Pathologic, immunologic, and clinical analysis of the microsatellite instability phenotype in endometrial carcinoma.

The objective of this study was to determine if quantifying the microsatellite instability (MSI) phenotype could serve as a biomarker for clinical and immunologic features of deficient mismatch repair (dMMR) endometrial cancer (EC). Patients with EC undergoing hysterectomy whose tumors demonstrated dMMR were included. Immunohistochemistry (IHC) of mismatch repair proteins and polymerase chain reaction analysis of NR27, BAT25, BAT26, NR24, and NR21 microsatellite loci were performed on each case. The MSI phenotype was quantified by subtracting the number of nucleotides of each microsatellite in tumor tissue from the corresponding microsatellite in paired normal tissue and summing the absolute differences. This was termed marker sum (MS) and is a novel quantification. Tumor-infiltrating lymphocytes (TILs) were identified by IHC for CD3, CD4, and CD8 and quantified with digital image analysis. Tumor infiltration of lymphocytes and clinical characteristics were stratified by MS. Four hundred fifty-nine consecutive patients with dMMR EC were analyzed. MS ranged from 1 to 32. Post hoc, 2 cohorts were defined using receiver operating characteristic curves (MS less than 13 and MS greater than 12). With the exception of tumor grade, all clinical and pathologic features, all tumor characteristics, and the numbers of TILs were similar between cohorts. The MSI phenotype is highly variable in dMMR EC, and no correlation between the immune profile and the severity of the MSI phenotype was observed.

Thyroid Oncocytic (Hürthle Cell) Nodules With Longitudinal Nuclear Grooves: Clinicopathologic, Immunohistochemical, and Molecular Genetic Study of 15 Cases.

CONTEXT.­: Thyroid nodules with longitudinal nuclear grooves have been widely regarded as synonymous with papillary thyroid carcinoma (PTC). OBJECTIVE.­: To study a series of cases of thyroid nodules that exhibited oncocytic (Hürthle cell) features and contained longitudinal nuclear grooves yet failed to display aggressive behavior or the full features of papillary thyroid carcinoma. DESIGN.­: The clinicopathologic, immunohistochemical, and molecular genetic features of 15 patients with these features were studied. Next-generation sequencing was performed to examine 161 genes for oncogenic driver alterations associated with thyroid neoplasia. RESULTS.­: The lesions occurred in 11 women and 4 men aged 27 to 80 years and measured 0.2 to 2.3 cm in diameter (mean, 1.1 cm). The tumors were well circumscribed and noninvasive and showed a proliferation of large cells with abundant granular cytoplasm and centrally placed nuclei displaying scattered longitudinal nuclear grooves. Immunohistochemical stains were negative for HBME-1, galectin-3, and CK19 in all cases. NRAS pQ61R was detected in 6 cases, KRAS p.Q61E in 1 case, and AKT2 p.E17K in 1 case. None of the genetic changes classically associated with conventional PTC or with high-grade thyroid malignant neoplasms were identified. Clinical follow-up in 9 patients showed no evidence of recurrence or metastases between 2 and 13 years (mean, 5.7 years). CONCLUSIONS.­: Longitudinal nuclear grooves can be occasionally encountered in oncocytic (Hürthle cell) tumors and should not lead to a diagnosis of PTC in the absence of other features supporting that diagnosis.

Performance of Idylla KRAS assay on extracted DNA and de-stained cytology smears: Can we rescue small sample?

OBJECTIVE: KRAS is a frequently mutated gene in cancers, and with recent FDA-approved targeted therapy for the G12C mutation, testing for KRAS variants is essential. We evaluated the performance of the Idylla KRAS assay on extracted DNA and cytology smears in order to expand the utility of the assay. METHODS: In total, fifty-seven human samples were analyzed. Idylla results from sixteen DNA extracted from formalin-fixed, paraffin-embedded tissues (FFPE DNA) and thirty cytology smears were compared to the reference method. We evaluated the performance of the Idylla assay using corresponding cytology smears to rescue cellblocks or surgical blocks that were quantity not sufficient (QNS) for next generation sequencing (NGS). RESULT: In the FFPE DNA cohort, 10 ng DNA input yielded valid results in all 16 samples, with 15 of 16 (93 %) concordant with NGS findings. In the cytology smear cohort, the Idylla KRAS assay demonstrated 100 % concordance with previous NGS results in 30 cases. In the QNS cohort, the assay was valid in all cases and KRAS mutations were identified in 3 of 11 cytology smears, including one G12C mutation. CONCLUSION: The Idylla KRAS assay is a high-performing, feasible, and convenient option for testing extracted DNA and cytology smears. It rescues QNS samples allowing it to be integrated into the molecular workflow as an initial screening test with remarkably quick turnaround times.

