endorsed by the World Health Organization for ...gastroenteropancreatic NETs classifies well-differentiated NETs into 2 categories based on the Ki67 labeling index: low (G1) and intermediate grades (G2). Tumor heterogeneity is a common finding in many tumors including NETs. Metastatic NETs to the liver are often diagnosed by radiographically guided needle core biopsy from which the Ki67 index is determined, which randomly samples the lesion without being targeted to regions that may show a higher proliferative rate. Whether the Ki67 index obtained from this type of limited material represents the whole tumor has been questioned. Forty-five surgically resected liver metastases of well-differentiated NETs were retrieved. A 9 core (3 core-triplets) tissue microarray (TMA) was constructed from the paraffin blocks of each tumor, each triplet considered to represent a virtual biopsy. Immunohistochemical staining for Ki67 was performed on TMA and whole slides, and the Ki67 labeling indices were determined by digital image analysis. Correlation of the Ki67 index with patient survival was analyzed. Forty-seven percent of cases showed intratumoral heterogeneity in Ki67 index that translated into discrepant grades among subsections on the whole slide. A similar trend was recapitulated on the virtual biopsies, although to a lesser degree. When the definitive grade of the tumor was based on the highest Ki67 index identified on the whole slide, the virtual biopsies perfectly predicted G1 cases (100%), but were much less accurate for G2 cases (47.8% with 3 biopsies and 34.8% with single biopsy). Accordingly, the predictive value for G1 on the virtual biopsies was low (64.7% and 59.5% for 3 and 1 biopsy, respectively), but was perfect for G2 (100%). By Kaplan-Meier survival analysis, there was a statistically significant difference between G1 and G2 in terms of overall survival, disease-free survival, and progression-free survival when graded on either whole-slide subsections or virtual biopsies. On the whole slides, the highest Ki67 grade showed a better correlation with overall survival than the mean Ki67 grade. In summary, by image analysis, we found that about half of the NETs metastatic to the liver show intratumoral heterogeneity resulting in discrepant Ki67 grade. In most cases, in particular G1, the virtual biopsy is representative of the whole slide, but for G2 the representation is <50%. Nevertheless, grades based on virtual biopsy had statistically significant prognostic values on patient survival, and there is no clear difference between the 3 and single virtual biopsy. Ki67 staining of core biopsies usually provides an adequately reliable method of proliferation assessment for prognosis of metastatic NETs to the liver, although the choice of treatment may be affected by intratumoral grade heterogeneity.
The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) ...and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.
Immune checkpoint inhibition is effective in a subset of patients with advanced gastric cancer. Genomic profiling has revealed the heterogeneity of gastric adenocarcinomas, but the immune ...microenvironment and predictors of immunotherapy response remain poorly understood. We aimed to better characterize the underlying immune response to gastric cancer. Retrospective review of a prospectively maintained institutional database was performed to identify patients who underwent curative intent resection of gastric adenocarcinoma from 2006 to 2016. Tumors were classified according to modified TCGA subtype: Epstein-Barr virus (EBV)-associated, microsatellite instability (MSI)-high, intestinal as a surrogate for chromosomal instability, diffuse as a surrogate for genomically stable. Tumor-infiltrating leukocytes were measured using immunohistochemistry. Forty-three patients were identified: 6 EBV, 11 MSI, 14 intestinal, 12 diffuse. The most prevalent tumor-infiltrating leukocytes were CD8 T lymphocytes and CD68 macrophages, comprising 15% and 13% of all tumor cells. EBV and MSI tumors were the most infiltrated, harboring 30% to 50% T cells and 20% macrophages. Intestinal tumors contained fewer T cells but disproportionately more macrophages. Diffuse tumors were the least infiltrated. Programmed cell death protein 1 was most frequently expressed in intestinal tumors, whereas 70% of EBV and MSI tumors expressed programmed death-ligand 1. We herein demonstrate a heterogenous immune response to gastric cancer, which varies by tumor subtype and has implications for future immunotherapy trials. Checkpoint inhibition is unlikely to be effective as single-agent therapy against intestinal and diffuse tumors lacking prominent T-cell infiltration or substantial programmed death-ligand 1 expression.
