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/.

A Critical Assessment of Diagnostic Criteria for the Tall Cell Subtype of Papillary Thyroid Carcinoma-How Much? How Tall? And When Is It Relevant?

Tall cell papillary thyroid carcinoma (TC-PTC) is considered adverse histology. However, previous studies are confounded by inconsistent criteria and strong associations with other adverse features. It is therefore still unclear if TC-PTC represents an independent prognostic factor in multivariate analysis and, if it does, what criteria should be employed for the diagnosis. We retrospectively reviewed 487 PTCs from our institution (where we have historically avoided the prospective diagnosis of TC-PTC) for both the height of tall cells (that is if the cells were two, or three, times as tall as wide) and the percentage of tall cells. On univariate analysis, there was significantly better disease free survival (DFS) in PTCs with no significant tall cell component (< 30%) compared to PTCs with cells two times tall as wide (p = 0.005). The proportion of tall cells (30-50% and > 50%) was significantly associated with DFS (p = 0.012). In a multivariate model including age, size, vascular space invasion, and lymph node metastasis, the current WHO tall cell criteria, met by 7.8% of PTCs, lacked statistical significance for DFS (p = 0.519). However, in the subset of tumours otherwise similar to the American Thyroid Association (ATA) guidelines low-risk category, WHO TC-PTC demonstrated a highly significant reduction in DFS (p = 0.004). In contrast, in intermediate to high-risk tumours, TC-PTC by WHO criteria lacked statistical significance (p = 0.384). We conclude that it may be simplistic to think of tall cell features as being present or absent, as both the height of the cells (two times versus three times) and the percentage of cells that are tall have different clinical significances in different contexts. Most importantly, the primary clinical significance of TC-PTC is restricted to PTCs that are otherwise low risk by ATA guidelines.

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.

The Presence of Typical "BRAFV600E-Like" Atypia in Papillary Thyroid Carcinoma is Highly Specific for the Presence of the BRAFV600E Mutation.

Papillary thyroid carcinomas (PTCs) are driven by a variety of molecular abnormalities including BRAF, RAS, ALK, RET, and NTRK alterations. PTCs driven by the BRAFV600E mutation, or tumours which demonstrate a similar gene expression profile to PTCs driven by this mutation, have been reported to demonstrate specific morphological features sometimes termed "BRAFV600E-like" atypia. BRAFV600E-like atypia is characterised by a well-developed papillary architecture, infiltrative growth, marked nuclear clearing, prominent intranuclear pseudoinclusions, abundant eosinophilic cytoplasm, and scattered psammoma bodies. We sought to investigate the sensitivity and specificity of these morphological features for the presence of BRAFV600E mutation in PTCs as determined by mutation specific immunohistochemistry. An unselected cohort of 495 PTCs was reviewed by a single pathologist and categorised into three groups: typical BRAFV600E-like atypia (145 cases, 29%), possible BRAFV600E-like atypia (166 cases, 33%) and little/no BRAFV600E-like atypia (184 cases, 37%). The specificity and sensitivity of typical BRAFV600E-like atypia for the BRAFV600E mutation was 97.2% and 44.3%, respectively. When typical and possible BRAFV600E-like atypia were analysed together, the specificity was 70.6% and the sensitivity was 81.7%. In the morphologically little/no BRAFV600E-like atypia group, 58 cases (31.5%) had a BRAFV600E mutation. We conclude that typical BRAFV600E-like atypia is highly specific for the presence of the BRAFV600E mutation; however, the absence of BRAFV600E-like atypia does not exclude this mutation.

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.

Expanding the clinicopathological spectrum of succinate dehydrogenase-deficient renal cell carcinoma with a focus on variant morphologies: a study of 62 new tumors in 59 patients.

