Challenges and future perspectives for the use of temozolomide in the treatment of SCLC.

Small-cell lung cancer (SCLC), accounting for 10-20 % of all lung tumors, represents the most aggressive high-grade neuroendocrine carcinoma. Most patients are diagnosed with extensive-stage SCLC (ES-SCLC), with brian metastases identified in âˆ¼ 80 % of cases during the disease cours, and the prognosis is dismal, with a 5-year survival rate of less than 5 %. Current available treatments in the second-line setting are limited, and topotecan has long been the only FDA-approved drug in relapsed or refractory ES-SCLC, until the recent approval of lurbinectedin, a selective inhibitor of RNA polymerase II. Temozolomide (TMZ) is an oral alkylating agent, which showed single-agent activity in SCLC, particularly among patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Several studies have revealed the synergistic activity of temozolomide with poly-ADP-ribose polymerase (PARP) inhibitors, that prevent repair of TMZ-induced DNA damage. This review focuses on the rationale for the use of TMZ in ES-SCLC and provides an overview of the main trials that have evaluated and are currently investigating its role, both as a single-agent and in combinations, in relapse or refractory disease.

Analysis of MDM2 and TP53 genes in canine liposarcoma.

Canine liposarcoma is an uncommon tumor that shares morphological similarities with its human counterpart. In dogs, the genetic features of this tumor are unknown and, based on immunohistochemical studies, amplification of the gene MDM2 and the mutation of TP53 are suspected. In this study 51 cases of primary liposarcomas were immunohistochemically stained for MDM2 and p53 and subjected to fluorescent in situ hybridization and next-generation sequencing to detect MDM2 amplification and TP53 mutations, respectively. MDM2 and p53 were expressed in 21 and 6 cases, respectively. MDM2 amplification and TP53 mutations were identified in 10 and 15 cases, respectively. Statistical analysis revealed an association of the myxoid subtype and the mitotic count with p53 expression and TP53 mutation. No association was found between MDM2 amplification and MDM2 expression or tumor subtype. These results suggest that despite morphological similarities, canine liposarcoma differs from its human counterpart, for which MDM2 amplification is diagnostic for well differentiated and de-differentiated variants, and TP53 mutations are more common in pleomorphic liposarcoma rather than the myxoid one as occur in our cases. Furthermore, canine myxoid liposarcoma likely represents a distinct disease rather than a mere morphological variant.

Beyond histology: A tissue algorithm predictive of post-surgical recurrence in hepatocellular carcinomas, including TERT promoter mutation.

Surgical resection for hepatocellular carcinoma (HCC) is burdened with a high recurrence rate and a lack of reliable prognostic factors. The aim of this study was to integrate the HCC pathological features with gene mutations to improve the prognostic role of pathological analysis. This is a monocentric prospective study, including 67 patients resected for HCC. All clinical data and histological features were collected, including tumor grade, architecture, margins, microvascular invasion, and microscopic portal vascular invasion (MPVI). Next-generation sequencing (NGS) was performed using a laboratory-developed multi-gene panel, allowing to amplify 330 amplicons (21.77 kb), covering the relevant targets for solid tumor analysis. The most represented mutations were TERT promoter (n = 41, 61.2%), TP53 (n = 18, 26.9%) and CTNNB1 (n = 17, 25.4%). At follow-up, 13 (19.4%) patients experienced HCC recurrence: at multivariate analysis, tumor dimensions (p = 0.040), MPVI (p = 0.010), and TERT mutation (p = 0.034) correlated with recurrence. Dimensions ≥ 4.5 cm (very close to AJCC stage pT3; 9 recurrences, p = 0.041, odd-ratio = 3.7), MPVI (9 recurrences, p = 0.062, OR = 3.3), and TERT (11 recurrences, p = 0.049, OR = 4.4) correlated with disease-free survival also at univariate analysis. The concomitant occurrence of these three variables was present in 7 cases, among which 5 recurred (p = 0.002, OR = 15.94). In conclusion, NGS analysis in resected HCC could not only be used for future therapies but should be integrated with histopathology to predict the risk of tumor recurrence after surgical resection: TERT mutation is among the strongest predictors of tumor recurrence, together with tumor stage (dimensions) and the occurrence of MPVI, which should always be reported separately from the classic MVI.

