Molecular characterization of diffuse large B-cell lymphomas associated with hepatitis C virus infection.

Hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) displays peculiar clinicopathological characteristics, but its molecular landscape is not fully elucidated. In this study, we investigated the clinicopathological and molecular features of 54 patients with HCV-associated DLBCL. The median age was 71 years. An underlying marginal zone lymphoma component was detected in 14.8% of cases. FISH analysis showed rearrangements involving BCL6 in 50.9% of cases, MYC in 11.3% and BCL2 in 3.7%. Lymph2Cx-based assay was successful in 38 cases, recognizing 16 cases (42.1%) as ABC and 16 cases as GCB subtypes, while six resulted unclassified. ABC cases exhibited a higher lymphoma-related mortality (LRM). Next-generation sequencing analysis showed mutations in 158/184 evaluated genes. The most frequently mutated genes were KMT2D (42.6%), SETD1B (33.3%), RERE (29.4%), FAS and PIM1 (27.8%) and TBL1XR1 (25.9%). A mutation in the NOTCH pathway was detected in 25.9% of cases and was associated with worst LRM. Cluster analysis by LymphGen classified 29/54 cases within definite groups, including BN2 in 14 (48.2%), ST2 in seven (24.2%) and MCD and EZB in four each (13.8%). Overall, these results indicate a preferential marginal zone origin for a consistent subgroup of HCV-associated DLBCL cases and suggest potential implications for molecularly targeted therapies.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

IDH1-mutated Crohn's disease-associated small bowel adenocarcinomas: Distinctive pathological features and association with MGMT methylation and serrated-type dysplasia.

AIMS: Patients with Crohn's disease (CrD) have an elevated risk for the development of small bowel adenocarcinomas (SBAs). Actionable isocitrate dehydrogenase 1 (IDH1) mutations have been reported to be more frequent in CrD-SBAs than in sporadic SBAs. The present study aimed to investigate the clinicopathological and immunophenotypical features, as well as methylation profiles, of IDH1-mutated CrD-SBAs. METHODS AND RESULTS: An international multicentre series of surgically resected CrD-SBAs was tested for IDH1 mutation. Clinicopathological features, immunophenotypical marker expression and O6-methylguanine-DNA methyltransferase (MGMT) and long interspersed nuclear element-1 (LINE-1) methylation were compared between IDH1-mutated and IDH1 wild-type CrD-SBAs. Ten (20%) of the 49 CrD-SBAs examined harboured an IDH1 mutation and all the mutated cancers harboured the R132C variant. Compared to IDH1 wild-type cases, IDH1-mutated CrD-SBAs showed significantly lower rates of cytokeratin 7 expression (P = 0.005) and higher rates of p53 overexpression (P = 0.012) and MGMT methylation (P = 0.012). All three dysplastic growths associated with IDH1-mutated SBAs harboured the same IDH1 variant (R132C) of the corresponding invasive cancer, and all were of non-conventional subtype (two serrated dysplastic lesions and one goblet cell-deficient dysplasia). In particular, non-conventional serrated dysplasia was significantly associated with IDH1-mutated CrD-SBAs (P = 0.029). No significant cancer-specific survival difference between IDH1-mutated CrD-SBA patients and IDH1 wild-type CrD-SBA patients was found (hazard ratio = 0.55, 95% confidence interval = 0.16-1.89; P = 0.313). CONCLUSIONS: IDH1-mutated CrD-SBAs, which represent approximately one-fifth of total cases, are characterised by distinctive immunophenotypical features and methylation profiles, with potential therapeutic implications. Moreover, IDH1-mutated non-conventional, serrated dysplasia is likely to represent a precursor lesion to such CrD-SBAs.

Mismatch repair deficiency as prognostic factor for stage III small bowel adenocarcinoma: A multicentric international study.