Most succinate dehydrogenase (SDH)-deficient renal cell carcinomas (RCCs) demonstrate stereotypical morphology characterized by bland eosinophilic cells with frequent intracytoplasmic inclusions. However, variant morphologic features have been increasingly recognized. We therefore sought to investigate the incidence and characteristics of SDH-deficient RCC with variant morphologies. We studied a multi-institutional cohort of 62 new SDH-deficient RCCs from 59 patients. The median age at presentation was 39 years (range 19-80), with a slight male predominance (M:F = 1.6:1). A relevant family history was reported in 9 patients (15%). Multifocal or bilateral tumors were identified radiologically in 5 patients (8%). Typical morphology was present at least focally in 59 tumors (95%). Variant morphologies were seen in 13 (21%) and included high-grade nuclear features and various combinations of papillary, solid, and tubular architecture. Necrosis was present in 13 tumors, 7 of which showed variant morphology. All 62 tumors demonstrated loss of SDHB expression by immunohistochemistry. None showed loss of SDHA expression. Germline SDH mutations were reported in all 18 patients for whom the results of testing were known. Among patients for whom follow-up data was available, metastatic disease was reported in 9 cases, 8 of whom had necrosis and/or variant morphology in their primary tumor. Three patients died of disease. In conclusion, variant morphologies and high-grade nuclear features occur in a subset of SDH-deficient RCCs and are associated with more aggressive behavior. We therefore recommend grading all SDH-deficient RCCs and emphasize the need for a low threshold for performing SDHB immunohistochemistry in any difficult to classify renal tumor, particularly if occurring at a younger age.

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.

PD-L1 Is Preferentially Expressed in PIT-1 Positive Pituitary Neuroendocrine Tumours.

Pituitary neuroendocrine tumours (PitNETs) cause lifelong morbidity, some requiring extensive surgical intervention, radiotherapy, or chemotherapy. A small percentage still cause debilitating disease, resistant to standard treatments, and may benefit from novel therapies. We assessed PD-L1 expression in a large cohort of PitNETs to investigate whether immunotherapy could represent a rational therapeutic choice. Unselected PitNETs undergoing surgical resection were reclassified according to the WHO 2017 system and underwent PD-L1 immunohistochemistry (clone SP263) in tissue microarray format. Membranous expression was scored as 0 (no expression), 1+ (< 50% expression) and 2+ (> 50% expression). A total of 265 PitNETs underwent PD-L1 immunohistochemistry. Prominent non-specific cytoplasmic staining was noted making assessment of true membrane expression difficult. Allowing for this, 40 of 264 (15%) PitNETs demonstrated strong staining (> 50% of neoplastic cells positive). These included 5/10 (50%) somatotrophs, 7/17 (41%) lactotrophs, 2/5 (40%) mammosomatotrophs, 4/8 (50%) mixed somatotroph-lactotrophs, 3/5 (60%) PIT-1 positive plurihormonal tumours with TSH expression, 10/28 (36%) of PIT-1 positive plurihormonal tumours, and 4/10 (40%) of PIT-1 positive tumours with no hormonal expression. Only 2/32 (6%) transcription factor triple negative, hormone negative tumours, 5/113 (4%) of gonadotrophs, and 0/6 thyrotrophs or 0/30 corticotrophs showed significant staining. We conclude that PD-L1 expression is common in somatotrophs, lactotrophs, and PIT-1 positive plurihormonal PitNETs but rare in transcription factor negative, hormone negative PitNETs, gonadotrophs, and corticotrophs. If the therapeutic role of immunotherapy is to be explored in PitNETs, it may be that it is of most benefit in the PD-L1 high subgroup.

DNA damage-inducible transcript 3 immunohistochemistry is highly sensitive for the diagnosis of myxoid liposarcoma but care is required in interpreting the significance of focal expression.

AIMS: Myxoid liposarcoma (MLPS) is characterised by DNA damage-inducible transcript 3 (DDIT3) gene rearrangements, confirmation of which is commonly used diagnostically. Recently, DDIT3 immunohistochemistry (IHC) has been reported to be highly sensitive and, when strict criteria are employed, specific for the diagnosis of MLPS. The aim of this study was to independently investigate DDIT3 IHC as a diagnostic marker for MLPS. METHODS AND RESULTS: DDIT3 IHC was performed on 52 MLPS and on 152 mimics on whole sections, and on 515 non-MLPS sarcomas in tissue microarray format. Only one MLPS (which had undergone acid-based decalcification) was completely negative. With inclusion of this case if any nuclear expression is considered to indicate positivity, the overall sensitivity of DDIT3 is 98% (51 of 52 cases) and the specificity is 94% (633 of 667 non-MLPS cases are negative). If a cut-off of >10% of neoplastic cells is required for positivity, then the sensitivity remains 98% (51/52) and the specificity is 98.5% (657 of 667 non-MLPS cases are negative). If a cut-off of >50% of cells is required for positivity, then the sensitivity is 96% (50 of 52 cases) but the specificity improves to 100%. CONCLUSIONS: Diffuse nuclear DDIT3 expression occurs in the overwhelming majority of MLPSs, and can be used to confirm the diagnosis in most cases without the need for molecular testing. A complete absence of expression argues strongly against MLPS, and almost completely excludes this diagnosis, particularly if there is consideration of technical factors such as decalcification. The significance of focal DDIT3 expression should be interpreted in the morphological and clinical context, although most tumours showing only focal expression are not MLPS.