Remote digital pathology allows healthcare systems to maintain pathology operations during public health emergencies. Existing Clinical Laboratory Improvement Amendments regulations require ...pathologists to electronically verify patient reports from a certified facility. During the 2019 pandemic of COVID-19 disease, caused by the SAR-CoV-2 virus, this requirement potentially exposes pathologists, their colleagues, and household members to the risk of becoming infected. Relaxation of government enforcement of this regulation allows pathologists to review and report pathology specimens from a remote, non-CLIA certified facility. The availability of digital pathology systems can facilitate remote microscopic diagnosis, although formal comprehensive (case-based) validation of remote digital diagnosis has not been reported. All glass slides representing routine clinical signout workload in surgical pathology subspecialties at Memorial Sloan Kettering Cancer Center were scanned on an Aperio GT450 at ×40 equivalent resolution (0.26 µm/pixel). Twelve pathologists from nine surgical pathology subspecialties remotely reviewed and reported complete pathology cases using a digital pathology system from a non-CLIA certified facility through a secure connection. Whole slide images were integrated to and launched within the laboratory information system to a custom vendor-agnostic, whole slide image viewer. Remote signouts utilized consumer-grade computers and monitors (monitor size, 13.3–42 in.; resolution, 1280 × 800–3840 × 2160 pixels) connecting to an institution clinical workstation via secure virtual private network. Pathologists subsequently reviewed all corresponding glass slides using a light microscope within the CLIA-certified department. Intraobserver concordance metrics included reporting elements of top-line diagnosis, margin status, lymphovascular and/or perineural invasion, pathology stage, and ancillary testing. The median whole slide image file size was 1.3 GB; scan time/slide averaged 90 s; and scanned tissue area averaged 612 mm2. Signout sessions included a total of 108 cases, comprised of 254 individual parts and 1196 slides. Major diagnostic equivalency was 100% between digital and glass slide diagnoses; and overall concordance was 98.8% (251/254). This study reports validation of primary diagnostic review and reporting of complete pathology cases from a remote site during a public health emergency. Our experience shows high (100%) intraobserver digital to glass slide major diagnostic concordance when reporting from a remote site. This randomized, prospective study successfully validated remote use of a digital pathology system including operational feasibility supporting remote review and reporting of pathology specimens, and evaluation of remote access performance and usability for remote signout.
Poorly differentiated neuroendocrine carcinomas (NECs) of the pancreas are rare malignant neoplasms with a poor prognosis. The aim of this study was to determine the clinicopathologic and genetic ...features of poorly differentiated NECs and compare them with other types of pancreatic neoplasms. We investigated alterations of KRAS, CDKN2A/p16, TP53, SMAD4/DPC4, DAXX, ATRX, PTEN, Bcl2, and RB1 by immunohistochemistry and/or targeted exomic sequencing in surgically resected specimens of 9 small cell NECs, 10 large cell NECs, and 11 well-differentiated neuroendocrine tumors (PanNETs) of the pancreas. Abnormal immunolabeling patterns of p53 and Rb were frequent (p53, 18 of 19, 95%; Rb, 14 of 19, 74%) in both small cell and large cell NECs, whereas Smad4/Dpc4, DAXX, and ATRX labeling was intact in virtually all of these same carcinomas. Abnormal immunolabeling of p53 and Rb proteins correlated with intragenic mutations in the TP53 and RB1 genes. In contrast, DAXX and ATRX labeling was lost in 45% of PanNETs, whereas p53 and Rb immunolabeling was intact in these same cases. Overexpression of Bcl-2 protein was observed in all 9 small cell NECs (100%) and in 5 of 10 (50%) large cell NECs compared with only 2 of 11 (18%) PanNETs. Bcl-2 overexpression was significantly correlated with higher mitotic rate and Ki67 labeling index in neoplasms in which it was present. Small cell NECs are genetically similar to large cell NECs, and these genetic changes are distinct from those reported in PanNETs. The finding of Bcl-2 overexpression in poorly differentiated NECs, particularly small cell NEC, suggests that Bcl-2 antagonists/inhibitors may be a viable treatment option for these patients.
Most well-differentiated neuroendocrine tumors (WD-NET) of the enteropancreatic system are low-intermediate grade (G1, G2). Elevated proliferation demonstrated by either a brisk mitotic rate (>20/10 ...high power fields) or high Ki-67 index (>20%) defines a group of aggressive neoplasms designated as high-grade (G3) neuroendocrine carcinoma (NEC). High-grade NEC is equated with poorly differentiated NEC (PD-NEC) and is associated with a dismal outcome. Progression of WD-NETs to a high-grade neuroendocrine neoplasm very rarely occurs and their clinicopathologic and molecular features need to be characterized.
We investigated 31 cases of WD-NETs with evidence of a component of a high-grade neoplasm. The primary sites included pancreas, small bowel, bile duct, and rectum. Histopathology of the cases was retrospectively reviewed and selected IHC and gene mutation analyses performed.
The high-grade component occurred either within the primary tumor (48%) or at metastatic sites (52%). The clinical presentation, radiographic features, biomarkers, and the genotype of these WD-NETs with high-grade component remained akin to those of G1-G2 WD-NETs. The median disease-specific survival (DSS) was 55 months (16-119 months), and 2-year and 5-year DSS was 88% and 49%, respectively-significantly better than that of a comparison group of true PD-NEC (DSS 11 months).
Mixed grades can occur in WD-NETs, which are distinguished from PD-NECs by their unique phenotype, proliferative indices, and the genotype. This phenomenon of mixed grade in WD-NET provides additional evidence to the growing recognition that the current WHO G3 category contains both WD-NETs as well as PD-NECs.
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