BACKGROUND: Small bowel adenocarcinoma (SBA) is a rare cancer with an aggressive behavior. No study has specifically addressed the putative prognostic role of mismatch repair status in stage III SBAs. AIMS: We aimed to investigate whether mismatch repair deficiency is associated with cancer-specific survival in a Western cohort of patients with stage III SBAs. METHODS: In this retrospective multicentric international cohort study, we enrolled 70 patients who underwent surgically resection for stage III SBAs and we analyzed the frequency of mismatch repair deficiency, tested by immunohistochemistry for mismatch repair proteins and by polymerase chain reaction for microsatellite instability, and its association with cancer-specific survival and other clinic-pathologic factors. RESULTS: We found sixteen (23%) patients with mismatch repair deficient adenocarcinoma, without discordance between immunohistochemical and polymerase chain reaction for microsatellite instability analyses. Mismatch repair deficiency proved to be associated with a better outcome both at univariable analysis (hazard ratio: 0.28, 95% confidence interval: 0.08-0.91, p: 0.035) and in bivariable models adjusted for patient age or gender, tumor site, pT4 stage, tumor budding, and perineural invasion. CONCLUSION: This study highlights the importance of testing mismatch repair status to improve prognostic stratification in stage III SBAs.

Molecular Landscape and Association With Crohn Disease of Poorly Cohesive Carcinomas of the Nonampullary Small Bowel.

OBJECTIVES: Poorly cohesive carcinomas (PCCs) are neoplasms defined by a predominantly dyshesive growth pattern with single cell or cord-like stromal infiltration. The -distinctive clinicopathologic and prognostic features of small bowel PCCs (SB-PCCs) in comparison with conventional-type small intestinal adenocarcinomas have only recently been characterized. However, as SB-PCCs' genetic profile is still unknown, we aimed to analyze the molecular landscape of SB-PCCs. METHODS: A next-generation sequencing analysis through Trusight Oncology 500 on a series of 15 nonampullary SB-PCCs was performed. RESULTS: The most frequently found gene alterations were TP53 (53%) and RHOA (13%) mutations and KRAS amplification (13%), whereas KRAS, BRAF, and PIK3CA mutations were not identified. Most SB-PCCs (80%) were associated with Crohn disease, including both RHOA-mutated SB-PCCs, which featured a non-SRC-type histology, and showed a peculiar appendiceal-type, low-grade goblet cell adenocarcinoma (GCA)-like component. Rarely, SB-PCCs showed high microsatellite instability, mutations in IDH1 and ERBB2 genes, or FGFR2 amplification (one case each), which are established or promising therapeutic targets in such aggressive cancers. CONCLUSIONS: SB-PCCs may harbor RHOA mutations, which are reminiscent of the diffuse subtype of gastric cancers or appendiceal GCAs, while KRAS and PIK3CA mutations, commonly involved in colorectal and small bowel adenocarcinomas, are not typical of such cancers.

Multimodal imaging approach in hyponatremic hypertensive syndrome. A rare case of pediatric unilateral hypoplasia of the main renal artery combined itself with stenosis and review of literature.

Renal artery stenosis (RAS) accounts for approximately 5%-10% of secondary renovascular hypertension in the pediatric population. It can occur as an isolated entity, or as a hypoplasia combined itself with stenosis. Hypoplasia, or long-segment developmental narrowing, is a rare cause of renovascular hypertension. Hyponatremic hypertensive syndrome (HHS) is a malignant complication of unilateral RAS and/or renal artery hypoplasia. Hyponatremia, hypokalemic hypochloremic metabolic alkalosis, nephrotic range proteinuria, polyuria, polydipsia, and weight loss are the most common findings. In particular, hypertension remains refractory despite aggressive antihypertensive therapy. Laboratory findings of elevated plasma levels of renin in most case suggest that the stimulation of renin release from the ischemic kidney plays an important pathophysiologic role. HHS is a diagnostic and therapeutic challenge in children. We report a case of a unilateral right renal artery hypoplasia, complicated by a segmental narrowing, in a 17-month-old male, clinically symptomatic for hypertension. We emphasize the role of ultrasound, computed tomography, and digital subtraction angiography that should be planned as reliable and non-invasive multimodal imaging approach.

Tumor Antigenicity and a Pre-Existing Adaptive Immune Response in Advanced BRAF Mutant Colorectal Cancers.

