Integration of the Molecular Classification of Endometrial Carcinoma to Select Patients for Fertility Sparing Strategies.

Fertility-sparing treatment (FST) for endometrial carcinoma (EC) is an option for a subgroup of young women with low-risk disease. The low-risk group comprises patients with endometrioid EC stage IA, grade 1, with or without focal lymphovascular invasion. In the era of molecular subtyping, treatment de-escalation for some EC subtypes is recommended. Recommendations for fertility-preserving treatments were developed regardless of the molecular classification of EC. However, few studies have focused on this topic. In this review, we summarize the actual data available in the literature and discuss the impact of some molecular subtypes of FST.

Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Novel prognostic nomogram for predicting recurrence-free survival in medullary thyroid carcinoma.

AIMS: Recently, there have been attempts to improve prognostication and therefore better guide treatment for patients with medullary thyroid carcinoma (MTC). In 2022, the International MTC Grading System (IMTCGS) was developed and validated using a multi-institutional cohort of 327 patients. The aim of the current study was to build upon the findings of the IMTCGS to develop and validate a prognostic nomogram to predict recurrence-free survival (RFS) in MTC. METHODS AND RESULTS: Data from 300 patients with MTC from five centres across the USA, Europe, and Australia were used to develop a prognostic nomogram that included the following variables: age, sex, AJCC stage, tumour size, mitotic count, necrosis, Ki67 index, lymphovascular invasion, microscopic extrathyroidal extension, and margin status. A process of 10-fold cross-validation was used to optimize the model's performance. To assess discrimination and calibration, the area-under-the-curve (AUC) of a receiver operating characteristic (ROC) curve, concordance-index (C-index), and dissimilarity index (D-index) were calculated. Finally, the model was externally validated using a separate cohort of 87 MTC patients. The model demonstrated very strong performance, with an AUC of 0.94, a C-index of 0.876, and a D-index of 19.06. When applied to the external validation cohort, the model had an AUC of 0.9. CONCLUSIONS: Using well-established clinicopathological prognostic variables, we developed and externally validated a robust multivariate prediction model for RFS in patients with resected MTC. The model demonstrates excellent predictive capability and may help guide decisions on patient management. The nomogram is freely available online at https://nomograms.shinyapps.io/MTC_ML_DFS/.

Immunodetection of NUT Protein: Implementation, Indications, and Results in a Tertiary Reference Center.

The immunodetection of NUT protein is a reliable tool to identify NUT carcinoma, a rare and still underdiagnosed tumor entity. The technique was implemented in 2017 in our department, a tertiary reference center with a large recruitment in all tumor types, including head and neck and thoracic tumors. We evaluated its use over a 6-year period (2017-2022) to (a) describe the indications for the technique, (b) determine the number of NUT carcinomas detected and confirmed by Fluorescence in situ hybridization, and (c) describe briefly the characteristics of these tumors. Over the study period, 382 NUT immunodetections were performed; the annual number of requests varied from 45 to 83. All 21 pathologists of the department made at least one request (range: 1 to 94; annual mean: 18.2). 54.7% of immunodetections were performed for internal cases, 37% for cases submitted for consultation, and 8.3% for cases submitted for confirmation of a suspected diagnosis. The main indications were poorly differentiated tumors of the head and neck region (39%) and the thorax (19.6%), and difficult-to-classify soft tissue tumors (11.8%). Twelve cases of NUT carcinoma were detected by immunohistochemistry and confirmed by Fluorescence in situ hybridization. Seven were from the head and neck region (4.7% of the tumors tested), 4 from lung or mediastinum (5.3%), 1 from an unknown primary at the time of diagnosis. In conclusion, the implementation of NUT immunodetection in the daily workflow of a pathology department improves the detection of NUT carcinoma. This becomes essential with the emergence of potential targeted therapies.

Impact of lymphovascular invasion on otherwise low-risk papillary thyroid carcinomas: a retrospective and observational study.

