Prevalence of CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT Pathogenic Variants in a Single-Center Retrospective Series of Patients With Melanoma and Personal or Family History Suggestive of Genetic Predisposition.

INTRODUCTION: Approximately 20%-45% of familial melanoma (FM) cases are associated with genetic predisposition. OBJECTIVES: This single-center retrospective study aimed to assess the frequency of pathogenic variants (PV) in the main melanoma-predisposing genes in patients with cutaneous melanoma and investigate the clinical predictors of genetic predisposition. METHODS: Patients included were those diagnosed with cutaneous melanoma at the Dermatology Unit of the University Hospital of Verona, Italy, from 2000 to 2022, presenting at least one of the followings: multiple melanomas (≥ 3); personal/family history of pancreatic cancer (PC) (up to 2nd-degree relatives); ≥ 2 1st-degree relatives with melanoma; ≥ 1 1st-degree relatives with early-onset (<45 years) melanoma and tested for CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT. RESULTS: During the study period, 35 out of 1320 patients (2.7%) underwent genetic testing. Four patients (11.4%) harbored a PV in a melanoma-predisposing gene, three in CDKN2A (8.6%), and one in MITF (2.9%). Variants currently classified as being of unknown clinical significance (VUS) were detected in CDKN2A (N = 1), MITF (N = 1), and ATM (N = 2). Family history of PC and ≥5 melanomas, personal history of ≥50 nevi, and ≥4 melanomas were significantly associated with PV in tested genes (P < 0.05). CONCLUSIONS: The prevalence of PV in predisposing genes in FM was lower than previously reported in Italian registries. Possible reasons include deleterious variants in untested intermediate/low-penetrance genes or yet-to-be-discovered high-penetrance genes and environmental risk factors. A family history of PC, a high number of nevi and melanomas predict a monogenic predisposition to melanoma.

mTOR eosinophilic renal cell carcinoma: a distinctive tumor characterized by mTOR mutation, loss of chromosome 1, cathepsin-K expression, and response to target therapy.

In the spectrum of oncocytic renal neoplasms, a subset of tumors with high-grade-appearing histologic features harboring pathogenic mutations in mammalian target of rapamycin (mTOR) and hitherto clinical indolent behavior has been described. Three cases (2F,1 M) with histologically documented metastases (lymph node, skull, and liver) were retrieved and extensively investigated by immunohistochemistry, FISH, and next-generation sequencing. Tumors were composed of eosinophilic cells with prominent nucleoli (G3 by ISUP/WHO) arranged in solid to nested architecture. Additionally, there were larger cells with perinuclear cytoplasmic shrinkage and sparse basophilic Nissl-like granules, superficially resembling the so-called spider cells of cardiac rhabdomyomas. The renal tumors, including the skull and liver metastases, showed immunoexpression PAX8, CK8-18, and cathepsin-K, and negativity for vimentin. NGS identified mTOR genetic alterations in the three cases, including the skull and liver metastases. One patient was then treated with Everolimus (mTOR inhibitors) with clinical response (metastatic tumor shrinkage). We present a distinct renal tumor characterized by high-grade eosinophilic cells, cathepsin-K immunohistochemical expression, and harboring mTOR gene mutations demonstrating a malignant potential and showing responsiveness to mTOR inhibitors.

First-line tepotinib for a very elderly patient with metastatic NSCLC harboring MET exon 14 skipping mutation and high PD-L1 expression.

Optimal treatment for metastatic non-small cell lung cancer (NSCLC) with mesenchymal epithelial transition gene (MET) exon 14 skipping mutation has not been established yet. MET inhibitors were demonstrated to be effective and tolerated in patients with this condition, while evidence on safety and efficacy of immunotherapy and/or chemotherapy in this population is limited. Here we report the case of an 86-year-old male with metastatic NSCLC harboring MET exon 14 skipping mutation and with high programmed cell death ligand 1 (PD-L1) expression (tumor proportion score ≥50%). The patient received the MET inhibitor tepotinib as first-line treatment, achieving a partial response, with G2 peripheral edema as adverse event that was successfully managed with temporary discontinuation, dose reduction, diuretics and physical therapy. After 31 months, the patient is still receiving tepotinib, with an ongoing response. Tepotinib is a valuable therapeutic option for first-line treatment of older patients with NSCLC harboring MET exon 14 skipping mutation, even in the presence of high PD-L1 expression.

TSC loss is a clonal event in eosinophilic solid and cystic renal cell carcinoma: a multiregional tumor sampling study.