The main hypothesis of this study is that gene expression profiles (GEPs) integrating both tumor antigenicity and a pre-existing adaptive immune response can be used to generate distinct immune-related signatures of BRAF mutant colorectal cancers (BRAF-CRCs) to identify actionable biomarkers predicting response to immunotherapy. GEPs of 89 immunotherapy-naïve BRAF-CRCs were generated using the Pan-Cancer IO 360 gene expression panel and the NanoString nCounter platform and were correlated with microsatellite instability (MSI) status and with CD8+ tumor-infiltrating lymphocyte (TIL) content. Hot/inflamed profiles were found in 52% of all cases, and high scores of Tumor Inflammation Signature were observed in 42% of the metastatic BRAF-CRCs. A subset of MSI tumors showed a cold profile. Antigen Processing Machinery (APM) signature was not differentially expressed in MSI tumors compared with MSS cases. By contrast, the APM signature was significantly upregulated in CD8+ BRAF-CRCs versus CD8- tumors. Our study demonstrates that a significant fraction of BRAF-CRCs may be a candidate for immunotherapy and that the simultaneous analysis of MSI status and CD8+ TIL content increases accuracy in identifying patients who can potentially benefit from immune checkpoint inhibitors. GEPs may be very useful in expanding the spectrum of patients with BRAF-CRCs who can benefit from immune checkpoint blockade.

Critical aspects of microsatellite instability testing in endometrial cancer: a comparison study.

The identification of mismatch repair deficient (dMMR) and microsatellite unstable (MSI) endometrial cancers (ECs) is important in screening, diagnosis, and therapeutic stratification of patients. We compared the diagnostic performance of 4 MSI molecular tests based on fragment length assay in capillary electrophoresis (OncoMate™ MSI assay, Promega) and in microcapillary electrophoresis (TapeStation 4200, Agilent); with high-resolution melting (HRM) analysis approaches (Idylla™ MSI Test, Biocartis; EasyPGX® ready MSI, Diatech Pharmacogenetics) on a series of 56 ECs, which was well characterized for MMR status with immunohistochemical approach (IHC, nonmolecular reference test). The concordance of fluorescence capillary electrophoresis with IHC (AUC 0.98) was higher respect to the other molecular methodologies. Otherwise, HRM approaches and microcapillary electrophoresis platform failed to detect MSI-ECs showing minimal microsatellite shifts. In conclusion, in colorectal site, several technologies are eligible for MSI test, whereas in ECs, MSI test should be based on fluorescent capillary electrophoresis as it identifies a higher proportion of cases that could be misdiagnosed with other strategies.

Poorly Cohesive Carcinoma of the Nonampullary Small Intestine: A Distinct Histologic Subtype With Prognostic Significance.

Poorly cohesive carcinomas (PCCs) are neoplasms characterized by a dyshesive cell invasion pattern featuring single-cell or cord-like stromal infiltration. Although they have been extensively studied in the stomach and other digestive system organs, limited data regarding nonampullary small bowel poorly cohesive carcinomas (SB-PCCs) are hitherto available. The aims of our study were to analyze the clinicopathologic and immunophenotypical features of SB-PCCs (PCC pattern accounting for >50% of the neoplasm) and to compare them with small bowel adenocarcinomas (SBAs), not otherwise specified (SBAs-NOS) and with cancers with a histologically distinct PCC component accounting for 10% to 50% of the neoplasm (mixed-poorly-cohesive-glandular-SBAs). Fifteen SB-PCCs were identified and compared with 95 SBAs-NOS and 27 mixed-poorly-cohesive-glandular-SBAs. Most SB-PCCs (67%) were composed of <10% of signet-ring cells, and all but 1 SB-PCCs exhibited loss of membranous expression of E-cadherin. Compared with SBAs-NOS, SB-PCCs showed a significantly younger patient age at diagnosis, and a stronger association with Crohn disease, and both SB-PCCs and mixed-poorly-cohesive-glandular-SBAs featured a higher rate of lymphovascular and perineural invasion and a lower percentage of mismatch repair-deficient cases. Importantly, the cancer-specific survival of SB-PCC (hazard ratio: 3.81; 95% confidence interval: 1.90-7.64; P<0.001) and mixed-poorly-cohesive-glandular-SBA (4.12; 2.20-7.71; P<0.001) patients was significantly worse compared with SBAs-NOS cases. This study provides objective evidence to the World Health Organization (WHO) 2019 introduction of SB-PCC as a distinctive subtype of nonampullary SBA, by virtue of its unique clinical and histologic features, and suggests that both SB-PCCs and mixed-poorly-cohesive-glandular-SBAs should be separated from SBAs-NOS.

