Molecular Classification Outperforms Histologic Classification in Prognostication of High-grade Endometrial Carcinomas With Undifferentiated and Sarcomatous Components.

Since the establishment of 4 molecular subgroups of endometrial carcinoma (EC), there has been significant interest in understanding molecular classification in the context of histologic features and diagnoses. ECs with undifferentiated, spindle, and/or sarcomatous components represent a diagnostically challenging subset of tumors with overlapping clinical and histologic features. We examined the clinicopathologic, morphologic, immunohistochemical, and molecular features of these tumors identified in our institutions' pathology databases using immunohistochemistry and targeted sequencing. Disease-specific survival (DSS) and progression-free survival (PFS) were analyzed using Kaplan-Meier curves and log-rank tests. One hundred sixty-two ECs were included: carcinosarcomas (UCS; n=96), dedifferentiated/undifferentiated EC (DDEC/UDEC; n=49), and grade 3 endometrioid EC with spindled growth (GR3spEEC) (n=17). All molecular subgroups were represented in all histologic subtypes and included 12 (7%) POLE-mutated (POLEmut), 43 (27%) mismatch repair-deficient (MMRd), 77 (48%) p53-abnormal (p53abn), and 30 (19%) no specific molecular profile (NSMP) tumors. However, the molecular classification (irrespective of histologic diagnosis) was a significant predictor for both DSS (P=0.008) and P≤0.0001). POLEmut EC showed an excellent prognosis with no recurrences or deaths from the disease. MMRd tumors also showed better outcomes relative to NSMP and p53abn tumors. In conclusion, molecular classification provides better prognostic information than histologic diagnosis for high-grade EC with undifferentiated and sarcomatous components. Our study strongly supports routine molecular classification of these tumors, with emphasis on molecular group, rather than histologic subtyping, in providing prognostication.

Extraskeletal Ewing Sarcoma of the Gastrointestinal and Hepatobiliary Tract: Deceptive Immunophenotype Commonly Leads to Misdiagnosis.

Ewing sarcoma (ES) is an uncommon mesenchymal neoplasm that typically develops as a bone mass, although up to 30% arise in extraskeletal sites. ES of the gastrointestinal (GI) and hepatobiliary tract is rare and may be misdiagnosed as other, more common neoplasms that occur in these sites. However, the correct classification of extraskeletal ES is important for timely clinical management and prognostication. We reviewed our experience of ES in the GI and hepatobiliary tract in order to further highlight the clinicopathologic features of these neoplasms and document the potential for misdiagnosis in this setting. The archives and consultation files of 6 academic institutions were retrospectively queried for cases of ES occurring in the GI and hepatobiliary tract. The histologic slides and ancillary studies were reviewed and clinical data were retrieved for each case through the electronic medical records, when available. Twenty-three patients with ES in the GI and/or hepatobiliary tract were identified from 2000 to 2022. Of these, 11 were women and 12 were men with a median age of 38 years (range, 2 to 64). Tumor locations included the pancreas (n=5), liver (n=2), stomach (n=3), colorectum (n=3), and small intestine (n=5), as well as tumors involving multiple organs, pelvis and retroperitoneum (n=5). Tumor size varied between 2 cm and 18 cm. Twenty were primary and 3 were metastases. Of the 23 cases, only 17% were initially diagnosed as ES. The most common misdiagnoses involved various forms of neuroendocrine neoplasia due to expression of synaptophysin and other neuroendocrine markers (22%). A wide variety of diagnoses including GI stromal tumor was considered due to aberrant CD117 expression (4%). The diagnosis of ES was ultimately confirmed by detection of the EWSR1 rearrangement in 22 cases. The remaining case was diagnosed using traditional immunohistochemistry. Follow-up information was available in 20 cases, with follow-up time varying between 2 and 256 months. Six patients with follow-up died of disease between 6 and 60 months following initial presentation. Our data indicate ES in the GI and hepatobiliary tract is commonly misdiagnosed leading to a delay in therapy. In light of the attendant therapeutic and prognostic implications, ES should be considered in the differential diagnosis of any GI or hepatobiliary tumor with epithelioid and/or small round cell morphology.

Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

Specific Pathology Features Enrich Selection of Endometrial Carcinomas for POLE Testing.