Eosinophilic, solid and cystic (ESC) renal cell carcinoma (RCC) is characterized by a solid and cystic architecture with cells showing abundant eosinophilic cytoplasm with hobnail arrangement and a cytokeratin 7-negative/cytokeratin 20-positive immunophenotype. Recent studies have suggested that bi-allelic events affecting TSC genes might play an important role for such tumors. However, only indirect evidence of the clonal origin of TSC mutation has been gathered so far. Therefore, in this paper we aimed to perform multi-regional tumor sampling molecular analysis in four ESC RCC cases that had been completely embedded, three sporadic and one occurring in a patient with tuberous sclerosis complex (TSC). Histologically, the 4 cases showed cystic and solid architecture and cells with abundant eosinophilic cytoplasm with cytoplasmic stippling and round to oval nuclei. Immunohistochemistry showed at least focal expression of cytokeratin 20 in all tissue samples and negative cytokeratin 7, as well as diffuse positivity for S100A1 and at least focal expression of cathepsin K in three out of four cases. The sporadic cases showed the same somatic TSC1 mutations in all tissue samples analyzed, while the TSC-associated case showed the same TSC1 alteration in both normal tissue and all tumor samples analyzed, proving the germline nature of the alteration. In conclusion, our data demonstrate that clonal TSC loss is a key event in ESC RCC and support considering ESC RCC as an entity given its distinct morphologic, immunophenotypical and molecular characteristics.

A Multi-Center, Real-Life Experience on Liquid Biopsy Practice for EGFR Testing in Non-Small Cell Lung Cancer (NSCLC) Patients.

BACKGROUND: circulating tumor DNA (ctDNA) is a source of tumor genetic material for EGFR testing in NSCLC. Real-word data about liquid biopsy (LB) clinical practice are lacking. The aim of the study was to describe the LB practice for EGFR detection in North Eastern Italy. METHODS: we conducted a multi-regional survey on ctDNA testing practices in lung cancer patients. RESULTS: Median time from blood collection to plasma separation was 50 min (20-120 min), median time from plasma extraction to ctDNA analysis was 24 h (30 min-5 days) and median turnaround time was 24 h (6 h-5 days). Four hundred and seventy five patients and 654 samples were tested. One hundred and ninety-two patients were tested at diagnosis, with 16% EGFR mutation rate. Among the 283 patients tested at disease progression, 35% were T790M+. Main differences in LB results between 2017 and 2018 were the number of LBs performed for each patient at disease progression (2.88 vs. 1.2, respectively) and the percentage of T790M+ patients (61% vs. 26%).

GNAS mutations as prognostic biomarker in patients with relapsed peritoneal pseudomyxoma receiving metronomic capecitabine and bevacizumab: a clinical and translational study.

BACKGROUND: There is lack of evidence about systemic treatment of pseudomyxoma peritonei (PMP) relapsing after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. There is also lack of biomarkers able to predict outcomes beyond known clinical and pathological prognostic features. METHODS: Fifteen patients with relapsed PMP and progressive disease within the last 6 months were included and received metronomic capecitabine (625 mg/mq/day b.i.d.) and bevacizumab (7.5 mg/Kg three-weekly) until progressive disease/unacceptable toxicity. The primary endpoint was progression-free survival (PFS). Ion Torrent(®) next generation sequencing technology (Hot-spot Cancer Panel) was used to characterize molecular features. RESULTS: At a median follow up of 12 months, median PFS was 8.2 months and 1-year overall survival was 91 %. Partial responses were observed in 20 % of cases, but a significant reduction of tumor markers in up to 79 %. Treatment was very well tolerated without no new safety signals. All tumor samples except one had KRAS mutations. Patients with GNAS mutations had a significantly shorter median PFS as compared to GNAS wild-type ones (5.3 months vs. not reached; p < 0.007). The results were externally validated on our previous series of PMP patients. GNAS mutations were rare in a parallel cohort of 121 advanced colorectal cancers (2.5 %), but were associated with peculiar clinical-pathological features and aggressive course. CONCLUSIONS: Metronomic capecitabine and bevacizumab is an active and well tolerated option in patients with relapsed PMP. The negative prognostic effect of GNAS mutations in gastrointestinal cancers warrants further confirmatory studies and may prompt the development of effective targeted strategies.

G48A, a New KRAS Mutation Found in Lung Adenocarcinoma.

A new somatic mutation in the coding region of Kirsten rat sarcoma viral oncogene homolog gene (KRAS), G48A, has been identified in a patient with non-small cell lung cancer (NSCLC). No other mutations were found by screening several genes known to be mutated in NSCLC. The patient responded to first-line therapy and is still under maintenance treatment 18 months after diagnosis. Normal and cancer cells were engineered to express the KRAS(G48A) mutation. KRAS(G48A) overexpression did not change the growth or the response to treatment compared with KRAS(wild type)-expressing cells. Analysis of the structure of the KRAS(G48A) mutant predicted altered interactions with other proteins. Analysis of KRAS binding to B-Raf proto-oncogene, serine/threonine kinase showed that the KRAS(G48A) mutant behaves more like a wild-type than a classical KRAS(G12) mutant. In conclusion, this new mutation in the coding region of KRAS, found in NSCLC, does not induce phenotypic changes similar to those induced by G12 mutants but presumably affects KRAS binding to proteins other than B-Raf proto-oncogene, serine/threonine kinase.