Mixed Neuroendocrine/Non-neuroendocrine Neoplasm (MiNEN) of the Ovary Arising from Endometriosis: Molecular Pathology Analysis in Support of a Pathogenetic Paradigm.

Primary ovarian neuroendocrine neoplasms (Ov-NENs) are infrequent and mainly represented by well-differentiated forms (neuroendocrine tumors - NETs - or carcinoids). Poorly differentiated neuroendocrine carcinomas (Ov-NECs) are exceedingly rare and only few cases have been reported in the literature. A subset of Ov-NECs are admixed with non-neuroendocrine carcinomas, as it occurs in other female genital organs, as well (mostly endometrium and uterine cervix), and may be assimilated to mixed neuroendocrine/non-neuroendocrine neoplasms (MiNENs) described in digestive and extra-digestive sites. Here, we present a case of large cell Ov-NEC admixed with an endometrioid carcinoma of the ovary, arising in the context of ovarian endometriosis, associated with a uterine endometrial atypical hyperplasia (EAH). We performed targeted next-generation sequencing analysis, along with a comprehensive immunohistochemical study and FISH analysis for TP53 locus, separately on the four morphologically distinct lesions (Ov-NEC, endometrioid carcinoma, endometriosis, and EAH). The results of our study identified molecular alterations of cancer-related genes (PIK3CA, CTNNB1, TP53, RB1, ARID1A, and p16), which were present with an increasing gradient from preneoplastic lesions to malignant proliferations, both neuroendocrine and non-neuroendocrine components. In conclusion, our findings underscored that the two neoplastic components of this Ov-MiNEN share a substantially identical molecular profile and they progress from a preexisting ovarian endometriotic lesion, in a patient with a coexisting preneoplastic proliferation of the endometrium, genotypically and phenotypically related to the ovarian neoplasm. Moreover, this study supports the inclusion of MiNEN in the spectrum ovarian and, possibly, of all gynecological NENs, among which they are currently not classified.

Methylation Drivers and Prognostic Implications in Sinonasal Poorly Differentiated Carcinomas.

BACKGROUND: Poorly differentiated sinonasal carcinomas (PDSNCs) are rare and aggressive malignancies, which include squamous cell carcinoma (SCC), sinonasal undifferentiated carcinoma (SNUC), and neuroendocrine carcinomas (NEC). Several epigenetic markers have been suggested to support the histopathological classification, predict prognosis, and guide therapeutic decision. Indeed, molecularly distinct subtypes of sinonasal carcinomas, including SMARCB1-INI1 or SMARCA4 deficient sinonasal carcinoma, isocitrate dehydrogenase (IDH)-mutant SNUC, ARID1A mutant PDSNCs, and NUT carcinomas, have recently been proposed as separate entities. Identification of aberrant DNA methylation levels associated with these specific epigenetic driver genes could be useful for prognostic and therapeutic purpose. METHODS: Histopathological review and immunohistochemical study was performed on 53 PDSNCs. Molecular analysis included mutational profile by NGS, Sanger sequencing, and MLPA analyses, and global DNA methylation profile using LINE-1 bisulfite-PCR and pyrosequencing analysis. RESULTS: Nine SWI/SNF complex defective cases and five IDH2 p.Arg172x cases were identified. A significant correlation between INI-1 or IDH2 defects and LINE-1 hypermethylation was observed (p = 0.002 and p = 0.032, respectively), which were associated with a worse prognosis (p = 0.007). CONCLUSIONS: Genetic and epigenetic characterization of PDSNCs should be performed to identify distinct prognostic entities, which deserved a tailored clinical treatment.

Evaluation of global and intragenic hypomethylation in colorectal adenomas improves patient stratification and colorectal cancer risk prediction.

