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/.

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.

A Detailed Histologic and Molecular Assessment of the Diffuse Sclerosing Variant of Papillary Thyroid Carcinoma.

Diffuse sclerosing variant papillary thyroid carcinoma (DS-PTC) is characterized clinically by a predilection for children and young adults, bulky neck nodes, and pulmonary metastases. Previous studies have suggested infrequent BRAFV600E mutation but common RET gene rearrangements. Using strict criteria, we studied 43 DS-PTCs (1.9% of unselected PTCs in our unit). Seventy-nine percent harbored pathogenic gene rearrangements involving RET, NTRK3, NTRK1, ALK, or BRAF; with the remainder driven by BRAFV600E mutations. All 10 pediatric cases were all gene rearranged (P = .02). Compared with BRAFV600E-mutated tumors, gene rearrangement was characterized by psammoma bodies involving the entire lobe (P = .038), follicular predominant or mixed follicular architecture (P = .003), pulmonary metastases (24% vs none, P = .04), and absent classical, so-called "BRAF-like" atypia (P = .014). There was no correlation between the presence of gene rearrangement and recurrence-free survival. Features associated with persistent/recurrent disease included pediatric population (P = .030), gene-rearranged tumors (P = .020), microscopic extrathyroidal extension (P = .009), metastases at presentation (P = .007), and stage II disease (P = .015). We conclude that DS-PTC represents 1.9% of papillary thyroid carcinomas and that actionable gene rearrangements are extremely common in DS-PTC. DS-PTC can be divided into 2 distinct molecular subtypes and all BRAFV600E-negative tumors (1.5% of papillary thyroid carcinomas) are driven by potentially actionable oncogenic fusions.

Gene expression profiling of pancreatic ductal adenocarcinomas in response to neoadjuvant chemotherapy.

AIM: Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all major cancers. Chemotherapy is the mainstay systemic therapy for PDAC, and chemoresistance is a major clinical problem leading to therapeutic failure. This study aimed to identify key differences in gene expression profile in tumors from chemoresponsive and chemoresistant patients. METHODS: Archived formalin-fixed paraffin-embedded tumor tissue samples from patients treated with neoadjuvant chemotherapy were obtained during surgical resection. Specimens were macrodissected and gene expression analysis was performed. Multi- and univariate statistical analysis was performed to identify differential gene expression profile of tumors from good (0%-30% residual viable tumor [RVT]) and poor (>30% RVT) chemotherapy-responders. RESULTS: Initially, unsupervised multivariate modeling was performed by principal component analysis, which demonstrated a distinct gene expression profile between good- and poor-chemotherapy responders. There were 396 genes that were significantly (p < 0.05) downregulated (200 genes) or upregulated (196 genes) in tumors from good responders compared to poor responders. Further supervised multivariate analysis of significant genes by partial least square (PLS) demonstrated a highly distinct gene expression profile between good- and poor responders. A gene biomarker of panel (IL18, SPA17, CD58, PTTG1, MTBP, ABL1, SFRP1, CHRDL1, IGF1, and CFD) was selected based on PLS model, and univariate regression analysis of individual genes was performed. The identified biomarker panel demonstrated a very high ability to diagnose good-responding PDAC patients (AUROC: 0.977, sensitivity: 82.4%; specificity: 87.0%). CONCLUSION: A distinct tumor biological profile between PDAC patients who either respond or not respond to chemotherapy was identified.

A novel prognostic nomogram for predicting survival in diffuse pleural mesothelioma.

Recent advances in the management of diffuse pleural mesothelioma (DPM) have increased interest in prognostication and risk stratification on the basis that maximum benefit of combination immunotherapy appears to be seen in patients who otherwise would have the worst prognosis. Various grading schemes have been proposed, including the recently published Mesothelioma Weighted Grading Scheme (MWGS). However, predictive modelling using deep learning algorithms is increasingly regarded as the gold standard in prognostication. We therefore sought to develop and validate a prognostic nomogram for DPM. Data from 369 consecutive patients with DPM were used as independent training and validation cohorts to develop a prognostic tool that included the following variables: age, sex, histological type, nuclear atypia, mitotic count, necrosis, and BAP1 immunohistochemistry. Patients were stratified into four risk groups to assess model discrimination and calibration. To assess discrimination, the area-under-the-curve (AUC) of a receiver-operator-curve (ROC), concordance-index (C-index), and dissimilarity index (D-index) were calculated. Based on the 5-year ROC analysis, the AUC for our model was 0.75. Our model had a C-index of 0.67 (95% CI 0.53-0.79) and a D-index of 2.40 (95% CI 1.69-3.43). Our prognostic nomogram for DPM is the first of its kind, incorporates well established prognostic markers, and demonstrates excellent predictive capability. As these factors are routinely assessed in most pathology laboratories, it is hoped that this model will help inform prognostication and difficult management decisions, such as patient selection for novel therapies. This nomogram is now freely available online at: https://nomograms.shinyapps.io/Meso_Cox_ML/.