When used together SS18-SSX fusion-specific and SSX C-terminus immunohistochemistry are highly specific and sensitive for the diagnosis of synovial sarcoma and can replace FISH or molecular testing in most cases.

AIMS: Synovial sarcoma is defined by recurrent t(X;18)(p11;q11) translocations creating SS18-SSX1, SS18-SSX2 or SS18-SSX4 fusions. Recently, a novel rabbit monoclonal antibody designed to identify these fusions (SS18-SSX, clone E9X9V) was proposed to be highly specific (100%), but not completely sensitive (95%) for this diagnosis. Another antibody designed to identify the C-terminal end of SSX (SSX_CT, clone E5A2C) was proposed to be highly sensitive (100%), but not completely specific (96%). We sought to validate these antibodies in an independent cohort. METHODS AND RESULTS: We performed immunohistochemistry for SS18-SSX and SSX_CT on 39 synovial sarcoma samples from 25 patients with confirmed gene rearrangements. Thirty-four (87%) and 36 (92%) were positive for SS18-SSX and SSX_CT, respectively. False-negative staining was associated with suboptimally handled small biopsies and decalcified specimens, even when staining was diffuse and strong in subsequent optimally processed excisions and non-decalcified areas. None of 580 non-synovial sarcoma tumours (76 whole sections, 504 TMA samples) were positive for SS18-SSX (100% specificity), whereas 39 (93% specificity) were positive for SSX_CT. CONCLUSIONS: SS18-SSX fusion-specific IHC is 87-95% sensitive for the diagnosis of synovial sarcoma and highly (perhaps perfectly) specific. Therefore, positive SS18-SSX staining definitively confirms the diagnosis of synovial sarcoma. SSX_CT is less specific (93-96%) but highly sensitive (92%, but approaching 100% when suboptimally processed biopsies and decalcified specimens are excluded). Negative SSX_CT staining may therefore have an ancillary role as a rule-out test for synovial sarcoma. We caution that both antibodies are prone to false-negative staining in decalcified specimens.

A Proposed Grading Scheme for Medullary Thyroid Carcinoma Based on Proliferative Activity (Ki-67 and Mitotic Count) and Coagulative Necrosis.

We investigated the prognostic value of a range of histologic parameters in medullary thyroid carcinoma (MTC) to design a grading system to predict overall survival. We assessed 76 patients with MTCs undergoing primary tumor resection for age, sex, tumor size, vascular space invasion, lymph node metastasis, multiple endocrine neoplasia type 2 (MEN2) status, mitotic count, Ki-67 proliferative index, spindled morphology, sheet-like growth pattern, coagulative necrosis, incipient necrosis, nuclear grade, multinucleation, prominent nucleoli, fibrosis, and amyloid deposition. In addition to the clinical features of age and the diagnosis of MEN2, the only histologic features that significantly predicted reduced overall survival were Ki-67 proliferative index, mitotic count, and the presence of coagulative necrosis. Using a combination of these 3 variables, we propose a 3-tiered grading system based solely on proliferative activity (Ki-67 proliferative index and mitotic count) and necrosis. There were 62 (82%) low-grade MTCs (low proliferative activity, no necrosis), 9 (12%) intermediate grade (low proliferative activity and necrosis present, or intermediate proliferative activity and no necrosis), and 5 (7%) high grade (intermediate proliferative activity and necrosis present, or high proliferative activity with or without necrosis). The mean overall survival was 193, 146, and 45 months, respectively (P=0.0001) for the 3 grades. The grading system remained prognostic when controlled for other factors associated with survival including age and known MEN2 syndrome. We conclude that this proposed grading system, which uses only a combination of proliferative activity (Ki-67 index, mitotic count) and coagulative necrosis, is a strong predictor of overall survival in MTC.

Utility of GATA-3 Expression in the Analysis of Pituitary Neuroendocrine Tumour (PitNET) Transcription Factors.