Identification of ultramutated/ POLE -mutated endometrial carcinomas ( POLEM ECs) has important implications given its association with better prognosis. However, POLE mutation testing is not widely available. Our objective was to evaluate POLEM ECs versus POLE wild-type ( POLEWT ) ECs, within a cohort of consultation cases with features suggestive of an ultramutated phenotype. Consultation cases of EC that had undergone POLE hotspot mutation testing over a 3.5-year period were included. Tumor morphology and immunohistochemistry were reviewed for both groups. Chi-square test and t test were used for statistical analysis. Of 25 consultation cases, 12 harbored a POLE mutation (48%) and 13 were wild-type (52%). Patients with POLEM ECs were younger (59 vs. 71.3 y; P =0.01). Ambiguous histomorphology (5/12 vs. 1/13; P =0.04) and the presence of more than rare bizarre nuclei (8/12 vs. 2/12; P =0.01) differed significantly between POLEM and POLEWT ECs, respectively. In the POLEM group, one case (1/12) demonstrated PMS2 loss, and one (1/12) showed subclonal MLH1/PMS2 loss. Among POLEWT ECs, 3/13 (23%) showed MLH1/PMS2 loss. p53 was subclonally overexpressed in 4/10 POLEM and 1/13 POLEWT cases ( P =0.06). Mutant p53 patterns were seen in 1/10 POLEM versus 6/13 of POLEWT ECs, respectively ( P =0.06). Within our cohort, the specificity of ambiguous histomorphology, bizarre nuclei, subclonal biomarker expression, and marked tumor-infiltrating lymphocytes for POLEM EC was 83%, 80%, 80%, and 71%, respectively. Where universal POLE testing is not available, these data suggest that morphologic screening (particularly ambiguous histomorphology and the presence of more than rare bizarre nuclei) can be useful for selective enrichment of ECs for POLE testing.

Synovial Sarcoma of the Gastrointestinal Tract.

We report the clinicopathologic and immunohistochemical features of 18 cases of confirmed primary synovial sarcoma of the gastrointestinal tract. The neoplasms arose in 10 women and 8 men ranging in age from 23 to 81 years (mean: 50; median: 57.5 years). The tumors for which size was known ranged from 1.8 to 15.0 cm (mean: 5.2; median: 5.1 cm). Microscopically, 14 synovial sarcomas were of the monophasic type, 2 were biphasic, and 2 were poorly differentiated. Immunohistochemical analysis of 4 cases showed strong, diffuse staining for SS18::SSX (4/4 cases). Pancytokeratin and EMA immunohistochemistry were performed on 13 and 9 tumors, respectively, and each showed patchy-to-diffuse staining. By reverse-transcription PCR, 3 cases were positive for the SS18::SSX1, and 2 cases were positive for the SS18::SSX2 gene fusion. Six cases contained an SS18 gene rearrangement by fluorescence in situ hybridization, and next-generation sequencing identified an SS18::SSX2 gene fusion in one case. Clinical follow-up information was available for 9 patients (4 months to 4.6 years; mean, 2.8 y; median: 29 months), and one patient had a recent diagnosis. Three patients died of disease within 41 to 72 months (mean, 56 months) of their diagnosis. Five patients were alive without evidence of disease 4 to 52 months (mean, 17.6 months) after surgery; of whom 1 of the patients received additional chemotherapy treatment after surgery because of recurrence of the disease. A single patient was alive with intraabdominal recurrence 13 months after surgery. We conclude that synovial sarcoma of the gastrointestinal tract is an aggressive tumor, similar to its soft tissue counterpart, with adverse patient outcomes. It is important to distinguish it from morphologically similar gastrointestinal tract lesions that may have different treatment regimens and prognoses.

S100 Protein Expression in Primary and Metastatic Neuroendocrine Neoplasms: A Specific Marker of Pancreatic Origin.

Neuroendocrine neoplasms can arise in a wide variety of anatomic sites including the gastrointestinal tract, pancreas, and lung, among others. Here, we report on the expression of S100 protein in a tissue microarray composed of 919 distinct primary and metastatic neuroendocrine neoplasms from 548 patients. S100 protein is a commonly used marker in many laboratories for the identification of neural and melanocytic neoplasms and occasionally used in the workup for neuroendocrine neoplasms when the diagnosis of paraganglioma is being considered. We show that strong S100 protein expression is highly specific to well-differentiated neuroendocrine tumors of pancreatic origin. This finding suggests potential diagnostic utility of this marker in cases of tumors of unknown origin, and emphasizes that S100 protein expression should not be an unexpected finding in neuroendocrine tumors of pancreatic origin.