Molecular Characterization of Advanced-Stage Melanomas in Clinical Practice Using a Laboratory-Developed Next-Generation Sequencing Panel.

Cutaneous melanoma is one of the most lethal tumors among skin cancers, characterized by complex genetic and molecular alterations that result in uncontrolled cell proliferation and metastatic spread. Next-generation sequencing (NGS) enables the simultaneous examination of numerous genes, making this molecular technique essential for melanoma diagnosis, prognostic stratification, and therapy planning. Herein, we present the experience with our laboratory-designed NGS panel for the routine assessment of advanced-stage melanoma. A total of 260 specimens of advanced-stage melanomas were evaluated utilizing a laboratory-developed multi-gene NGS panel, which allowed the investigation of 229 amplicons in 25 oncogene/oncosuppressor genes. The NGS panel proved to be a reliable tool, failing to produce results in only 1.2% of the samples tested. BRAF and TERT were the two more commonly altered genes in 44.0% and 59.9% of samples, respectively. In 59.3% of the mutated cases, at least two concomitant variants were detected. In eight cases, both primary lesion and metastatic disease were analyzed by NGS. In all specimens (8/8, 100%), a perfect concordance in variants harbored by the primary and recurrence lesions was observed. Finally, this study described the validity of a laboratory-developed multi-gene NGS panel built specifically for advanced-stage melanomas in ordinary clinical practice.

Accuracy of World Health Organisation-grade parameters (necrosis and mitotic activity) and foci of vascular invasion in predicting prognosis of papillary thyroid carcinoma. A case-control validation study.

AIMS: Tumour necrosis and/or increased mitoses define high-grade papillary thyroid carcinoma (PTC). It is unclear whether angioinvasion is prognostic for PTC. Cut-offs at five or more mitoses/2 mm2 and four or more angioinvasive foci have been empirically defined based upon data from all forms of aggressive non-anaplastic thyroid carcinomas. Performance of tumour necrosis, mitoses and vascular invasion in predicting distant metastases when specifically applied to PTC is undefined. METHODS: We analysed 50 consecutive PTC cases with distant metastases (DM-PTC): 16 synchronous and 34 metachronous. A total of 108 non-metastatic PTC (N-DM-PTC, 15.0-year median follow-up) were used as controls. Invasive encapsulated follicular variant PTC was excluded. Necrosis, mitoses and angioinvasion were quantified. Receiver operating characteristics (ROC) and area under the curve (AUC) analyses determined best sensitivity and specificity cut-offs predictive of distant metastases. RESULTS: Metastases correlated with necrosis (any extent = 43.8% all DM-PTC, 53.1% metachronous DM-PTC versus 5% N-DM-PTC; P < 0.001), mitoses (P < 0.001) and angioinvasion (P < 0.001). Mitoses at five or more per 2 mm2 was the best cut-off correlating with distant metastases: sensitivity/specificity 42.9%/97.2% all DM-PTC (AUC = 0.78), 18.8%/97.2% synchronous DM-PTC (AUC = 0.63), 54.6%/97.2% metachronous DM-PTC (AUC = 0.85). Angioinvasive foci at five or more was the best cut-off correlating with distant metastases: sensitivity/specificity 36.2%/91.7% all DM-PTC (AUC = 0.75), 25%/91.7% synchronous DM-PTC (AUC = 0.79) and 41.9%/91.7% metachronous DM-PTC (AUC = 0.73). Positive/negative predictive values (PPV/NPV) were: necrosis 22.6%/98.2%; five or more mitoses 32.3%/98.2%; five or more angioinvasive foci 11.8%/97.9%. After multivariable analysis, only necrosis and mitotic activity remained associated with DM-PTC. CONCLUSION: Our data strongly support PTC grading, statistically validating World Health Organisation (WHO) criteria to identify poor prognosis PTC. Angioinvasion is not an independent predictor of DM-PTC.