BACKGROUND: Aberrant DNA hypomethylation of the long interspersed nuclear elements (LINE-1 or L1) has been recognized as an early event of colorectal transformation. Simultaneous genetic and epigenetic analysis of colorectal adenomas may be an effective and rapid strategy to identify key biological features leading to accelerated colorectal tumorigenesis. In particular, global and/or intragenic LINE-1 hypomethylation of adenomas may represent a helpful tool for improving colorectal cancer (CRC) risk stratification of patients after surgical removal of polyps. To verify this hypothesis, we analyzed a cohort of 102 adenomas derived from 40 high-risk patients (who developed CRC in a post-polypectomy of at least one year) and 43 low-risk patients (who did not develop CRC in a post-polypectomy of at least 5 years) for their main pathological features, the presence of hotspot variants in driver oncogenes (KRAS, NRAS, BRAF and PIK3CA), global (LINE-1) and intragenic (L1-MET) methylation status. RESULTS: In addition to a significantly higher adenoma size and an older patients' age, adenomas from high-risk patients were more hypomethylated than those from low-risk patients for both global and intragenic LINE-1 assays. DNA hypomethylation, measured by pyrosequencing, was independent from other parameters, including the presence of oncogenic hotspot variants detected by mass spectrometry. Combining LINE-1 and L1-MET analyses and profiling the samples according to the presence of at least one hypomethylated assay improved the discrimination between high and low risk lesions (p = 0.005). Remarkably, adenomas with at least one hypomethylated assay identified the patients with a significantly (p < 0.001) higher risk of developing CRC. Multivariable analysis and logistic regression evaluated by the ROC curves proved that methylation status was an independent variable improving cancer risk prediction (p = 0.02). CONCLUSIONS: LINE-1 and L1-MET hypomethylation in colorectal adenomas are associated with a higher risk of developing CRC. DNA global and intragenic hypomethylation are independent markers that could be used in combination to successfully improve the stratification of patients who enter a colonoscopy surveillance program.

Prognostic relevance and putative histogenetic role of cytokeratin 7 and MUC5AC expression in Crohn's disease-associated small bowel carcinoma.

Most Crohn's disease-associated small bowel carcinomas (CrD-SBCs) are diagnosed in advanced stage and have poor prognosis. To improve diagnosis and therapy, a better knowledge of tumour precancerous lesions, histotypes and prognostic factors is needed. We investigated histologically and immunohistochemically 52 CrD-SBCs and 51 small bowel carcinomas unrelated to inflammatory disease, together with their tumour-associated mucosa, looking for Crohn-selective changes. Histologic patterns and phenotypic markers potentially predictive of CrD-SBC histogenesis and prognosis were analysed. Cytokeratin 7 or MUC5AC-positive metaplastic changes were found in about half of investigated CrD-SBCs, significantly more frequently than in CrD-unrelated SBCs. They correlated with metaplastic changes of their associated mucosa, while being absent in normal ileal mucosa. Histologic patterns suggestive for progression of some cytokeratin 7 and/or MUC5AC-positive metaplastic lesions into cancer of the same phenotype were also observed. Patient survival analyses showed that tumour cytokeratin 7 or MUC5AC expression and non-cohesive histotype were adverse prognostic factors at univariable analysis, while cytokeratin 7 and non-cohesive histotype were also found to predict worse survival in stage- and age-inclusive multivariable analyses. Besides conventional dysplasia, hyperplasia-like non-conventional lesions were observed in CrD-SBC-associated mucosa, with patterns suggestive for a histogenetic link with adjacent cancer. In conclusion the cytokeratin 7 and/or MUC5AC-positive metaplastic foci and the non-conventional growths may have a role in cancer histogenesis, while tumour cytokeratin 7 and non-cohesive histotype may also predict poor patient survival. Present findings are worth being considered in future prospective histogenetic and clinical studies.

Gene Expression Profiling of Pancreas Neuroendocrine Tumors with Different Ki67-Based Grades.