The diagnostic utility of RNA-based fusion panel testing ordered by pathologists in challenging cases.

INTRODUCTION: Gene fusion identification by RNA-based next-generation sequencing (NGS) provides important information for cancer patients. NGS is commonly initiated by treating oncologists to identify therapeutic options. However, the implications of large fusion panels on tumor classification and diagnosis are underappreciated. We investigated the extent to which these tests aid diagnosis when ordered by pathologists. METHODS: We retrospectively reviewed the results of a validated Archer FusionPlex panel ordered by surgical pathologists at our institution, excluding cases tested for therapeutic targets. One hundred thirty-five cases of solid tumors from October 2020 and September 2021 were included. We compared the initial diagnosis to the final diagnosis, which incorporated fusion gene results. We classified the cases into groups based on the degree of contribution of the RNA fusion panel to the final diagnosis. RESULTS: Among 135 cases, a fusion event was identified in 47 cases, and no fusion event was identified in 88 cases. The results changed the diagnosis in 4 of 135 fusion positive cases (3%). Twenty-one cases (15%) provided a more specific diagnosis, and original diagnosis was confirmed in 17 cases (13%). In the remaining 5 cases (4%), the results identified fusion events of unknown clinical significance. CONCLUSIONS: RNA-based NGS provides significant benefit as an ancillary diagnostic tool. In our cohort, fusion analysis provided a more definitive diagnosis in 25 cases (19%). Our findings demonstrate an important role for pathologists in appropriate utilization of molecular testing, and diagnostic workflows integrating RNA-based NGS will lead to more accurate diagnosis and better patient care.

Lymphocyte-Rich Spindle Cell Thymoma: Clinicopathologic, Immunohistochemical, Ultrastructural and Molecular Genetic Study of 80 Cases.

A study of 80 cases of spindle cell thymoma in which the spindle cell component was overshadowed by massive numbers of stromal lymphocytes is presented. The patients were 38 women and 42 men, aged 8 to 81 years (mean=54 y). All tumors presented as an anterior mediastinal mass; 5 patients had myasthenia gravis and one had Good syndrome. The tumors were well-circumscribed, encapsulated, and measured 2.9 to 26.0 cm in greatest diameter (mean=7.3 cm). Using modified Masaoka staging, 66 tumors were stage I, 10 were stage IIa, 2 were stage III and 1 was stage IV. Histologically the tumors were characterized by a predominant lymphocytic population admixed with scattered small spindle epithelial cells. The neoplastic spindle cells in these tumors demonstrated 2 major growth patterns: in 33 cases, the tumors were exclusively composed of dense sheets of lymphocytes containing scattered spindle cells resembling a lymphocyte-rich thymoma (WHO type B1); in the remaining cases the tumors showed admixtures of a predominantly lymphocytic component with areas that were lymphocyte-poor and contained a pure spindle cell population similar to WHO type A. Immunohistochemical stains and electron microscopy corroborated the spindle cell morphology in both types. The GTF2I p.L424H variant was identified in 53 of 63 (84%) cases analyzed. Clinical follow-up in 27 cases showed that most of the tumors behaved in an indolent manner. Our study expands the spectrum of spindle cell thymoma by demonstrating the existence of cases that are predominantly composed of lymphocyte-rich elements and lack areas with a pure (lymphocyte poor) spindle cell morphology.

The Interpretation of Sequence Variants in Myeloid Neoplasms.

OBJECTIVES: To provide an overview of the challenges encountered during the interpretation of sequence variants detected by next-generation sequencing (NGS) in myeloid neoplasms, as well as the limitations of the technology with the goal of preventing the over- or undercalling of alterations that may have a significant effect on patient management. METHODS: Review of the peer-reviewed literature on the interpretation, reporting, and technical challenges of NGS assays for myeloid neoplasms. RESULTS: NGS has been integrated widely and rapidly into the standard evaluating of myeloid neoplasms. Review of the literature reveals that myeloid sequence variants are challenging to detect and interpret. Large insertions and guanine-cytosine-heavy areas prove technically challenging while frameshift and truncating alterations may be classified as variants of uncertain significance by tertiary analysis informatics pipelines due to their absence in the literature and databases. CONCLUSIONS: The analysis and interpretation of NGS results in myeloid neoplasia are challenging due to the varied number of detectable gene alterations. Familiarity with the genomic landscape of myeloid malignancies and knowledge of the tools available for the interpretation of sequence variants are essential to facilitate translation into clinical and therapy decisions.

Increased SPARC expression is associated with neoadjuvant therapy in resectable pancreatic ductal adenocarcinoma.