Primary vulvar adenocarcinoma of intestinal type: Report of two cases showing molecular similarity with colorectal adenocarcinoma.

Primary vulvar adenocarcinoma is a particularly rare tumor with poorly understood histogenesis and unclear clinical characteristics and prognosis. Vulvar adenocarcinoma of intestinal type (VAIt) is a very uncommon subtype of primary vulvar adenocarcinoma and only 27 cases have been described in the literature in the past. Of these cases, two have been described as human papillomavirus (HPV)-associated VAIt. The current report presents two additional cases of primary VAIt showing variants in the KRAS, TP53, and DPYD genes and no evidence of HPV DNA by real-time polymerase chain reaction (RT-PCR). Next-generation sequencing (NGS) revealed TP53 pathogenic variants in both cases, but only one case had aberrant p53 protein immunohistochemical characteristics. KRAS and DPYD mutations were identified separately in the two cases. Due to their capacity to imitate the spread of more prevalent gastrointestinal carcinomas, these tumors may present diagnostic issues. Additional cases can contribute to a better understanding of the pathophysiology and prognosis of VAIt.

Association of the Genomic Profile of Medullary Thyroid Carcinoma with Tumor Characteristics and Clinical Outcomes in an International Multicenter Study.

Purpose: The prognostic importance of RET and RAS mutations and their relationship to clinicopathologic parameters and outcomes in medullary thyroid carcinoma (MTC) need to be clarified. Experimental Design: A multicenter retrospective cohort study was performed utilizing data from 290 patients with MTC. The molecular profile was determined and associations were examined with clinicopathologic data and outcomes. Results: RET germ line mutations were detected in 40 patients (16.3%). Somatic RET and RAS mutations occurred in 135 (46.9%) and 57 (19.8%) patients, respectively. RETM918T was the most common somatic RET mutation (n = 75). RET somatic mutations were associated with male sex, larger tumor size, advanced American Joint Committee Cancer (AJCC) stage, vascular invasion, and high International Medullary Thyroid Carcinoma Grading System (IMTCGS) grade. When compared with other RET somatic mutations, RETM918T was associated with younger age, AJCC (eighth edition) IV, vascular invasion, extrathyroidal extension, and positive margins. RET somatic or germ line mutations were significantly associated with reduced distant metastasis-free survival on univariate analysis, but there were no significant independent associations on multivariable analysis, after adjusting for tumor grade and stage. There were no significant differences in outcomes between RET somatic and RET germ line mutations, or between RETM918T and other RET mutations. Other recurrent molecular alterations included TP53 (4.2%), ARID2 (2.9%), SETD2 (2.9%), KMT2A (2.9%), and KMT2C (2.9%). Among them, TP53 mutations were associated with decreased overall survival (OS) and disease-specific survival (DSS), independently of tumor grade and AJCC stage. Conclusions: RET somatic mutations were associated with high-grade, aggressive primary tumor characteristics, and decreased distant metastatic-free survival but this relationship was not significant after accounting for tumor grade and disease stage. RETM918T was associated with aggressive primary tumors but was not independently associated with clinical outcomes. TP53 mutation may represent an adverse molecular event associated with decreased OS and DSS in MTC, but its prognostic value needs to be confirmed in future studies.

KIT/PDGFRA Variant Allele Frequency as Prognostic Factor in Gastrointestinal Stromal Tumors (GISTs): Results From a Multi-Institutional Cohort Study.