AKT1 and BRAF mutations in pediatric aggressive fibromatosis.

Aside from the CTNNB1 and adenomatous polyposis coli (APC) mutations, the genetic profile of pediatric aggressive fibromatosis (AF) has remained poorly characterized. The aim of this study was to shed more light on the mutational spectrum of pediatric AF, comparing it with its adult counterpart, with a view to identifying biomarkers for use as prognostic factors or new potential therapeutic targets. CTNNB1, APC, AKT1, BRAF TP53, and RET Sanger sequencing and next-generation sequencing (NGS) with the 50-gene Ion AmpliSeq Cancer Hotspot Panel v2 were performed on formalin-fixed samples from 28 pediatric and 33 adult AFs. The prognostic value of CTNNB1, AKT1, and BRAF mutations in pediatric AF patients was investigated. Recurrence-free survival (RFS) curves were estimated with the Kaplan-Meier method and statistical comparisons were drawn using the log-rank test. In addition to the CTNNB1 mutation (64%), pediatric AF showed AKT1 (31%), BRAF (19%), and TP53 (9%) mutations, whereas only the CTNNB1 mutation was found in adult AF. The polymorphism Q472H VEGFR was identified in both pediatric (56%) and adult (40%) AF. Our results indicate that the mutational spectrum of pediatric AF is more complex than that of adult AF, with multiple gene mutations involving not only CTNNB1 but also AKT1 and BRAF. This intriguing finding may have clinical implications and warrants further investigations.

Deciphering intra-tumor heterogeneity of lung adenocarcinoma confirms that dominant, branching, and private gene mutations occur within individual tumor nodules.

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Dissecting Pulmonary Large-Cell Carcinoma by Targeted Next Generation Sequencing of Several Cancer Genes Pushes Genotypic-Phenotypic Correlations to Emerge.

INTRODUCTION: Little is known about genotypic and phenotypic correlations in undifferentiated large-cell carcinoma (LCC) of the lung. METHODS: Thirty LCC were dissected by unsupervised targeted next generation sequencing analysis for 50 cancer-associated oncogenes and tumor suppressor genes. Cell differentiation lineages were unveiled by using thyroid transcription factor-1 (TTF1) for adenocarcinoma (ADC) and p40 for squamous cell carcinoma (SQC), dichotomizing immunohistochemistry (IHC) results for TTF1 as negative or positive (whatever its extent) and for p40 as negative, positive, or focal (if <10% of reactive tumor cells). RESULTS: Three LCC were wild type (all TTF1+/p40-), whereas the remaining 27 (90%) tumors had at least one gene mutation. Twenty-four cases featuring TTF1+/p40-, TTF1+/p40±, TTF1-/p40±, or TTF1-/p40- phenotypes comprised ATM, BRAF, CDKN2A, EGFR, ERBB4, FBXW7, FLT3, KRAS, NRAS, PIK3CA, PTPN11, RET, SMAD4, SMO, STK11, or TP53 mutations in keeping with ADC lineage, whereas three tumors showing TTF1-/p40+ phenotype harbored TP53 only and no ADC-related mutations in keeping with SQC lineage. Single, double, triple, quadruple, and quintuple mutations occurred in 16, 6, 2, 2, and 1 patient, respectively. The occurrence of three mutations or more but not any immunohistochemistry categorization predicted shorter overall survival (OS, p = 0.001) and disease-free survival (DFS, p = 0.007), independent of age, sex, and tumor stage. CONCLUSIONS: Albeit preliminary also because of the relatively small number of LCC under evaluation, this targeted next generation sequencing study, however, revealed gene mutation heterogeneity in LCC with some genotypic-phenotypic correlations. Negativity or focal occurrence of p40 made SQC diagnosis unlikely on molecular grounds, but suggested ADC confirming validity of the axiom "no p40, no squamous."

Different clinical effects upon separate inhibition of coexisting EGFR and PI3KCA mutations in a lung adenocarcinoma patient.

We present a very interesting case of lung adenocarcinoma carrying an uncommon EGFR exon 19 insertion with concomitant PIK3CA mutation showing dramatic and persisting improvement upon erlotinib therapy, after progression during PI3K inhibitor.