Secreted Protein Acid and Rich in Cysteine (SPARC) is an extracellular glycoprotein secreted by fibroblasts and osteoblasts in normal tissues. SPARC overexpression occurs in multiple tumors including pancreatic ductal adenocarcinoma (PDAC) and may predict favorable response to nab-paclitaxel. The prognostic significance of SPARC expression in PDAC is unclear - some reports indicate SPARC overexpression associates with poor outcomes and others find no correlation. Considering neoadjuvant therapy enhances the stromal fibrosis of PDAC and taking into account that SPARC is a component of PDAC stromal fibrosis, we hypothesized that SPARC expression would be greater in neoadjuvant-treated versus treatment-naive PDAC. Quantitative immunohistochemistry was used to measure SPARC expression in resected PDAC in 74 cases of neoadjuvant treated PDAC and 95 cases of treatment-naïve PDAC. SPARC expression was increased 54% in neoadjuvant treated PDAC compared to treatment-naïve PDAC. These data indicate that increased SPARC expression correlates with neoadjuvant therapy in PDAC.

A-C Estrogens as Potent and Selective Estrogen Receptor-Beta Agonists (SERBAs) to Enhance Memory Consolidation under Low-Estrogen Conditions.

Estrogen receptor-beta (ERß) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERß agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the ß over α isoform and with EC50s of 20-30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound's ability to drive the productive conformational change needed to activate transcription. The compound also shows in vivo efficacy after microinfusion into the dorsal hippocampus and after intraperitoneal injection (0.5 mg/kg) or oral gavage (0.5 mg/kg). This simple yet novel A-C estrogen is selective, brain penetrant, and facilitates memory consolidation.

Antral atrophy, intestinal metaplasia, and preneoplastic markers in Mexican children with Helicobacter pylori-positive and Helicobacter pylori-negative gastritis.

Chronic inflammation and infection are major risk factors for gastric carcinogenesis in adults. As chronic gastritis is common in Mexican children, diagnosis of Helicobacter pylori and other causes of gastritis are critical for the identification of children who would benefit from closer surveillance. Antral biopsies from 82 Mexican children (mean age, 8.3 ± 4.8 years) with chronic gastritis (36 H pylori+, 46 H pylori-) were examined for gastritis activity, atrophy, intestinal metaplasia (IM), and immunohistochemical expression of gastric carcinogenesis biomarkers caudal type homeobox 2 (CDX2), ephrin type-B receptor 4 (EphB4), matrix metalloproteinase 3 (MMP3), macrophage migration inhibitory factor (MIF), p53, ß-catenin, and E-cadherin. Atrophy was diagnosed in 7 (9%) of 82, and IM, in 5 (6%) of 82 by routine histology, whereas 6 additional children (7%) (3 H pylori+) exhibited aberrant CDX2 expression without IM. Significant positive correlations were seen between EphB4, MMP3, and MIF (P<.0001). Atrophy and follicular pathology were more frequent in H pylori+ biopsies (P<.0001), whereas IM and CDX2 expression showed no significant correlation with H pylori status. Antral biopsies demonstrating atrophy, IM, and/or aberrant CDX2 expression were seen in 21.95% (18/82) of the children, potentially identifying those who would benefit from closer surveillance and preventive dietary strategies. Biomarkers CDX2, EphB4, MMP3, and MIF may be useful in the workup of pediatric gastritis.

Identification of histologically distinct conventional adenomas that arise predominately in patients with sessile serrated adenomas.

We have recently shown that a study population of patients with at least 1 sessile serrated adenoma (SSA) are 4 times more likely to harbor synchronous serrated polyps [SSAs, traditional serrated adenomas (TSAs) and right sided hyperplastic polyps] than a unselected population of patients. However, 35% of the polyps in the study patients were conventional adenomas (CAds). We hypothesized that the CAds in these study patients would have histologic and molecular differences compared with CAds from a control population without sessile serrated adenomas. To this end, 104 study and 79 control CAds were analyzed according to 9 histologic criteria. A subset of these polyps was also screened for BRAF mutations, KRAS mutations, CpG island methylation, and MUC6 expression. A total of 31 study CAds and 2 control CAds had atypical histologic features (bright cytoplasmic eosinophilia +/- focal serrations and crypt dilatation). None of the adenomas tested had mutations in BRAF or KRAS. Evidence of low levels of CpG island methylation was seen in 35% of the atypical CAds and in only 4.5% of the typical CAds. In addition, these atypical CAds were more likely to express MUC6. Thus, the presence of cytoplasmic eosinophilia with or without focal serrations and crypt dilatation identifies a subset of CAds with characteristics of the serrated neoplasia pathway. These atypical CAds occur more commonly in patients predisposed to developing SSAs and suggest the presence of a mucosal field defect in these patients.