With the introduction of the WHO 2017 classification of endocrine neoplasms, the use of the pituitary transcription factors PIT-1, Tpit and SF-1 has become the standard of care. However, immunohistochemistry for these transcription factors is not available in all institutions, and their reliability has been questioned. We read with interest the findings of Mete et al. that GATA-3 expression was detected in some pituitary neuroendocrine tumours (PitNET). We therefore sort to validate this in our large cohort of PitNETs. We searched the database of Royal North Shore Hospital for PitNETs between 1998 and 2012, constructed a tissue microarray and reclassified these entities based on their expression for PIT-1, Tpit and SF-1. We then scored the expression of GATA-3 immunohistochemistry on a scale of 0-2, where 0 was no staining, 1 was patchy or weak staining and 2 was strong and diffuse staining. 265 of 346 tumours were able to be classified into a specific tumour subtype, and 263 tumours had tissue available for GATA-3 immunohistochemistry. 89% of gonadotrophs and 93% of triple-negative tumours with expression for luteinising hormone and follicle-stimulating hormone were positive for GATA-3. In the triple-negative group, GATA-3 was positive in 1 mammosomatotroph and 80% of tumours with thyroid-stimulating hormone expression. In the triple-negative hormone-negative group, 21 of 33 tumours were positive (64%). The results demonstrate that GATA-3 is a useful marker to supplement the existing pituitary transcription factors, albeit slightly less sensitive and specific than previously reported. GATA-3 may be employed in addition to the current array of immunohistochemical transcription factors, especially in the resource poor setting. However, given its potential cross-reactivity with other entities of the Sella, positive staining should be interpreted with caution and in the morphological and clinical context.

NTRK gene rearrangements are highly enriched in MLH1/PMS2 deficient, BRAF wild-type colorectal carcinomas-a study of 4569 cases.

NTRK gene rearrangements are important to identify as predictors of response to targeted therapy in many malignancies. Only 0.16-0.3% of colorectal carcinomas (CRCs) harbor these fusions making universal screening difficult. We therefore investigated whether pan-Trk immunohistochemistry (IHC), mismatch repair deficiency (MMRd), and BRAFV600E mutation status could be used to triage molecular testing for NTRK gene rearrangements in CRC. CRCs from 4569 unselected patients underwent IHC in TMA format with two different anti-pan-Trk rabbit monoclonal antibodies. All positive cases were confirmed on whole sections and underwent RNA-sequencing. Pan-Trk IHC was positive in 0.2% of CRCs (9/4569). Both antibodies demonstrated similar staining characteristics with diffuse positive staining in all neoplastic cells. Of note 8/9 (89%) IHC positive cases were both MMRd (all showing MLH1/PMS2 loss) and lacked BRAFV600E mutation. That is, IHC was positive in 5.3% (8/152) MLH1/PMS2/BRAFV600E triple negative CRCs, but only 0.02% (1/4417) not showing this phenotype. All nine IHC positive CRCs demonstrated gene rearrangements (LMNA-NTRK1 in 5 CRCs, TPR-NTRK1, STRM-NTRK1, MUC2-NTRK2, and NTRK1 with an unknown partner in one each), suggesting close to 100% specificity for IHC in this sub-population. NTRK fusions were associated with right sided (p = 0.02), larger tumors (p = 0.029) with infiltrative growth (p = 0.021). As a part of universal Lynch syndrome screening many institutions routinely test all CRCs for MMRd, and then proceed to reflex BRAFV600E mutation testing in MLH1/PMS2 negative CRCs. We conclude that performing pan-Trk IHC on this preselected subgroup of MLH1/PMS2/BRAFV600E triple negative CRCs (only 3.3% of all CRC patients) is a resource effective approach to identify the overwhelming majority of CRC patients with NTRK gene fusions.

Parafibromin-deficient (HPT-JT Type, CDC73 Mutated) Parathyroid Tumors Demonstrate Distinctive Morphologic Features.