BACKGROUND: The patient selection for optimal adjuvant therapy in gastrointestinal stromal tumors (GISTs) is provided by nomogram based on tumor size, mitotic index, tumor location, and tumor rupture. Although mutational status is not currently used to risk assessment, tumor genotype showed a prognostic influence on natural history and tumor relapse. Innovative measures, such as KIT/PDGFRA-mutant-specific variant allele frequency (VAF) levels detection from next-generation sequencing (NGS), may act as a surrogate of tumor burden and correlate with prognosis and overall survival of patients with GIST, helping the choice for adjuvant treatment. PATIENTS AND METHODS: This was a multicenter, hospital-based, retrospective/prospective cohort study to investigate the prognostic role of KIT or PDGFRA-VAF of GIST in patients with radically resected localized disease. In the current manuscript, we present the results from the retrospective phase of the study. RESULTS: Two-hundred (200) patients with GIST between 2015 and 2022 afferent to 6 Italian Oncologic Centers in the EURACAN Network were included in the study. The receiver operating characteristic (ROC) curves analysis was used to classify "low" vs. "high" VAF values, further normalized on neoplastic cellularity (nVAF). When RFS between the low and high nVAF groups were compared, patients with GIST with KIT/PDGFRA nVAF > 50% showed less favorable RFS than patients in the group of nVAF ≤ 50% (2-year RFS, 72.6% vs. 93%, respectively; P = .003). The multivariable Cox regression model confirmed these results. In the homogeneous sub-population of intermediate-risk, patients with KIT-mutated GIST, the presence of nVAF >50% was statistically associated with higher disease recurrence. CONCLUSION: In our study, we demonstrated that higher nVAF levels were independent predictors of GIST prognosis and survival in localized GIST patients with tumors harboring KIT or PDGFRA mutations. In the cohort of intermediate-risk patients, nVAF could be helpful to improve prognostication and the use of adjuvant imatinib.

TERT promoter mutations and the outcome of patients with advanced urothelial carcinoma treated by platinum-based chemotherapy or pembrolizumab.

BACKGROUND: TERT promoter mutation is one of the most common genomic alterations in urothelial carcinoma (UC). Its prognostic role on patients' outcomes is still not clear. METHODS: We performed a single-center retrospective analysis on patients with advanced UC treated with platinum-based chemotherapy or immunotherapy to assess the presence of somatic TERT-124[C>T] and TERT-146[C>T] mutations and their association with clinicopathologic factors and survival outcomes. Patients were assessed for Overall Survival (OS), Progression-Free Survival (PFS), and Overall Response Rate (ORR). RESULTS: We analyzed 45 UC tumors; 38 of them received first-line chemotherapy and 21 second-line pembrolizumab; 6 patients (13%) harbored -146 C > T TERTp mutation and 25 patients (56%)-124 C > T. The presence of TERT promoter mutations was associated with a higher rate of lower tract UC and a lower rate of synchronous or lymph node metastases. TERT wild-type patients showed higher 12- and 24-months OS-rates in the chemotherapy subgroup and 6-, 12- and 24-months OS rates in the pembrolizumab subgroup. The presence of TERT promoter mutations was also associated with a lower 6 months-PFS rate in patients receiving chemotherapy and in all the three time points in those treated by pembrolizumab. The ORRs of pembrolizumab were 21% and 71% in patients with or without TERT promoter mutations, respectively (p < 0.001). CONCLUSIONS: Our analysis suggests that the presence of TERT promoter mutations could negatively affect the outcome of UC patients treated by chemotherapy or pembrolizumab. This hypothesis should be further evaluated in wider cohorts.

Molecular pathology of endocrine gland tumors: genetic alterations and clinicopathologic relevance.

Tumors of the endocrine glands are common. Knowledge of their molecular pathology has greatly advanced in the recent past. This review covers the main molecular alterations of tumors of the anterior pituitary, thyroid and parathyroid glands, adrenal cortex, and adrenal medulla and paraganglia. All endocrine gland tumors enjoy a robust correlation between genotype and phenotype. High-throughput molecular analysis demonstrates that endocrine gland tumors can be grouped into molecular groups that are relevant from both pathologic and clinical point of views. In this review, genetic alterations have been discussed and tabulated with respect to their molecular pathogenetic role and clinicopathologic implications, addressing the use of molecular biomarkers for the purpose of diagnosis and prognosis and predicting response to molecular therapy. Hereditary conditions that play a key role in determining predisposition to many types of endocrine tumors are also discussed.