Pan-TRK immunohistochemistry in gynaecological mesenchymal tumours: diagnostic implications and pitfalls.

AIMS: NTRK-rearranged sarcomas of the female genital tract mainly occur in the uterus (more commonly cervix than corpus) and are characterized by a "fibrosarcoma-like" morphology and NTRK gene rearrangements. These neoplasms may exhibit histological overlap with other entities and can present diagnostic difficulties without molecular confirmation. Pan-TRK immunohistochemistry was developed to identify tumours harbouring NTRK rearrangements. The aim of this study was to characterize pan-TRK immunohistochemical expression in a large cohort of gynaecological mesenchymal neoplasms and investigate the utility of pan-TRK immunohistochemistry to distinguish NTRK-rearranged sarcoma from its mimics. METHODS AND RESULTS: A total of 473 gynaecological mesenchymal tumours (461 without known NTRK fusions and 12 NTRK-rearranged sarcomas) were selected. Pan-TRK immunohistochemistry (EPR17341, Abcam) was performed on whole tissue sections and tissue microarrays. Molecular interrogation of pan-TRK positive tumours was performed by RNA sequencing or fluorescence in situ hybridization (FISH). Of the 12 NTRK-rearranged sarcomas, 11 (92%) exhibited diffuse (≥70%) cytoplasmic pan-TRK staining with moderate/marked intensity, while the other was negative. Eleven (2.4%) additional tumours also exhibited pan-TRK immunohistochemical expression: three low-grade endometrial stromal sarcomas, seven high-grade endometrial stromal sarcomas, and an undifferentiated uterine sarcoma. Molecular confirmation of the absence of NTRK rearrangements was possible in nine of these tumours. Of these nine neoplasms, seven exhibited focal/multifocal (<70%) pan-TRK cytoplasmic staining with weak/moderate intensity. CONCLUSION: Even though pan-TRK immunohistochemical expression is not entirely sensitive or specific for NTRK-rearranged sarcomas, these neoplasms tend to exhibit diffuse staining of moderate/strong intensity, unlike its mimics. Pan-TRK should be performed in monomorphic uterine (corpus and cervix) spindle cell neoplasms that are negative for smooth muscle markers and hormone receptors and positive for CD34 and/ or S100. Ultimately, the diagnosis requires molecular confirmation.

SMARCA4/BRG1-deficient Uterine Neoplasm With Hybrid Adenosarcoma and Carcinoma Features: Expanding the Molecular-morphologic Spectrum of SMARCA4-driven Gynecologic Malignancies.

SMARCA4 gene encodes BRG1, a member of the SWItch/sucrose non-fermentable protein family involved in epigenetic transcriptional regulation of important cellular processes. In the uterine corpus, SMARCA4/BRG1 deficiency is associated with a novel class of undifferentiated uterine sarcomas, characterized by younger age onset, rhabdoid histology, focal phyllodiform architecture, high-risk pathologic findings, and dismal prognosis. Herein, we report a case of a 34-year-old Asian woman with a SMARCA4/BRG1-deficient uterine tumor fulfilling the clinicopathologic features of an undifferentiated uterine sarcoma. However, the tumor exhibited several unique features that have not been previously emphasized, including (1) conspicuous phyllodiform architecture recapitulating conventional adenosarcoma, (2) rhabdoid tumor cells forming cords and keratin-positive cohesive epithelial islands, and (3) cooccurrence with a spatially distinct and discrete endometrial complex atypical hyperplasia from the rest of the proliferation. By immunohistochemistry, the tumor cells were diffusely positive for synaptophysin, whereas BRG1 was lost. Pertinent molecular findings included frameshift mutations in the SMARCA4 gene, mutations in histone modification and chromatin remodeling genes, including KMT2C, ARID1B, KAT6A, and NCOR1, and mutations in Wnt signaling involving APC and CTNNB1. Copy number gain in MDM2 and CDK4 were also identified. The tumor mutation burden was intermediate (6.8/MB) and it was microsatellite stable. On balance, our case exhibited morphologic and molecular features that overlap with (1) an undifferentiated uterine sarcoma, (2) an adenosarcoma with sarcomatous overgrowth, and (3) a mixed adenosarcoma and undifferentiated endometrial carcinoma. These hybrid features further expand the molecular-morphologic spectrum of SMARCA4/BRG1-deficient uterine neoplasms.