The gene CDC73 (previously known as HRPT2) encodes the protein parafibromin. Biallelic mutation of CDC73 is strongly associated with malignancy in parathyroid tumors. Heterozygous germline mutations cause hyperparathyroidism jaw tumor syndrome,which is associated with a high life-time risk of parathyroid carcinoma. Therefore loss of parafibromin expression by immunohistochemistry may triage genetic testing for hyperparathyroidism jaw tumor syndrome and be associated with malignant behavior in atypical parathyroid tumors. We share our experience that parafibromin-negative parathyroid tumors show distinctive morphology. We searched our institutional database for parathyroid tumors demonstrating complete loss of nuclear expression of parafibromin with internal positive controls. Forty-three parafibromin-negative tumors from 40 (5.1%) of 789 patients undergoing immunohistochemistry were identified. Thirty-three (77%) were external consultation cases; the estimated incidence in unselected tumors was 0.19%. Sixteen (37.2%) fulfilled World Health Organization 2017 criteria for parathyroid carcinoma and 63% had serum calcium greater than 3mmol/L. One of 27 (3.7%) noninvasive but parafibromin-negative tumors subsequently metastasized. Parafibromin-negative patients were younger (mean, 36 vs. 63 y; P<0.001) and had larger tumors (mean, 3.04 vs. 0.62 g; P<0.001). Not all patients had full testing, but 26 patients had pathogenic CDC73 mutation/deletions confirmed in tumor (n=23) and/or germline (n=16). Parafibromin-negative tumors demonstrated distinctive morphology including extensive sheet-like rather than acinar growth, eosinophilic cytoplasm, nuclear enlargement with distinctive coarse chromatin, perinuclear cytoplasmic clearing, a prominent arborizing vasculature, and, frequently, a thick capsule. Microcystic change was found in 21 (48.8%). In conclusion, there are previously unrecognized morphologic clues to parafibromin loss/CDC73 mutation in parathyroid tumors which, given the association with malignancy and syndromic disease, are important to recognize.

ATRX loss is an independent predictor of poor survival in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms accounting for 1% to 2% of all pancreatic tumors. The biological behavior of PanNETs is heterogeneous and unpredictable, adding to the difficulties of clinical management. The DAXX (death domain associated protein) and ATRX (α-thalassemia/mental retardation syndrome X-linked) genes encode proteins involved in SWI/SNF-like chromatin remodeling. Somatic inactivating mutations in DAXX and ATRX are frequent in PanNETs, mutually exclusive, and associated with telomere dysfunction, resulting in genomic instability and alternate lengthening of telomeres. We sought to assess the clinical significance of the loss of the ATRX and DAXX proteins as determined by immunohistochemistry (IHC) in patients with PanNET. From an unselected cohort of 105 patients, we found ATRX loss in 10 tumors (9.5%) and DAXX loss in 16 (15.2%). DAXX and ATRX losses were confirmed mutually exclusive and associated with other adverse clinicopathological variables and poor survival in univariate analysis. In addition, ATRX loss was also associated with higher AJCC stage and infiltrative tumor borders. However, only ATRX loss, lymphovascular invasion, and perineural spread were independent predictors of poor overall survival in multivariate analysis. In conclusion, loss of expression of ATRX as determined by IHC is a useful independent predictor of poor overall survival in PanNETs. Given its relative availability, ATRX loss as determined by IHC may have a role in routine clinical practice to refine prognostication in patients with PanNET.

The epithelioid BAP1-negative and p16-positive phenotype predicts prolonged survival in pleural mesothelioma.

AIMS: Mesothelioma is a relatively uncommon but highly malignant neoplasm. Most patients die of disease within 1 year of diagnosis, but some have prolonged survival. Prospective identification of these longer-term survivors may help to guide treatment. We therefore sought to investigate the role of p16 immunohistochemistry (IHC) both alone and in combination with other markers as a potential predictor of prolonged survival in mesothelioma. METHODS AND RESULTS: P16 IHC was performed on unselected pleural mesotheliomas biopsied from 1991 to 2014; 153 of 208 (74%) cases were p16-negative, which correlated significantly with poor overall survival in both univariate (median survival 7.6 versus 13.6 months; P = 0.001) and multivariate analysis [hazard ratio (HR): 1.632; 95% confidence interval (CI): 1.103-2.415; P = 0.014]. Other independent factors associated with prolonged survival included loss of expression of BAP1 and epithelioid morphology. We therefore stratified patients further based on these three independent prognostic variables and demonstrated an unusually prolonged survival in mesotheliomas which were epithelioid, BAP1 IHC negative and p16 IHC positive (12% of cases, median survival 31.7 months, P < 0.0001). CONCLUSIONS: In conclusion, p16 IHC is an independent prognostic biomarker in pleural mesothelioma. When used in combination with BAP1 IHC and morphological subtyping, patients with exceptionally prolonged survival can potentially be identified.