Hepatocellular Carcinomas with Concomitant Mutations of TERT, TP53, and CTNNB1: Is There a Role for Artificial Intelligence?

TP53, CTNNB1, and TERT-promoter mutations are the most common driver mutations in hepatocellular carcinoma (HCC). The morphological and genetical HCC heterogeneities are difficult to discriminate by the eye of the pathologist. Here, we describe two rare cases of HCC with simultaneous co-mutation of all three of genes, which represent a poorly described occurrence in the literature. In these two cases, areas with different tumor grade and different ß-catenin and Glutamine Synthetase expression (performed by automated immunohistochemistry) were observed. NGS analysis was performed in these different areas, to check for potential diversity of mutation burden on the different regions, but no differences were found: all micro-areas analyzed showed the co-presence of mutations in TP53, CTNNB1, and TERT. The evidence that all mutations were found in all the different areas analyzed by NGS leads to hypothesize that the tumor is not composed of different clones harboring different mutations. All the variants are harbored by the same neoplastic clone, albeit leading to different phenotypes. Mutation prediction Artificial Intelligence models could help the morpho-genetic classification of HCC in the future, since they can find variabilities not obvious to the human eye, with increased sensitivity, specificity and reproducibility.

Evaluation of an institutional series of low-grade oncocytic tumor (LOT) of the kidney and review of the mutational landscape of LOT.

The 2022 WHO classification of urinary and male genital tumors introduced several novel kidney entities exhibiting eosinophilic/oncocytic features with specific mutational backgrounds. Thus, molecular techniques, such as next-generation sequencing (NGS), became more commonly used for their evaluation. We studied 12 low-grade oncocytic tumors (LOT) of the kidney (from 11 patients), identified in a cohort of 210 eosinophilic/oncocytic renal tumors, diagnosed in our institution between October 2019 and May 2023, which represented 5.7% (12/210) of all eosinophilic/oncocytic renal tumors during this period. We reviewed their clinicopathologic, histologic, and immunohistochemical features, as well as their mutational profiles. We also reviewed the literature on NGS-derived data of LOT, by selecting papers in which LOT diagnosis was rendered according to the criteria proposed initially. Median age was 65 years (mean: 63.5; range 43-79) and median tumor size was 2.0 cm (mean: 2.2; range: 0.9-3.1). All tumors were positive for PAX8, CK7, and GATA3, and negative or focally positive for CD117/KIT. We found the following gene mutations: MTOR ((6/11), 54.5%)), TSC1 ((2/11), 18.2%)), and 1 had both NOTCH1 and NOTCH4 ((1/11), 9.1%)). Wild-type status was found in 2/11 (18.2%) patients and one tumor was not analyzable. A review of 8 previous studies that included 79 LOTs revealed frequent mutations in the genes that regulate the mammalian target of rapamycin (mTOR) pathway: MTOR (32/79 (40.5%)), TSC1 (21/79 (26.6%)), and TSC2 (9/79 (11.4%)). Other mutated genes included PIK3CA, NF2, and PTEN, not typically known to affect the mTOR pathway, but potentially acting as upstream and downstream effectors. Our study shows that LOT is increasingly diagnosed in routine practice when applying the appropriate diagnostic criteria. We also confirm that the mTOR pathway is strongly implicated in the pathogenesis of this tumor mainly through MTOR, TCS1, and TSC2 mutations, but other genes could also be involved in the pathway activation, especially in LOTs without "canonical" mutations.

NRAS-mutated oncocytic benign liver lesion in an organ donor: Pitfalls and troubles in frozen section diagnosis and risk assessment.