Desmoplastic stromal signatures predict patient outcomes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death. Hallmarks include desmoplasia with variable extracellular matrix (ECM) architecture and a complex microenvironment with spatially defined tumor, stromal, and immune populations. Nevertheless, the role of desmoplastic spatial organization in patient/tumor variability remains underexplored, which we elucidate using two technologies. First, we quantify ECM patterning in 437 patients, revealing architectures associated with disease-free and overall survival. Second, we spatially profile the cellular milieu of 78 specimens using codetection by indexing, identifying an axis of pro-inflammatory cell interactions predictive of poorer outcomes. We discover that clinical characteristics, including neoadjuvant chemotherapy status, tumor stage, and ECM architecture, correlate with differential stromal-immune organization, including fibroblast subtypes with distinct niches. Lastly, we define unified signatures that predict survival with areas under the receiver operating characteristic curve (AUCs) of 0.872-0.903, differentiating survivorship by 655 days. Overall, our findings establish matrix ultrastructural and cellular organizations of fibrosis linked to poorer outcomes.

In Vivo Ultrasound Molecular Imaging in the Evaluation of Complex Ovarian Masses: A Practical Guide to Correlation with Ex Vivo Immunohistochemistry.

Ovarian cancer is the fifth leading cause of cancer-related deaths in women and the most lethal gynecologic cancer. It is curable when discovered at an early stage, but usually remains asymptomatic until advanced stages. It is crucial to diagnose the disease before it metastasizes to distant organs for optimal patient management. Conventional transvaginal ultrasound imaging offers limited sensitivity and specificity in the ovarian cancer detection. With molecularly targeted ligands addressing targets, such as kinase insert domain receptor (KDR), attached to contrast microbubbles, ultrasound molecular imaging (USMI) can be used to detect, characterize and monitor ovarian cancer at a molecular level. In this article, the authors propose a standardized protocol is proposed for the accurate correlation between in- vivo transvaginal KDR-targeted USMI and ex vivo histology and immunohistochemistry in clinical translational studies. The detailed procedures of in vivo USMI and ex vivo immunohistochemistry are described for four molecular markers, CD31 and KDR with a focus on how to enable the accurate correlation between in vivo imaging findings and ex vivo expression of the molecular markers, even if not the entire tumor could can be imaged by USMI, which is not an uncommon scenario in clinical translational studies. This work aims to enhance the workflow and the accuracy of characterization of ovarian masses on transvaginal USMI using histology and immunohistochemistry as reference standards, which involves sonographers, radiologists, surgeons, and pathologists in a highly collaborative research effort of USMI in cancer.

The Evolving Spectrum of Endometrial Glandular Proliferations With Corded and Hyalinized Features.

We present the clinicopathologic and immunohistochemical features of 14 endometrial glandular proliferations with conspicuous corded and hyalinized (CH) features comprised entirely or predominantly of endometrial hyperplasia. Endometrial glandular lesions ranged in severity from endometrial hyperplasia with and without cytologic atypia (5/14 [36%]) to hyperplasia with architectural complexity bordering on well-differentiated endometrioid adenocarcinoma (3/14 [21%]) to frank corded and hyalinized endometrial carcinoma ("CHEC") (6/14 [43%]). In addition to sex cord-like growth and hyalinized stroma, other common histologic features included prominent spindle cells (11/14 [79%]), keratinizing and/or morular squamous differentiation (10/14 [71%]), and osseous metaplasia (6/14 [43%]). Immunohistochemical characterization revealed aberrant nuclear beta-catenin in all cases (14/14 [100%]); additionally, all cases demonstrated positive estrogen receptors, intact PTEN, PMS2 and MSH6, and wild-type p53 expression. Patients ranged in age from 24 to 58 (mean 38) years. Of 5 patients with hyperplasia with CH features, 2 experienced complete resolution after progestin therapy and none progressed to adenocarcinoma (mean follow-up 15.6 mo, range 2 to 64). By contrast, of 2 patients with hyperplasia bordering on CHEC and with available follow-up, both subsequently developed adenocarcinoma, suggesting that even focal increased architectural complexity may predict an elevated risk of malignancy. We conclude that CH morphology is not limited to endometrioid carcinoma and may occur across a spectrum of neoplastic proliferations, including those without sufficient architectural complexity or cytologic atypia to warrant classification as adenocarcinoma. We propose the term "corded and hyalinized endometrial hyperplasia" to describe this precursor lesion and report favorable outcomes with conservative treatment.