Pancreatic neuroendocrine tumors (PanNETs) display variable aggressive behavior. A major predictor of survival is tumor grade based on the Ki67 proliferation index. As information on transcriptomic profiles of PanNETs with different tumor grades is limited, we investigated 29 PanNETs (17 G1, 7 G2, 5 G3) for their expression profiles, mutations in 16 PanNET relevant genes and LINE-1 DNA methylation profiles. A total of 3050 genes were differentially expressed between tumors with different grades (p < 0.05): 1279 in G3 vs. G2; 2757 in G3 vs. G1; and 203 in G2 vs. G1. Mutational analysis showed 57 alterations in 11 genes, the most frequent being MEN1 (18/29), DAXX (7/29), ATRX (6/29) and MUTYH (5/29). The presence and type of mutations did not correlate with the specific expression profiles associated with different grades. LINE-1 showed significantly lower methylation in G2/G3 versus G1 tumors (p = 0.007). The expression profiles of matched primaries and metastasis (nodal, hepatic and colorectal wall) of three cases confirmed the role of Ki67 in defining specific expression profiles, which clustered according to tumor grades, independently from anatomic location or patient of origin. Such data call for future exploration of the role of Ki67 in tumor progression, given its involvement in chromosomal stability.

Small Bowel Epithelial Precursor Lesions: A Focus on Molecular Alterations.

The wider use of gastrointestinal endoscopic procedures has led to an increased detection of small intestinal preneoplastic and neoplastic epithelial lesions, most of which are identified in the duodenum and ampullary region. Like their malignant counterparts, small intestinal glandular precursor lesions, which include adenomas and hamartomas, may arise sporadically or be associated with hereditary tumor syndromes, such as familial adenomatous polyposis, MUTYH-associated polyposis, Lynch syndrome, Peutz-Jeghers syndrome, juvenile polyposis syndrome, and Cowden syndrome. In addition, dysplastic, preinvasive lesions have been observed adjacent to small bowel adenocarcinomas complicating immune-related disorders, such as celiac or Crohn's disease. Adenomatous lesions may exhibit an intestinal-type, gastric-type, or, very rarely, serrated differentiation, related to different molecular pathogenetic mechanisms. Finally, in the background of multiple endocrine neoplasia 1 syndrome, precursor neuroendocrine growths have been described. In this review we offer a comprehensive description on the histo-molecular features of the main histotypes of small bowel epithelial precursors lesions, including: (i) sporadic adenomas (intestinal-type and gastric-type; non-ampullary and ampullary); (ii) syndromic adenomas; (iii) small bowel dysplasia in celiac and Crohn's disease; (iv) serrated lesions; (v) hamartomatous lesions; and (vi) neuroendocrine precursor lesions.

MYD88L265P Detection in IgM Monoclonal Gammopathies: Methodological Considerations for Routine Implementation.

In IgM monoclonal gammopathies MYD88L265P is a prognostic and predictive biomarker of therapy response. MYD88L265P detection is mainly performed by allele-specific quantitative PCR (ASqPCR), however recently, droplet digital PCR (ddPCR) has been proved to be suitable for MYD88L265P screening and minimal residual disease monitoring (MRD). This study compared ASqPCR and ddPCR to define the most sensitive method for MYD88L265P detection in bone marrow (BM), peripheral blood (PB) sorted or unsorted CD19+ cells, and in plasma cell-free DNA (cfDNA). Overall, the analysis showed a good concordance rate (74%) between the two methods, especially in BM samples, while discordances (26%) were mostly in favor of ddPCR (ddPCR+ vs. ASqPCR-) and were particularly evident in samples with low mutational burden, such as PB and cfDNA. This study highlights ddPCR as a feasible approach for MYD88L265P detection across different specimen types (including cfDNA). Interestingly, its high sensitivity makes CD19+ selection dispensable. On the other hand, our results showed that MYD88L265P detection on PB samples, especially with ASqPCR, is suboptimal for screening and MRD analysis. Finally, significantly different MYD88L265P mutational levels observed between Waldenström Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance patients suggest the need for further studies in order to identify possible correlations between mutational levels and risk of progression to Waldenström.

Histologic heterogeneity and syndromic associations of non-ampullary duodenal polyps and superficial mucosal lesions.

