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Aims: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS‐associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAFV600EIHC and BRAF molecular analysis in a large, mono‐institutional CRC whole‐slide, case series with laboratory validation. Methods and results: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAFV600EIHC were performed on all unselected cases of surgically resected CRCs (2018–2023). An in‐house validation study for BRAFV600EIHC was performed in order to obtain optimal IHC stains. BRAFVV600EIHC was considered negative (score 0), positive (scores 2–3), and equivocal (score 1). Interobserver differences in BRAFV600EIHC scoring were noted in the first 150 cases prospectively collected. Nine‐hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAFV600EIHC (BRAFV600EIHC‐positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAFV600EIHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAFV600EIHC expression. Among the 12 IHC‐equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF‐mutated and nine BRAF‐wildtype. Conclusion: BRAFV600EIHC can be used as a reliable surrogate of molecular testing after stringent in‐house validation. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures. [ABSTRACT FROM AUTHOR]

Featured Application: Molecular barcoding NGS workflow for a high sensitive and accurate mutational profiling of circulating tumor DNA (ctDNA) in non-small cell lung cancer (NSCLC) patients. Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories. [ABSTRACT FROM AUTHOR]

Background: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab.Patients and Methods: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab.Results: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006).Conclusions: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory. [ABSTRACT FROM AUTHOR]

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use. [ABSTRACT FROM AUTHOR]

Aims: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS-associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAF V600E IHC and BRAF molecular analysis in a large, mono-institutional CRC whole-slide, case series with laboratory validation., Methods and Results: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAF V600E IHC were performed on all unselected cases of surgically resected CRCs (2018-2023). An in-house validation study for BRAF V600E IHC was performed in order to obtain optimal IHC stains. BRAFV V600E IHC was considered negative (score 0), positive (scores 2-3), and equivocal (score 1). Interobserver differences in BRAF V600E IHC scoring were noted in the first 150 cases prospectively collected. Nine-hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAF V600E IHC (BRAF V600E IHC-positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAF V600E IHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAF V600E IHC expression. Among the 12 IHC-equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF-mutated and nine BRAF-wildtype., Conclusion: BRAF V600E IHC can be used as a reliable surrogate of molecular testing after stringent in-house validation., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Purpose: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole., Experimental Design: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method., Results: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026)., Conclusions: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization., (©2023 The Authors; Published by the American Association for Cancer Research.)

Deficiency of glycosylphosphatidylinositol (GPI)-anchored molecules on blood cells accounts for most features of paroxysmal nocturnal hemoglobinuria (PNH) but not for the expansion of PNH (GPI(-)) clone(s). A plausible model is that PNH clones expand by escaping negative selection exerted by autoreactive T cells against normal (GPI(+)) hematopoiesis. By a systematic analysis of T-cell receptor beta (TCR-beta) clonotypes of the CD8+ CD57+ T-cell population, frequently deranged in PNH, we show recurrent clonotypes in PNH patients but not in healthy controls: 11 of 16 patients shared at least 1 of 5 clonotypes, and a set of closely related clonotypes was present in 9 patients. The presence of T-cell clones bearing a set of highly homologous TCR-beta molecules in most patients with hemolytic PNH is consistent with an immune process driven by the same (or similar) antigen(s)-probably a nonpeptide antigen, because patients sharing clonotypes do not all share identical HLA alleles. These data confirm that CD8+ CD57+ T cells play a role in PNH pathogenesis and provide strong new support to the hypothesis that the expansion of the GPI(-) blood cell population in PNH is due to selective damage to normal hematopoiesis mediated by an autoimmune attack against a nonpeptide antigen(s) that could be the GPI anchor itself.

Background: We describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) for detecting MUTYH mutations, which are associated with colorectal adenomas and colorectal cancer., Methods: We designed specific T-ARMS-PCR assays for 6 mutations (Y165C, G382D, 1395&#95;7delGGA, Y90X, 1103delC, and R231H) selected on the basis of the frequency of their occurrence. We also designed a set of 3 multiplex T-ARMS PCR assays, each for detection of 2 mutations. We tested DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations., Results: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing., Conclusions: T-ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 common MUTYH mutations with use of as few as 3 single tube PCR reactions. It could be useful to carry out large population-based epidemiologic studies.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a large clonal population of blood cells deriving from hematopoietic stem cells (HSCs) deficient in glycosylphosphatidylinositol (GPI)-anchored surface molecules. A current model postulates that PNH arises through negative selection against normal HSCs exerted by autoreactive T cells, whereas PNH HSCs escape damage. We have investigated the inhibitory receptor superfamily (IRS) system in 13 patients with PNH. We found a slight increase in the proportion of T cells expressing IRS. In contrast to what applies to healthy donors, the engagement of IRS molecules on T cells from patients with PNH elicited a powerful cytolytic activity in a redirected killing assay, indicating that these IRSs belong to the activating type. This was confirmed by clonal analysis: 50% of IRS+ T-cell clones in patients with PNH were of the activating type, while only 5% were of the activating type in healthy donors. Moreover, the ligation of IRS induces (1) production of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) and (2) brisk cytolytic activity against cells bearing appropriate IRS counter-ligands. In addition, these IRS+ T cells show natural killer (NK)-like cytolytic activity to which GPI- cells were less sensitive than GPI+ cells. Thus, T cells with NK-like features, expressing the activating isoforms of IRS, may include effector cells involved in the pathogenesis of PNH.