Lessons learnt from implementation of a Lynch syndrome screening program for patients with gynaecological malignancy.

Despite a trend towards universal testing, best practice to screen patients presenting with gynaecological malignancy for Lynch syndrome (LS) is uncertain. We report our institutional experience of a co-ordinated gynaecological LS screening program. All patients with endometrial carcinoma or carcinosarcoma, or gynaecological endometrioid or clear cell carcinomas undergo reflex four panel immunohistochemistry (IHC) for MLH1, PMS2, MSH2 and MSH6 followed by cascade somatic hypermethylation analysis of the MLH1 promoter locus for dual MLH1/PMS2 negative tumours. On the basis of these results, genetic counselling and targeted germline mutation testing is then offered to patients considered at high risk of LS. From 1 August 2013 to 31 December 2015, 124 patients were screened (mean age 64.6 years). Thirty-six (29.0%) demonstrated abnormal MMR IHC: 26 (72.2%) showed dual loss of MLH1/PMS2, five (13.9%) dual loss of MSH2/MSH6, three (8.3%) isolated loss of MSH6, and two (5.6%) isolated loss of PMS2. Twenty-five of 26 (96.1%) patients with dual MLH1/PMS2 loss demonstrated MLH1 promoter methylation. Therefore, 11 (8.9%) patients screened were classified as high risk for LS, of whom nine (81.8%) accepted germline mutation testing. Three (2.4% of total screened) were confirmed to have LS, two with germline PMS2 and one with germline MSH2 mutation. Massive parallel sequencing of tumour tissue demonstrated somatic mutations which were concordant with the IHC results in the remainder. Interestingly, the one MLH1/PMS2 IHC negative but not hypermethylated tumour harboured only somatic MLH1 mutations, indicating that universal cascade methylation testing in MLH1/PMS2 IHC negative tumours is very low yield and could be reconsidered in a resource-poor setting. In conclusion, universal screening for LS in patients presenting with gynaecological malignancy using the algorithm described above identified LS in three of 124 (2.4%) of our population. Only three of nine (33.3%) patients considered at high risk for LS by combined IHC and hypermethylation analysis were proven to have LS. Only one of the LS patients was less than 50 years of age and none of these patients would have been identified had more restrictive Amsterdam or Bethesda criteria been applied.

Inflammatory Myofibroblastic Tumors of the Female Genital Tract Are Under-recognized: A Low Threshold for ALK Immunohistochemistry Is Required.

Inflammatory myofibroblastic tumor (IMT) of the female genital tract is under-recognized. We investigated the prevalence of ALK-positive IMT in lesions previously diagnosed as gynecologic smooth muscle tumors. Immunohistochemistry (IHC) for ALK was performed on tissue microarrays of unselected tumors resected from 2009 to 2013. Three of 1176 (0.26%) "leiomyomas" and 1 of 44 (2.3%) "leiomyosarcomas" were ALK IHC positive, confirmed translocated by fluorescence in situ hybridization (FISH) and therefore more appropriately classified as IMT. On review significant areas of all 4 tumors closely mimicked smooth muscle tumors morphologically, but all showed at least subtle/focal features suggesting IMT. Recognizing that the distinction between IMT and leiomyoma/leiomyosarcoma can be subtle, we then reviewed 1 hematoxylin and eosin slide from each patient undergoing surgery for "leiomyoma" from 2014 to 2017 and selected cases for ALK IHC with a low threshold. Of these, 30 of 571 (5.3%) underwent IHC. Two were confirmed to be IHC positive and FISH rearranged. Of the 6 IMTs, only 1 tumor with a previous diagnosis of leiomyosarcoma, an infiltrative margin and equivocal necrosis, metastasized. Of note it demonstrated a less aggressive clinical course compared with most metastatic leiomyosarcomas (alive with disease at 6 y). The patient was subsequently offered crizotinib to which she responded rapidly. In conclusion, IMTs may closely mimic gynecologic smooth muscle tumors. IMTs account for at least 5 of 1747 (0.3%) tumors previously diagnosed as leiomyoma and 1 of 44 (2.3%) as leiomyosarcoma. These tumors may be recognized prospectively with awareness of subtle/focal histologic clues, coupled with a low threshold for ALK IHC.