Segmentation of human functional tissue units in support of a Human Reference Atlas.

The Human BioMolecular Atlas Program (HuBMAP) aims to compile a Human Reference Atlas (HRA) for the healthy adult body at the cellular level. Functional tissue units (FTUs), relevant for HRA construction, are of pathobiological significance. Manual segmentation of FTUs does not scale; highly accurate and performant, open-source machine-learning algorithms are needed. We designed and hosted a Kaggle competition that focused on development of such algorithms and 1200 teams from 60 countries participated. We present the competition outcomes and an expanded analysis of the winning algorithms on additional kidney and colon tissue data, and conduct a pilot study to understand spatial location and density of FTUs across the kidney. The top algorithm from the competition, Tom, outperforms other algorithms in the expanded study, while using fewer computational resources. Tom was added to the HuBMAP infrastructure to run kidney FTU segmentation at scale-showcasing the value of Kaggle competitions for advancing research.

Organization of the human intestine at single-cell resolution.

The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.

Randomized Study of Temozolomide or Temozolomide and Capecitabine in Patients With Advanced Pancreatic Neuroendocrine Tumors (ECOG-ACRIN E2211).

PURPOSE: Patients with advanced pancreatic neuroendocrine tumors (NETs) have few treatment options that yield objective responses. Retrospective and small prospective studies suggest that capecitabine and temozolomide are associated with high response rates (RRs) and long progression-free survival (PFS). PATIENTS AND METHODS: E2211 was a multicenter, randomized, phase II trial comparing temozolomide versus capecitabine/temozolomide in patients with advanced low-grade or intermediate-grade pancreatic NETs. Key eligibility criteria included progression within the preceding 12 months and no prior temozolomide, dimethyl-triazeno-imidazole-carboxamide or dacarbazine, capecitabine or fluorouracil. The primary end point was PFS; secondary endpoints were overall survival, RR, safety, and methylguanine methyltransferase (MGMT) by immunohistochemistry and promoter methylation. RESULTS: A total of 144 patients were enrolled between April 2013 and March 2016 to temozolomide (n = 72) or capecitabine and temozolomide (n = 72); the primary analysis population included 133 eligible patients. At the scheduled interim analysis in January 2018, the median PFS was 14.4 months for temozolomide versus 22.7 months for capecitabine/temozolomide (hazard ratio = 0.58), which was sufficient to reject the null hypothesis for the primary end point (stratified log-rank P = .022). In the final analysis (May 2021), the median overall survival was 53.8 months for temozolomide and 58.7 months for capecitabine/temozolomide (hazard ratio = 0.82, P = .42). MGMT deficiency was associated with response. CONCLUSION: The combination of capecitabine/temozolomide was associated with a significant improvement in PFS compared with temozolomide alone in patients with advanced pancreatic NETs. The median PFS and RR observed with capecitabine/temozolomide are the highest reported in a randomized study for pancreatic NETs. MGMT deficiency was associated with response, and although routine MGMT testing is not recommended, it can be considered for select patients in need of objective response (ClinicalTrials.gov identifier: NCT01824875).

DICER1-associated Tumors in the Female Genital Tract: Molecular Basis, Clinicopathologic Features, and Differential Diagnosis.

DICER1 syndrome is a tumor predisposition syndrome in which patients are at an increased risk of developing a wide variety of benign and malignant neoplasms with a hallmark constellation of pediatric pleuropulmonary blastoma, cystic nephroma, and thyroid lesions. DICER1 encodes an RNA endoribonuclease that is crucial to the processing of microRNA and may play a role in the maturation of Müllerian tissue. Within the gynecologic tract, germline mutations in DICER1 are associated with an array of rare tumors, including Sertoli-Leydig cell tumor, embryonal rhabdomyosarcoma of the cervix, gynandroblastoma, and juvenile granulosa cell tumor, which typically present in childhood, adolescence, or early adulthood. In addition, somatic DICER1 mutations have been described in rare gynecologic tumors such as adenosarcoma, Sertoli cell tumor, ovarian fibrosarcoma, cervical primitive neuroectodermal tumor, carcinosarcoma, and germ cell tumors. In light of the significant association with multiple neoplasms, genetic counseling should be considered for patients who present with a personal or family history of these rare DICER1-associated gynecologic tumors. This review highlights the most current understanding of DICER1 genetic alterations and describes the clinical, histopathologic, and immunohistochemical features and differential diagnoses for gynecologic tumors associated with DICER1 mutation.