A Clinicopathologic and Molecular Analysis of Fumarate Hydratase-deficient Pheochromocytoma and Paraganglioma.

Up to 40% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are hereditary. Germline mutations/deletions in fumarate hydratase ( FH ) cause hereditary leiomyomatosis and renal cell carcinoma syndrome which manifests predominantly with FH-deficient uterine/cutaneous leiomyomas and renal cell carcinomas (RCCs)-tumors characterized by loss of immunohistochemical (IHC) expression of FH and/or positive staining for S-(2-succino)-cysteine. Occasional patients develop PCC/PGL. We investigated the incidence, morphologic, and clinical features of FH-deficient PCC/PGL. We identified 589 patients with PCC/PGLs that underwent IHC screening for FH and/or S-(2-succino)-cysteine. Eight (1.4%) PCC/PGLs were FH deficient (1.1% in an unselected population). The median age for FH-deficient cases was 55 (range: 30 to 77 y) with 50% arising in the adrenal. All 4 with biochemical data were noradrenergic. Two (25%) metastasized, 1 dying of disease after 174 months. Germline testing was performed on 7 patients, 6 of whom had FH missense mutations. None were known to have a significant family history before presentation or developed cutaneous leiomyomas, or FH-deficient RCC at extended follow-up. The patient wild-type for FH on germline testing was demonstrated to have somatic FH mutation and loss of heterozygosity corresponding to areas of subclonal FH deficiency in her tumor. One patient did not undergo germline testing, but FH mutation was demonstrated in his tumor. We conclude that FH-deficient PCC/PGL are underrecognized but can be identified by IHC. FH-deficient PCC/PGL are strongly associated with germline missense mutations but are infrequently associated with leiomyoma or RCC, suggesting there may be a genotype-phenotype correlation. FH-deficient PCC/PGL may have a higher metastatic risk.

What are the problems with the current staging of discontinuous tumour nodules (DTNs) in colorectal carcinoma? Is there a better way?

Discontinuous extramural tumour nodules (DTNs) are deposits of metastatic carcinoma in soft tissue not associated with lymph nodes. Although they are established as an adverse prognostic factor in colorectal carcinoma (CRC), under the AJCC eighth edition staging system their presence does not upstage patients who also have lymph node metastases. Counterintuitively, in some situations the presence of lymph node metastases may in effect downstage a patient with DTNs from pN1c to pN1a/b. Therefore, we sought to critically assess the significance of DTNs in a large unselected single institution cohort of patients undergoing surgical resection for CRC. Of 3822 CRC patients undergoing surgical resection from 2005 to 2021, DTNs were present in 686 (18.0%). In univariate (HR=2.687, 95CI 2.355-3.065; p<0.001) and multivariate analysis (HR=1.805, 95CI 1.529-2.132; p<0.001) in a model including age, gender, stage, grade, location, lymph node ratio and apical lymph node status, DTNs were associated with worse overall survival (OS). N1c patients (DTN present but no nodal metastasis) demonstrated worse OS compared to the current pN1a group (p<0.001) and were least different to the current pN2a group (p=0.571). Within the current N1a (p=0.013), N1b (p=0.004) and N2a (p=0.002) groups, patients who also had DTNs had worse OS. DTNs were associated with worse OS for all stage III CRCs combined (p<0.001), and for stage IIIB (p<0.001) and stage IIIC (p=0.007) individually. We conclude that DTNs are an independent adverse prognostic factor that should be considered in the staging system in a way that is additional to (rather than integrated with) the number of involved lymph nodes. We then assess a simple suggestion for how this could be achieved by increasing the overall stage by one group in the presence of DTNs (requiring the creation of a new stage IIID).

Deep Sequencing of Early T Stage Colorectal Cancers Reveals Disruption of Homologous Recombination Repair in Microsatellite Stable Tumours with High Mutational Burdens.

