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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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

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.

Background: Up to 60% of patients who undergo curative-intent pancreatic ductal adenocarcinoma (PDAC) resection experience disease recurrence within six months. We recently published a systematic review of prognostic immunohistochemical biomarkers in PDAC and shortlisted a panel of those reported with the highest level of evidence, including p53, p16, Ca-125, S100A4, FOXC1, EGFR, mesothelin, CD24 and UPAR. This study aims to discover and validate the prognostic significance of a combinatorial panel of tumor biomarkers in patients with resected PDAC., Methods: Patients who underwent PDAC resection were included from a single institution discovery cohort and a multi-institutional validation cohort. Tumors in the discovery cohort were stained immunohistochemically for all nine shortlisted biomarkers. Biomarkers significantly associated with overall survival (OS) were reevaluated as a combinatorial panel in both discovery and validation cohorts for its prognostic significance., Results: 224 and 191 patients were included in the discovery and validation cohorts, respectively. In both cohorts, S100A4, Ca-125 and mesothelin expression were associated with shorter OS. In both cohorts, the number of these biomarkers expressed was significantly associated with OS (discovery cohort 36.8 vs. 26.4 vs 16.3 vs 12.8 months, P < 0.001; validation cohort 25.2 vs 18.3 vs 13.6 vs 11.9 months, P = 0.008 for expression of zero, one, two and three biomarkers, respectively). On multivariable analysis, expression of at least one of three biomarkers was independently associated with shorter OS., Conclusion: Combinations of S100A4, Ca-125 and mesothelin expression stratify survival after resection of localized PDAC. Co-expression of all three biomarkers is associated with the poorest prognostic outcome., (Copyright © 2018. Published by Elsevier Ltd.)

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., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

A quarter of patients with medullary thyroid carcinoma (MTC) have germline mutations in the RET proto-oncogene indicating MEN2. Therefore genetic testing is recommended for all patients presenting with MTC. Approximately 40% of MTCs have somatic RET mutations. Somatic mutations in the RAS genes are the next most common driver mutations and appear to be mutually exclusive with germline RET mutation. The single most common somatic RAS mutation is HRASQ61R (c.182A>G), reported in 4.6% to 11% of all MTCs. Mutation-specific immunohistochemistry (IHC) initially developed to identify the NRASQ61R mutation in melanoma (clone SP174) has proven highly sensitive and specific. Because the amino acid sequences for the HRAS and NRAS proteins at codon 61 are identical, we postulated that SP174 IHC would also identify the somatic HRASQ61R mutation. IHC with SP174 was performed on a tissue microarray of 68 patients with MTC including 13 (22.8%) with molecularly confirmed MEN2. Seven (10.3%) MTCs demonstrated positive staining. Six of these patients had already undergone germline RET mutation testing as part of clinical care and were all confirmed to be wild type, excluding the diagnosis of MEN2. All SP174 immunohistochemically positive MTCs were proven to have HRASQ61R mutation (and lack KRASQ61R and NRASQ61R) by Sanger sequencing. All MEN2 patients showed negative staining. We conclude that IHC with SP174 is highly specific for the HRASQ61R mutation in MTC. Because current data suggest that this mutation is mutually exclusive with germline RET mutation, IHC may also have a role in triaging formal genetic testing for MEN2.

SMARCB1 is a tumor suppressor gene that encodes for the protein INI1. SMARCB1 is commonly inactivated and INI1 correspondingly shows loss of expression in a range of malignant neoplasms including rhabdoid tumors, renal medullary carcinomas, and epithelioid sarcomas. Loss of INI1 expression has recently been reported in occasional gastrointestinal adenocarcinomas. We sought to investigate the incidence and clinicopathological significance of INI1 loss in colorectal adenocarcinoma (CRC). Immunohistochemistry for INI1 was performed in tissue microarray (TMA) format on a well-characterized and unselected cohort of CRCs undergoing surgical resection. If staining was negative or equivocal in the TMA sections, immunohistochemistry was repeated on whole sections. Focal or widespread negative staining for INI1 was identified in whole sections from 14 (0.46%) of 3051 CRCs. In 7 (50%) of 14 negative cases, the loss of staining was focal, whereas the remainder were characterized by negative staining in all neoplastic cells in whole sections. In the cases with focal staining, loss of staining was frequently found in areas of poor differentiation. Global or focal INI1 loss was strongly associated with higher histological grade, larger tumor size and poor overall survival (P<.001). We conclude that INI1 loss occurs rarely (0.46% when screened by TMA) in CRC, where it is associated with higher grade, larger tumor size, poorer survival, mismatch repair deficiency, and BRAFV600E mutation., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Despite the established role of Ki67 labeling index in prognostic stratification of adrenocortical carcinomas and its recent integration into treatment flow charts, the reproducibility of the assessment method has not been determined. The aim of this study was to investigate interobserver variability among endocrine pathologists using a web-based virtual microscopy approach. Ki67-stained slides of 76 adrenocortical carcinomas were analyzed independently by 14 observers, each according to their method of preference including eyeballing, formal manual counting, and digital image analysis. The interobserver variation was statistically significant (P<0.001) in the absence of any correlation between the various methods. Subsequently, 61 static images were distributed among 15 observers who were instructed to follow a category-based scoring approach. Low levels of interobserver (F=6.99; Fcrit=1.70; P<0.001) as well as intraobserver concordance (n=11; Cohen κ ranging from -0.057 to 0.361) were detected. To improve harmonization of Ki67 analysis, we tested the utility of an open-source Galaxy virtual machine application, namely Automated Selection of Hotspots, in 61 virtual slides. The software-provided Ki67 values were validated by digital image analysis in identical images, displaying a strong correlation of 0.96 (P<0.0001) and dividing the cases into 3 classes (cutoffs of 0%-15%-30% and/or 0%-10%-20%) with significantly different overall survivals (P<0.05). We conclude that current practices in Ki67 scoring assessment vary greatly, and interobserver variation sets particular limitations to its clinical utility, especially around clinically relevant cutoff values. Novel digital microscopy-enabled methods could provide critical aid in reducing variation, increasing reproducibility, and improving reliability in the clinical setting.