Single-cell analyses define a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer.

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts. In the cancerous state, we observe T cell exhaustion, RUNX1-regulated cancer-associated fibroblasts and increasing accessibility associated with HNF4A motifs in epithelia. DNA methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

Percent Agreement Between Immunohistochemistry and Next-Generation Sequencing in Testing Patients for Mismatch Repair Deficiency.

The presence of mismatch repair deficiency is frequently assessed in gastrointestinal and gynecologic neoplasms by surgical pathologists using immunohistochemical methods. Targeted next-generation sequencing (NGS) covering some genes in the mismatch repair complex is used with increasing frequency, however, the percent positive and negative agreement of immunohistochemical methods and NGS of mismatch repair genes is not well-described in the literature. We sought to compare performance of immunohistochemistry (IHC) and NGS of mismatch repair genes on our institutional targeted panel. We evaluated the concordance of immunohistochemical and panel-based gene sequencing methods in a retrospective cohort study of patients evaluated at our center with both immunohistochemical and panel-based sequencing. Our NGS panel covers only MLH1 and MSH2, whereas our immunohistochemical panel assesses for expression of MLH1, PMS2, MSH2, and MSH6. We identified 68 unique patients with both immunohistochemical evaluation of mismatch repair protein expression and NGS panel sequencing, of which 67 were suitable for analysis given the patterns of immunohistochemical loss of expression observed. The percent positive agreement for NGS with IHC was 50%, albeit with very rare positive cases (n=2/4). Percent negative agreement was also high at 100% (n=63/63). One case with loss of MLH1, PMS2, and MSH6 expression by IHC and no pathogenic variants by NGS exhibited MLH1 promoter hypermethylation. Percent negative agreement between immunohistochemical and NGS gene sequencing is high, although firm conclusions regarding percent positive agreement between NGS and IHC are limited by low numbers of positive cases in our cohort. In general, we consider the findings to support continued use of immunohistochemical methods to screen for the presence of mismatch repair deficiency and consider additional testing by NGS likely to add little diagnostic value in the context of intact immunohistochemical expression of mismatch repair proteins.

Surgery for Hereditary Diffuse Gastric Cancer: Long-Term Outcomes.

INTRODUCTION: Gastric cancer is inherited as an autosomal dominant condition in hereditary diffuse gastric cancer (HDGC). The gene associated with HDGC is an E-cadherin gene CDH1. At the time of initiation of this study, it was estimated that 70% of patients who inherited the CDH1 gene mutation would develop gastric cancer. We hypothesized that the rate of signet ring cell cancer in asymptomatic patients with CDH1 mutations may be higher than anticipated and that the surgery could be conducted with acceptable short-term and long-term complications suggesting that the quality of life with the surgery is acceptable. METHODS: We prospectively studied the role of total gastrectomy in symptomatic and asymptomatic patients with CDH1 mutations. A total of 43 patients with mutations of the CDH1 gene were studied prospectively, including 8 with symptoms and 35 without symptoms. Total gastrectomy was recommended to each. Quality of life was assessed in patients who underwent prophylactic gastrectomy. Proportions are compared with Fisher's exact test. RESULTS: In total, 13 (30%) asymptomatic patients declined surgery. Total gastrectomy was performed in 8 symptomatic patients and 22 asymptomatic patients of whom only 3 asymptomatic patients (14%) had endoscopically proven signet ring cell cancer preoperatively, while 21 of 22 (95%) had it on final pathology (p = 0.05). Each asymptomatic patient was T1, N0, while seven out of eight symptomatic patients had T3-T4 tumors and six had positive lymph nodes. None had operative complications or operative death. The median follow-up was 7 years. Five (63%) symptomatic patients died, while only one (95%) prophylactic patient died of a non-gastric cancer- or surgery-related issue (p = 0.05). A total of 15 prophylactic patients had long-term follow-up. Each had significant weight loss (mean 23%) but all had a normal body mass index. In total, 40% had bile reflux gastritis controlled with sucralfate. Each returned to work and, if given the choice, said that they would undergo the surgery again. CONCLUSIONS: Total gastrectomy is indicated for patients who have an inherented CDH1 mutation. Endoscopic screening is not reliable for diagnosing signet ring cell stomach cancer. If patients wait for symptoms, they will have a more advanced disease and significantly reduced survival. Operative complications of prophylactic gastrectomy are minimal, and long-term quality of life is acceptable.