PURPOSE: Presence of venous vascular invasion is a criterion of intermediate risk of recurrence in papillary thyroid carcinoma (PTC). However, the presence and type of vascular invasion (lymphatic or venous) is often underreported and its impact on PTCs without other risk features remains unknown. The aim of this study was to evaluate the impact of both lymphatic and venous invasion on the risk of recurrence/persistence on otherwise low-risk PTCs. METHODS: Retrospective study including patients with otherwise low-risk PTCs but with vascular invasion, diagnosed between 2013 and 2019. The persistence/recurrence during the follow-up was evaluated. Pathology was reviewed to confirm the presence of lymphovascular invasion and determine the type of invasion. RESULTS: A total of 141 patients were included. Lymphovascular invasion was confirmed in 20.6%. After surgery, 48.9% (N = 69) of the patients received radioactive iodine (RAI). The median follow-up time was 4 [3-6] years. Overall, 6 (4.2%) patients experienced persistent/recurrent disease in the neck, including 3 with lymphovascular invasion, confirmed as "only lymphatic". Overall, patients with tumors harboring lymphovascular invasion had sensibly more persistent/recurrence disease compared with those without lymphovascular invasion (10.3% vs 2.7%, p = 0.1), especially in the subgroup of patients not treated with RAI (20% vs 1.6%, p = 0.049) [OR 15.25, 95% CI 1.24-187.85, p = 0.033]. CONCLUSION: Lymphovascular invasion, including lymphatic invasion only, is associated with a sensibly higher risk of persistent/recurrent disease in otherwise low-risk PTCs, namely in patients not treated with RAI. Lymphatic invasion could have a role in risk-stratification systems for decision making.

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.

Patterns of Treatment Failure After Selective Rearranged During Transfection (RET) Inhibitors in Patients With Metastatic Medullary Thyroid Carcinoma.

PURPOSE: Medullary thyroid cancer (MTC) harbors frequent mutations in RET oncogene. Selective RET inhibitors (RETi) have emerged as effective treatments. However, resistance almost invariably occurs. METHODS: MTC patients who were initiated on RETi between 2018 and 2022 were included. Baseline characteristics, RET mutational status, RETi response, available tumor tissue and molecular profiles sampled pre- and post-RETi were analyzed. RESULTS: Among 46 MTC patients on RETi during the study period, 26 patients had discontinued at data cut-off because of progression (n = 16), death (n = 4), and toxicity (n = 6). The most frequent RET mutations at baseline were p.M918T (n = 29), and p.C634X (n = 6). Pre- and post-RETi molecular profiles were available in 14 patients. There was no primary resistance on pre-RETi samples. Post-RETi profiles revealed a bypass mechanism of resistance in 75% of the cases including RAS genes mutations (50%), FGFR2 and ALK fusions and and MYC p.P44L. RET solvent from and hinge region mutations was the only resistance mechanisms in 25% of the cases. Tumor samples from initial thyroidectomy, pre- and post-RETi, from six patients, showed an increase of the mean Ki 67-index of 7%, 17% and 40% respectively (P = 0.037) and a more aggressive poorly differentiated histology in three patients. DISCUSSION: Bypass resistance may be the most frequent mechanism of progression under RETi. A more aggressive histology may arise following RETi and warrants further investigation.

Implementing patient derived organoids in functional precision medicine for patients with advanced colorectal cancer.

BACKGROUND: Patient Derived Organoids (PDOs) emerged as the best technology to develop ex vivo tumor avatars. Whether drug testing on PDOs to identify efficient therapies will bring clinical utility by improving patient survival remains unclear. To test this hypothesis in the frame of clinical trials, PDO technology faces three main challenges to be implemented in routine clinical practices: i) generating PDOs with a limited amount of tumor material; ii) testing a wide panel of anti-cancer drugs; and iii) obtaining results within a time frame compatible with patient disease management. We aimed to address these challenges in a prospective study in patients with colorectal cancer (CRC). METHODS: Fresh surgical or core needle biopsies were obtained from patients with CRC. PDOs were established and challenged with a panel of 25 FDA-approved anti-cancer drugs (chemotherapies and targeted therapies) to establish a scoring method ('chemogram') identifying in vitro responders. The results were analyzed at the scale of the cohort and individual patients when the follow-up data were available. RESULTS: A total of 25 PDOs were successfully established, harboring 94% concordance with the genomic profile of the tumor they were derived from. The take-on rate for PDOs derived from core needle biopsies was 61.5%. A chemogram was obtained with a 6-week median turnaround time (range, 4-10 weeks). At least one hit (mean 6.16) was identified for 92% of the PDOs. The number of hits was inversely correlated to disease metastatic dissemination and the number of lines of treatment the patient received. The chemograms were compared to clinical data obtained from 8 patients and proved to be predictive of their response with 75% sensitivity and specificity. CONCLUSIONS: We show that PDO-based drug tests can be achieved in the frame of routine clinical practice. The chemogram could provide clinicians with a decision-making tool to tailor patient treatment. Thus, PDO-based functional precision oncology should now be tested in interventional trials assessing its clinical utility for patients who do not harbor activable genomic alterations or have developed resistance to standard of care treatments.

Primary Colorectal Tumor Displays Differential Genomic Expression Profiles Associated with Hepatic and Peritoneal Metastases.