Early T stage colorectal cancers (CRC) that invade lymph nodes (Stage IIIA) are rare and greatly under-represented in large-scale genomic mapping projects. We retrieved 10 Stage IIIA CRC cases, matched these to 16 Stage 1 CRC cases (T1 depth without lymph node metastasis) and carried out deep sequencing of 409 genes using the IonTorrent system. Tumour mutational burdens (TMB) ranged from 2.4 to 77.2/Mb sequenced. No stage-related mutational differences were observed, consistent with reanalysis of The Cancer Genome Atlas (TCGA) Stage I and IIIA datasets. We next examined mutational burdens and observed that the top five cancers were microsatellite stable (MSS) genotypes (mean TMB 49.3/Mb), while the other 16 MSS cancers had a mean TMB of 5.9/Mb. To facilitate comparison with TCGA hypermutator CRC, we included four microsatellite instability-high (MSI-H) samples with the high mutation burden MSS cases to form a TMB-High group. Comparison of TMB-High with TMB-Low groups revealed differences in mutational frequency of ATM, ALK, NSD1, UBR5, BCL9, CARD11, KDM5C, MN1, PTPRT and PIK3CA, with ATM and UBR5 validated in reanalysis of TCGA hypermutator Stages I and IIIA samples. Variants in ATM were restricted to the TMB-High group, and in four of five MSS specimens, we observed the co-occurrence of mutations in homologous recombination repair (HRR) genes in either two of ATM, CDK12, PTEN or ATR, with at least one of these being a likely pathogenic truncating mutation. No MSI-H specimens carried nonsense mutations in HRR genes. These findings add to our knowledge of early T stage CRC and highlight a potential therapeutic vulnerability in the HRR pathway of TMB-H MSS CRC.

The Anatomy of Nerve Transfers Used in Tetraplegic Hand Reconstruction.

PURPOSE: To evaluate the anatomy of nerve transfers used to reconstruct wrist extension, hand opening, and hand closing in tetraplegic patients. METHODS: Nerve transfers were completed on 18 paired cadaveric upper limbs. The overlap of donor and recipient nerves was measured, as well as the distance to the target muscle. Axons were counted in each nerve and branch, with the axon percentage calculated by dividing the donor nerve count by that of the recipient. RESULTS: Transfers with overlap of the donor and recipient nerve were from the radial nerve branch to extensor carpi radialis brevis to anterior interosseous nerve (AIN) and from the branch(es) to supinator to posterior interosseous nerve. The extensor carpi radialis brevis to AIN had the shortest distance to the target, with the branch to brachialis to AIN being the longest. The nerve transfers for wrist extension had the highest axon percentage. Of the transfers for hand closing, the brachialis to AIN had the highest axon percentage, and the branch to brachioradialis to AIN had the lowest. CONCLUSIONS: The anatomical features of nerve transfers used in tetraplegic hand reconstruction are variable. Differences may help explain clinical outcomes. CLINICAL RELEVANCE: This study demonstrates which nerve transfers may be anatomically favorable for restoring hand function in tetraplegic patients.

Switch/sucrose-non-fermentable (SWI/SNF) complex (SMARCA4, SMARCA2, INI1/SMARCB1)-deficient colorectal carcinomas are strongly associated with microsatellite instability: an incidence study in 4508 colorectal carcinomas.

AIMS: Loss of expression of mammalian switch/sucrose-non-fermentable (SWI/SNF) [BRG1/BRM-associated factor (BAF)] complex subunits, including SMARCA4, SMARCA2 and INI1/SMARCB1 (termed SWI/SNF complex deficiency), has been reported in colorectal carcinomas (CRCs) but its frequency and clinical significance are uncertain. METHODS AND RESULTS: We performed immunohistochemistry for SMARCA4, SMARCA2 and SMARCB1 on 4508 consecutive resected CRCs. Loss of SMARCA4 expression was found in 13 cases (0.3%), loss of SMARCA2 expression was found in 59 cases (1.3%), and loss of SMARCB1 expression was found in 21 cases (0.4%). Some CRCs showed loss of expression of more than one subunit, so that 84 CRCs (1.7%) were deficient for at least one component. SWI/SNF complex deficiency was associated with higher grade, a right-sided location, mismatch repair deficiency, and BRAF V600E mutation (P < 0.05); 5.8% of mismatch repair-deficient (MMRd) cases and 5.4% of BRAF V600E-mutant cases were SWI/SNF complex-deficient, as compared with 0.9% and 0.4% of mismatch repair-proficient and BRAF-wild-type cases (P < 0.001). Any loss of SMARCB1 expression and global loss of SMARCA2 expression were associated with statistically significant worse overall survival, whereas SMARCA4-deficient cases showed a trend only towards poor overall survival (P = 0.121). In multivariate analysis, any loss of SMARCA4 expression and global loss of SMARCA2 expression were associated with worse survival [odds ratio (OR) 3.33, P = 0.019; and OR 3.39, P < 0.001]. Of particular note, among the subgroup of cases that were MMRd and BRAF V600E-mutated (otherwise considered to be a good prognostic group), loss of SMARCA4 expression was associated with much worse median survival (10.5 months versus 110.9 months; P = 0.003). CONCLUSIONS: SWI/SNF complex deficiency is rare in CRC but is enriched in MMRd cases. Identifying these cases has morphological associations and prognostic significance, and in the future may have potential therapeutic implications.