BACKGROUND: In the transplant setting, the definition of the risk of neoplastic transmission from donor to recipient often requires intraoperative pathological evaluation on frozen sections. Although most lesions can be easily classified into acceptable or unacceptable risk according to the Italian National Guidelines, there are cases in which unusual histologic features cannot be further investigated because of the lack of ancillary techniques on frozen sections. CASE PRESENTATION: Here we present a case of a liver lesion in a 51-year-old male donor, subjected to histopathological on-call examination. The frozen sections showed a well-demarcated lesion consisting of epithelioid cells disposed in laminar structures and intermingled with a dense lymphocytic population: this led to organ discard with interruption of the donation process. The definitive histological analysis required an extensive immunohistochemical (IHC) investigation: the final diagnosis was "bile duct adenoma with oncocytic features", eventually confirmed by a strongly positive anti-mitochondrial IHC. Finally, an NGS panel analysis was performed, which revealed NRAS mutation. DISCUSSION: To the best of our knowledge, this is the first case of oncocytic bile duct adenoma confirmed by anti-mitochondrial IHC and with NRAS mutation. The most challenging aspect of this case was represented by the transplant setting. In fact, the oncocytic features and the dense lymphocytic infiltrate represented concomitant unusual histological features that led to the halt of the organ donation procedures.

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer.

Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC.

Prognostic Impact of Pathologic Features in Molecular Subgroups of Endometrial Carcinoma.

The molecular characterization of endometrial carcinoma (EC) has recently been included in the ESGO/ESTRO/ESP guidelines. The study aims to evaluate the impact of integrated molecular and pathologic risk stratification in the clinical practice and the relevance of pathologic parameters in predicting prognosis in each EC molecular subgroup. ECs were classified using immunohistochemistry and next-generation sequencing into the four molecular classes: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). According to the WHO algorithm, 219 ECs were subdivided into the following molecular subgroups: 7.8% POLE, 31% MMRd, 21% p53abn, 40.2% NSMP. Molecular classes as well as ESGO/ESTRO/ESP 2020 risk groups were statistically correlated with disease-free survival. Considering the impact of histopathologic features in each molecular class, stage was found to be the strongest prognostic factor in MMRd ECs, whereas in the p53abn subgroup, only lymph node status was associated with recurrent disease. Interestingly, in the NSMP tumor, several histopathologic features were correlated with recurrence: histotype, grade, stage, tumor necrosis, and substantial lymphovascular space invasion. Considering early-stage NSMP ECs, substantial lymphovascular space invasion was the only independent prognostic factor. Our study supports the prognostic importance of EC molecular classification and demonstrated the essential role of histopathologic assessment in patients' management.

"Paradoxical" p16 overexpression in cutaneous melanoma: Molecular and immunohistochemical analysis of a rare phenomenon with a focus on cell cycle regulatory molecules.

BACKGROUND: One of the most relevant genetic alterations in cutaneous melanoma (CM) is the biallelic inactivation/loss-of-heterozygosis (LOH) of cyclin-dependent kinase inhibitor 2 A (CDKN2A), which results in the immunohistochemical loss of p16 frequently found in CM. However, we recently described a rare case of dermal/deep-seated melanoma arising in giant congenital nevus (DDM-GCN) with p16 overexpression combined with p53 loss and tumor protein 53 (TP53) mutation. Herein, we reported a case series of CM with p16 overexpression and analyzed their clinicopathologic features, immunohistochemical expression of the cell cycle regulatory molecules (CCRM: p53, p21, Cyclin D1, Rb), and mutational landscape. METHODS: We retrospectively tested for p16 all cases of CM diagnosed at our institution between January 1st 2019-April 1st 2022. In CM with p16 overexpression, we reported clinicopathologic features, immunohistochemical results for melanocytic markers and CCRM, and mutational landscape investigated with a next-generation sequencing (NGS) panel. In cases with zonal p16 overexpression, the immunohistochemical assessment for melanocytic markers and CCRM, as well as the NGS analysis have been performed in both components {with and without p16 overexpression [p16(+)c and p16(-)]}. RESULTS: Overexpression of p16 was found in 10/2879 (0.35%) CM [5/10 (50%) diffuse and 5/10 (50%) zonal]. We combined the immunohistochemical results for CCRM and molecular data to classify the cases as follows: a) Group 1 with altered expression of at least one CCRM but no TP53 mutations [3/10 (30%), all with Rb altered/lost]; b) Group 2 with altered expression of at least one CCRM and TP53 mutations [4/10 (40%), all with p53 altered]; c) Group 3 with normal expression of CCRM and no TP53 mutations [3/10 (30%), all with mutations in MAPK pathway genes (NRAS and BRAF)]. In CM with zonal p16 overexpression, the histologic appearance of p16(+)c was heterogeneous, whereas combining CCRM profiles and molecular data the cases could be categorized as follows: a) cases with the same CCRM and molecular profiles in both p16(+)c and p16(-)c; b) cases with p16(+)c showing additional genetic mutations and/or modifications of CCRM expression. CONCLUSIONS: p16 overexpression is a rare event, occurring in advanced-stage, clinically- and histologically-heterogeneous CM. These lesions may be classified into three different groups based on CCRM expression and mutational profiles (including TP53 mutation). The analysis of CM with zonal p16 overexpression suggests that, at least in a subset of cases, this phenomenon could represent a sign of "molecular progression" due to the acquisition of additional genetic mutations and/or modifications of the CCRM profile.

Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice.

Introduction: The European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class. Methods: The cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). Results: Immuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between "unknown molecular classification" and "known," the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients. Conclusion: Application of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification.

Expanding the Spectrum of BRAF Non-V600E Mutations in Thyroid Nodules: Evidence-Based Data from a Tertiary Referral Centre.

The BRAF p.V600E mutation represents the most specific marker for papillary thyroid carcinoma and is potentially related to aggressive behavior and persistent disease. BRAF alterations other than the p.V600E are less common in thyroid carcinoma and represent an alternative mechanism of BRAF activation with unclear clinical significance. The study aims to describe the frequency and clinicopathologic characteristics of BRAF non-V600E mutations in a large cohort (1654 samples) of thyroid lesions characterized by next-generation sequencing. BRAF mutations have been found in 20.3% (337/1654) of thyroid nodules, including classic (p.V600E) mutation in 19.2% (317/1654) of samples and non-V600E variants in 1.1% of cases (19/1654). BRAF non-V600E alterations include 5 cases harboring p.K601E, 2 harboring p.V600K substitutions, 2 with a p.K601G variant, and 10 cases with other BRAF non-V600E alterations. BRAF non-V600E mutations have been reported in one case of follicular adenoma, three cases of conventional papillary thyroid carcinoma, eight cases of follicular variant of papillary carcinomas, one case of columnar cell variant papillary thyroid carcinoma, one case of oncocytic follicular carcinoma, and two bone metastasis of follicular thyroid carcinoma. We confirm that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential. However, in both aggressive cases, the BRAF mutations were concomitant with other molecular alterations, such as TERT promoter mutation.

Case report: Dramatic response to pralsetinib in an elderly patient with advanced RET-fusion positive papillary thyroid carcinoma.

We are recently faced with a progressive evolution of the therapeutic paradigm for radioiodine refractory differentiated thyroid cancer (RAI-R DTC), since the advent of tissue agnostic inhibitors. Thus, tumor genotype assessment is always more relevant and is playing a crucial role into clinical practice. We report the case of an elderly patient with advanced papillary thyroid carcinoma (PTC) harboring RET-CCDC6 fusion with four co-occurring mutations involving PI3KCA, TP53, and hTERT mutations, treated with pralsetinib under a compassionate use program. Despite the high histological grade and the coexistence of aggressive RET co-mutations, an impressive metabolic and structural tumor response has been obtained, together with a patient's prolonged clinical benefit. A timely comprehensive molecular testing of those cases wild-type for the common thyroid carcinoma BRAF V600E-like and RAS-like driver mutations may uncover actionable gene rearrangements that can be targeted by highly selective inhibitors with great potential benefit for the patients.