BACKGROUND: Despite improvements in characterization of CRC heterogeneity, appropriate risk stratification tools are still lacking in clinical practice. This study aimed to elucidate the primary tumor transcriptomic signatures associated with distinct metastatic routes. METHODS: Primary tumor specimens obtained from CRC patients with either isolated LM (CRC-Liver) or PM (CRC-Peritoneum) were analyzed by transcriptomic mRNA sequencing, gene set enrichment analyses (GSEA) and immunohistochemistry. We further assessed the clinico-pathological associations and prognostic value of our signature in the COAD-TCGA independent cohort. RESULTS: We identified a significantly different distribution of Consensus Molecular Subtypes between CRC-Liver and CRC-peritoneum groups. A transcriptomic signature based on 61 genes discriminated between liver and peritoneal metastatic routes. GSEA showed a higher expression of immune response and epithelial invasion pathways in CRC-Peritoneum samples and activation of proliferation and metabolic pathways in CRC-Liver samples. The biological relevance of RNA-Seq results was validated by the immunohistochemical expression of three significantly differentially expressed genes (ACE2, CLDN18 and DUSP4) in our signature. In silico analysis of the COAD-TCGA showed that the CRC-Peritoneum signature was associated with negative prognostic factors and poor overall and disease-free survivals. CONCLUSIONS: CRC primary tumors spreading to the liver and peritoneum display significantly different transcriptomic profiles. The implementation of this signature in clinical practice could contribute to identify new therapeutic targets for stage IV CRC and to define individualized follow-up programs in stage II-III CRC.

Ki67 proliferation index in medullary thyroid carcinoma: a comparative study of multiple counting methods and validation of image analysis and deep learning platforms.

AIMS: The International Medullary Thyroid Carcinoma Grading System, introduced in 2022, mandates evaluation of the Ki67 proliferation index to assign a histological grade for medullary thyroid carcinoma. However, manual counting remains a tedious and time-consuming task. METHODS AND RESULTS: We aimed to evaluate the performance of three other counting techniques for the Ki67 index, eyeballing by a trained experienced investigator, a machine learning-based deep learning algorithm (DeepLIIF) and an image analysis software with internal thresholding compared to the gold standard manual counting in a large cohort of 260 primarily resected medullary thyroid carcinoma. The Ki67 proliferation index generated by all three methods correlate near-perfectly with the manual Ki67 index, with kappa values ranging from 0.884 to 0.979 and interclass correlation coefficients ranging from 0.969 to 0.983. Discrepant Ki67 results were only observed in cases with borderline manual Ki67 readings, ranging from 3 to 7%. Medullary thyroid carcinomas with a high Ki67 index (≥ 5%) determined using any of the four methods were associated with significantly decreased disease-specific survival and distant metastasis-free survival. CONCLUSIONS: We herein validate a machine learning-based deep-learning platform and an image analysis software with internal thresholding to generate accurate automatic Ki67 proliferation indices in medullary thyroid carcinoma. Manual Ki67 count remains useful when facing a tumour with a borderline Ki67 proliferation index of 3-7%. In daily practice, validation of alternative evaluation methods for the Ki67 index in MTC is required prior to implementation.

Alkylating Agent-Induced High Tumor Mutational Burden in Medullary Thyroid Cancer and Response to Immune Checkpoint Inhibitors: Two Case Reports.

Background: Patients with metastatic medullary thyroid cancer (MTC) who progressed under tyrosine kinase inhibitors can benefit from an alkylating agent such as dacarbazine or temozolomide. Patient Findings: We describe two patients with metastatic MTC who developed a hypermutant phenotype after alkylating agent treatment. This phenotype was characterized by a high tumor mutational burden (TMB) and a mutational signature indicative of alkylating agent mutagenesis (single-base substitution 11). Both patients received immune checkpoint inhibitors, with partial morphological responses, clinical benefit, and progression-free survival of 6 and 9 months, respectively. Summary and Conclusions: Based on the described observations, we suggest that a hypermutant phenotype may be induced after alkylating agent treatment for MTC and the sequential use of immunotherapy should be further explored as a treatment option for MTC patients with increased TMB.

NF1-Associated Inflammatory Polyp of the Colon: First Report of a Sporadic Case.