Molecular Features of Lymph Node Metastasis in T1/2 Colorectal Cancer from Formalin-Fixed Paraffin-Embedded Archival Specimens.

Histological risk factors for lymph node metastasis (LNM) in early-stage colorectal cancers (CRC) have been described, although the predictive utility of these factors varies. Improved LNM risk assessment based on findings in endoscopic colon and rectal excisions is necessary for optimal surgical management of CRC patients with pathologic T1- /T2-staged invasive depth (i.e., tumor not invading beyond the muscularis propria layer); as the current system is overly conservative, and results in many unnecessary radical surgeries. To identify molecular features in early CRC with elevated LNM potential, we carried out proteomic and gene expression profiling to compare T1 lymph node (LN) negative with T1/2 LN positive CRC tumors from formalin-fixed paraffin-embedded (FFPE) specimens. Using a data-independent acquisition mass spectrometry workflow, we detected over 7400 proteins and quantified over 4400 in all 21 specimens. Proteins from tumors with LN metastasis were enriched with effectors of epithelial-mesenchymal transition (EMT) and gene expression profiling confirmed activation of key transcription factors, SNAI1 and ZEB1, as well as a reduction in E-cadherin expression. Toward an implementation pathway, we investigated immunohistochemistry assays targeting four EMT-related proteins. While MS could reliably discern twofold protein abundance changes, we found the semiquantitative nature of IHC scoring limited confirmation of this degree of protein expression difference. This study demonstrated that EMT effectors are associated with locoregional metastasis in T1/T2 CRC and could be used to augment metastatic risk assessment, although further developments are required to enable routine implementation.

Tissue biomarker panel as a surrogate marker for squamous subtype of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been recently classified into four subtypes based on the gene expression levels, with squamous subtype having worst prognostic outcomes. However, gene expression analysis for each individual patient is not clinically feasible due to very high associated cost. We previously reported that levels of three biomarkers (S100A4, Ca-125 and Mesothelin) can be used to classify PDAC patients based on their survival outcomes. This project aimed to determine if this novel biomarker panel can be used as a surrogate to identify squamous PDAC subtype. METHODS: Using the Nanostring gene expression platform, tumor tissue from 24 PDAC patients were analysed for our novel biomarkers and markers associated with four PDAC subtypes. RESULTS: Gene expression of our biomarker panel (S100A4, Ca-125 and Mesothelin) closely clustered together with markers for squamous PDAC subtype. CONCLUSION: These results highlight the potential of our biomarkers to be utilized for identification of squamous PDAC subtype.

Loss of expression of BAP1 is a useful adjunct, which strongly supports the diagnosis of mesothelioma in effusion cytology.

Although most mesotheliomas present with pleural effusions, it is controversial whether mesothelioma can be diagnosed with confidence in effusion cytology. Therefore, an ancillary marker of malignant mesothelial cells applicable in effusions would be clinically valuable. BRCA-1-associated protein (BAP1) is a tumor suppressor gene, which shows biallelic inactivation in approximately half of all mesotheliomas. We investigated whether loss of BAP1 expression by immunohistochemistry can be used to support a diagnosis of mesothelioma in effusion cytology. Immunohistochemistry for BAP1 was performed on cell blocks and interpreted blinded. 43 of 75 (57%) effusions associated with confirmed mesothelioma showed negative staining with positive internal controls. Of 57 effusions considered to have atypical mesothelial cells in the absence of a definitive diagnosis of mesothelioma, 8 cases demonstrated negative staining for BAP1. On follow-up six of these patients received a definitive diagnosis of mesothelioma in the subsequent 14 months (two were lost to follow-up immediately, and mesothelioma could not be excluded). Only 5 of 100 consecutive benign effusions were interpreted as BAP1 negative. One of these patients died soon after and mesothelioma could not be excluded. On unblinded review the four other patients with apparently negative BAP1 staining but no malignancy lacked convincing positive staining in non-neoplastic cells suggesting that BAP1 immunohistochemistry may have initially been misinterpreted. 47 effusions with adenocarcinoma were BAP1 positive. We conclude that loss of BAP1 expression, while not definitive, can be used to support the diagnosis of mesothelioma in effusion cytology. We caution that interpretation of BAP1 immunohistochemistry on cell block may be difficult and that convincing positive staining in non-neoplastic cells is required before atypical cells are considered negative. We also note that BAP1 loss is not a sensitive test as it occurs in only half of all mesotheliomas and cannot be used to exclude the diagnosis.