BACKGROUND: Duodenal polyps and superficial mucosal lesions (DP/SMLs) are poorly characterised. AIMS: To describe a series of endoscopically-diagnosed extra-ampullary DPs/SMLs. METHODS: This is a retrospective study conducted in a tertiary referral Endoscopy Unit, including patients who had DPs or SMLs that were biopsied or removed in 2010-2019. Age, gender, history of familial polyposis syndromes, DP/SML characteristics were recorded. Histopathological, immunohistochemical and molecular analyses were performed. RESULTS: 399 non-ampullary DP/SMLs from 345 patients (60.6% males; median age 67 years) were identified. Gastric foveolar metaplasia represented the most frequent histotype (193 cases, 48.4%), followed by duodenal adenomas (DAs; 77 cases, 19.3%). Most DAs (median size 6 mm) were sessile (Paris Is; 48%), intestinal-type (96.1%) with low-grade dysplasia (93.5%). Among syndromic DAs (23%), 15 lesions occurred in familial adenomatous polyposis 1, two were in MUTYH-associated polyposis and one was in Peutz-Jeghers syndrome (foveolar-type, p53-positive, low-grade dysplasia). Only one (3.3%) tubular, low-grade DA showed mismatch repair deficiency (combined loss of MLH1 and PMS2, heterogeneous MSH6 expression), and it was associated with a MLH1 gene germline mutation (Lynch syndrome). CONCLUSION: DPs/SMLs are heterogeneous lesions, most of which showing foveolar metaplasia, followed by low-grade, intestinal-type, non-syndromic DAs. MMR-d testing may identify cases associated with Lynch syndrome.

Small Bowel Adenocarcinomas Featuring Special AT-Rich Sequence-Binding Protein 2 (SATB2) Expression and a Colorectal Cancer-Like Immunophenotype: A Potential Diagnostic Pitfall.

Special AT-rich sequence-binding protein 2 (SATB2) is a transcription factor expressed by colonic cryptic epithelium and epithelial neoplasms of the lower gastrointestinal (GI) tract, as well as by small bowel adenocarcinomas (SBAs), though at a lower rate. Nevertheless, up to now, only small SBA series, often including a very limited number of Crohn's disease-associated SBAs (CrD-SBAs) and celiac disease-associated SBAs (CD-SBA), have been investigated for SATB2 expression. We evaluated the expression of SATB2 and other GI phenotypic markers (cytokeratin (CK) 7 and CK20, caudal type homeobox 2 (CDX2) and alpha-methylacyl-CoA racemase (AMACR)), as well as mismatch repair (MMR) proteins, in 100 SBAs, encompassing 34 CrD-SBAs, 28 CD-SBAs and 38 sporadic cases (Spo-SBAs). Any mutual association and correlation with other clinico-pathologic features, including patient prognosis, were searched. Twenty (20%) SATB2-positive SBAs (4 CrD-SBAs, 7 CD-SBAs and 9 Spo-SBAs) were identified. The prevalence of SATB2 positivity was lower in CrD-SBA (12%) in comparison with both CD-SBAs (25%) and Spo-SBAs (24%). Interestingly, six SBAs (two CD-SBAs and four Spo-SBAs) displayed a full colorectal carcinoma (CRC)-like immunoprofile (CK7-/CK20+/CDX2+/AMACR+/SATB2+); none of them was a CrD-SBA. No association between SATB2 expression and MMR status was observed. Although SATB2-positive SBA patients showed a more favorable outcome in comparison with SATB2-negative ones, the difference did not reach statistical significance. When cancers were stratified according to CK7/CK20 expression patterns, we found that CK7-/CK20- SBAs were enriched with MMR-deficient cases (71%) and patients with CK7-/CK20- or CK7-/CK20+ SBAs had a significantly better survival rate compared to those with CK7+/CK20- or CK7+/CK20+ cancers (p = 0.002). To conclude, we identified a small (6%) subset of SBAs featuring a full CRC-like immunoprofile, representing a potential diagnostic pitfall in attempts to identify the site of origin of neoplasms of unknown primary site. In contrast with data on colorectal carcinoma, SATB2 expression is not associated with MMR status in SBAs. CK patterns influence patient survival, as CK7-/CK20- cancers show better prognosis, a behavior possibly due to the high rate of MMR-deficient SBAs within this subgroup.