"Juvenile-like (hyperplastic/inflammatory) mucosal polyp" is a term proposed for rare benign mesenchymal lesions of the gastro-intestinal tract so far reported only in patients with type 1 neurofibromatosis (NF1). We report here a first sporadic case of NF1-associated mucosal inflammatory polyp of the colon. The diagnosis was made in a 53-year old female patient with a large polypoid tumor of the cecum. The lesion was predominantly mucosal, made of fibroblast-like cells associated with inflammatory infiltrates rich in eosinophils and containing entrapped, distorted epithelial glands, responsible for the juvenile-like appearance. Whole exome sequencing showed a pathogenic variant of NF1. The patient had no evidence of NF1; no NF1 mutation was detected in normal tissues. Our observation may support the existence of juvenile-like inflammatory polyps associated with NF1 alterations, either germline or somatic. This justifies to test NF1 in difficult-to-classify gastrointestinal mesenchymal tumors.

International Medullary Thyroid Carcinoma Grading System: A Validated Grading System for Medullary Thyroid Carcinoma.

PURPOSE: Medullary thyroid carcinoma (MTC) is an aggressive neuroendocrine tumor (NET) arising from the calcitonin-producing C cells. Unlike other NETs, there is no widely accepted pathologic grading scheme. In 2020, two groups separately developed slightly different schemes (the Memorial Sloan Kettering Cancer Center and Sydney grade) on the basis of proliferative activity (mitotic index and/or Ki67 proliferative index) and tumor necrosis. Building on this work, we sought to unify and validate an internationally accepted grading scheme for MTC. PATIENTS AND METHODS: Tumor tissue from 327 patients with MTC from five centers across the United States, Europe, and Australia were reviewed for mitotic activity, Ki67 proliferative index, and necrosis using uniform criteria and blinded to other clinicopathologic features. After reviewing different cutoffs, a two-tiered consensus grading system was developed. High-grade MTCs were defined as tumors with at least one of the following features: mitotic index ≥ 5 per 2 mm2, Ki67 proliferative index ≥ 5%, or tumor necrosis. RESULTS: Eighty-one (24.8%) MTCs were high-grade using this scheme. In multivariate analysis, these patients demonstrated decreased overall (hazard ratio [HR] = 11.490; 95% CI, 3.118 to 32.333; P < .001), disease-specific (HR = 8.491; 95% CI, 1.461 to 49.327; P = .017), distant metastasis-free (HR = 2.489; 95% CI, 1.178 to 5.261; P = .017), and locoregional recurrence-free (HR = 2.114; 95% CI, 1.065 to 4.193; P = .032) survivals. This prognostic power was maintained in subgroup analyses of cohorts from each of the five centers. CONCLUSION: This simple two-tiered international grading system is a powerful predictor of adverse outcomes in MTC. As it is based solely on morphologic assessment in conjunction with Ki67 immunohistochemistry, it brings the grading of MTCs in line with other NETs and can be readily applied in routine practice. We therefore recommend grading of MTCs on the basis of mitotic count, Ki67 proliferative index, and tumor necrosis.

Cancer Induces a Stress Ileopathy Depending on ß-Adrenergic Receptors and Promoting Dysbiosis that Contributes to Carcinogenesis.

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of ß-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies. SIGNIFICANCE: Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a ß-adrenergic receptor-dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.-related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy. This article is highlighted in the In This Issue feature, p. 873.

Identification of Specific Tumor Markers in Vulvar Carcinoma Through Extensive Human Papillomavirus DNA Characterization Using Next Generation Sequencing Method.

OBJECTIVES: A subset of vulvar carcinomas (VC) are associated with human papillomavirus (HPV) DNA. This trait can be used to identify tumor markers for patient's follow-up. A large diversity of HPV prevalence in VC has been reported, but no data are available concerning the insertional HPV status in this tumor type. Therefore, we have used an innovative next generation sequencing (NGS)-based CaptHPV method able to provide an extensive characterization of HPV DNA in tumors. MATERIAL AND METHODS: Tumor tissue specimens from 55 patients with VC were analyzed using p16 immunohistochemistry, in situ hybridization, polymerase chain reaction, and CaptHPV-NGS assays. RESULTS: Our analyses showed that 8 (14.5%) of 55 cases were associated with HPV 16 DNA. No other HPV genotypes were identified. The HPV genome was in a free episomal state only in one case and both episomal and integrated into the tumor cell genome in 7. There was a single insertion in 5 cases and multiple sites, scattered at different chromosomal loci in two. ISH data suggest that some of these might reflect tumor heterogeneity. Viral integration targeted cellular genes among which were TP63, CCDC148, LOC100133091, PKP1, and POLA2. Viral integration at the PKP1 locus was associated with partial gene deletion, and no PKP1 protein was detected in tumor tissue. CONCLUSIONS: Using the NGS-based innovative capture-HPV approach, we established a cartography of HPV 16 DNA in 8 VC cases and identified novel genes targeted by integration that may be used as specific tumor markers. In addition, we established a rationale strategy for optimal characterization of HPV status in VC.