Patients with secondary or tertiary hyperparathyroidism due to chronic renal failure who fail medical treatment require sub-total or total parathyroidectomy. The histological characteristics of parathyroid glands resected in this setting are not well studied; however, there is awareness that the findings may mimic malignancy. We conducted a detailed clinicopathological study of parathyroid resection specimens from an unselected Royal North Shore Hospital (RNSH) cohort (n=110) and an external consultation cohort (n=44). The most common atypical features observed in the RNSH cohort were fibrous bands (43%) and fibrous capsule (29%), while sheet-like/trabecular architecture (12%), invasive/pseudoinvasive growth pattern (4%) and mitotic activity (9%) were less prevalent. Clinical follow-up data were available for these cases, and none showed recurrence or metastasis. In the external consultation cohort, fibrous bands (84%) and fibrous capsule (66%) were also the most frequently observed features; however, invasive/pseudoinvasive growth pattern (34%) and sheet-like/trabecular architecture (34%) ​were more commonly seen in this group. One case in this cohort demonstrated unequivocal vascular invasion, fulfilling the diagnostic criteria for parathyroid carcinoma. However, at 5-year ​follow-up, there was no evidence of recurrence or metastasis. In summary, atypical histological features are common in parathyroid glands resected for secondary or tertiary hyperparathyroidism and should not be misinterpreted as atypical parathyroid tumour or carcinoma. Parathyroid carcinoma is exceedingly rare in this context, and even when strict diagnostic histological criteria are met, the long-term prognosis may be favourable.

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/ .

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.

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.

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.

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.

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.

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.

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.

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.

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.

Aims Synovial sarcoma is defined by recurrent t(X;18)(p11;q11) translocations creatingSS18-SSX1,SS18-SSX2orSS18-SSX4fusions. 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.

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.

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.

Somatostatin analogues (including octreotide, lanreotide and pasireotide) are first line medical therapy for acromegaly secondary to growth hormone (GH) secreting pituitary adenomas.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar Somatostatin analogues function by binding to and activating the somatostatin receptor (SSTR) which in turn suppresses adenylate cyclase activity and inhibits proliferation and hormone secretion.2Fougner S.L. Casar-Borota O. Heck A. et al.Adenoma granulation pattern correlates with clinical variables and effect of somatostatin analogue treatment in a large series of patients with acromegaly.Clin Endocrinol. 2012; 76: 96-102Google Scholar There are five SSTR subtypes, SSTR1, SSTR2A, SSTR3, SSTR4 and SSTR5. Octreotide and lanreotide preferentially activate SSTR2A, whereas pasireotide has greater tropism for SSTR5.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar, 3Elston M.S. Meyer-Rochow G.Y. Conaglen H.M. et al.Increased SSTR2A and SSTR3 expression in succinate dehydrogenase-deficient pheochromocytomas and paragangliomas.Hum Pathol. 2015; 46: 390-396Google Scholar The clinical response to somatostatin analogues can be highly variable4Casar-Borota O. Heck A. Schulz S. et al.Expression of SSTR2a, but not of SSTRs 1, 3, or 5 in somatotroph adenomas assessed by monoclonal antibodies was reduced by octreotide and correlated with the acute and long-term effects of octreotide.J Clin Endocrinol Metab. 2013; 98: E1730-E1739Google Scholar and it has been suggested that immunohistochemical demonstration of SSTR2A and SSTR5 receptors using next generation antibodies may predict both the response to somatostatin analogues and also the overall outcome in neuroendocrine neoplasms.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar, 3Elston M.S. Meyer-Rochow G.Y. Conaglen H.M. et al.Increased SSTR2A and SSTR3 expression in succinate dehydrogenase-deficient pheochromocytomas and paragangliomas.Hum Pathol. 2015; 46: 390-396Google Scholar, 5Mehta S. de Reuver P.R. Gill P. et al.Somatostatin receptor SSTR-2a expression is a stronger predictor for survival than Ki-67 in pancreatic neuroendocrine tumors.Medicine. 2015; 94: 1-6Google Scholar Therefore, we sought to investigate the expression of SSTR2A and SSTR5 as determined by immunohistochemistry in a large cohort of pituitary adenomas. We obtained tissue microarray (TMA) slides from a large unselected cohort of pituitary adenomas. The details of this cohort have previously been described in detail.6Gill A.J. Toon C.W. Clarkson A. et al.Succinate dehydrogenase deficiency is rare in pituitary adenomas.Am J Surg Pathol. 2014; 38: 560-566Google Scholar, 7Farzin M. Toon C.W. Toon C.W. Clarkson A. Sioson L. Gill A.J. BRAF V600E mutation specific immunohistochemistry with clone VE1 is not reliable in pituitary adenomas.Pathology. 2014; 46: 79-80Google Scholar Briefly, the TMAs included two 1 mm cores of all available pituitary adenomas resected at Royal North Shore Hospital, Sydney, Australia, during the calendar years 1998–2012. During this period at the time of primary reporting it was routine practice to perform immunohistochemistry for the six pituitary hormones: growth hormone (GH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), follicle stimulating hormone (FSH), luteinising hormone (LH) and thyroid stimulating hormone (TSH). Therefore, we were able to classify the tumours based on the immunohistochemical (IHC) evidence of hormone expression into seven groups: somatotroph, lactotroph, thyrotroph, corticotroph, gonadotroph, plurihormonal, or null cell adenomas. Unfortunately, serum hormone levels and reliable data on clinical features were not available. IHC was performed on the TMA sections using highly sensitive and specific rabbit monoclonal antibodies for both SSTR2A (Clone UMB-1; Epitomics, USA) and SSTR5 (Clone UMB-4; Epitomics) at dilutions of 1 in 100 (detailed methods have been previously reported3Elston M.S. Meyer-Rochow G.Y. Conaglen H.M. et al.Increased SSTR2A and SSTR3 expression in succinate dehydrogenase-deficient pheochromocytomas and paragangliomas.Hum Pathol. 2015; 46: 390-396Google Scholar, 5Mehta S. de Reuver P.R. Gill P. et al.Somatostatin receptor SSTR-2a expression is a stronger predictor for survival than Ki-67 in pancreatic neuroendocrine tumors.Medicine. 2015; 94: 1-6Google Scholar). The IHC was scored by a blinded observer using a scheme similar to that reported by Korner et al.8Korner M. Waser B. Schonbrunn A. et al.Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumours suitable for in vitro somatostatin receptor targeting.Am J Surg Pathol. 2012; 36: 242-252Google Scholar That is, cases were scored as 0 (negative) if no neoplastic cells demonstrated positive staining, and then semiquantitatively if positive from 1+ (weak staining at ×100 magnification), to 2+ (moderate staining, i.e., strong staining at ×100 magnification, but non-circumferential staining at ×400 magnification), to 3+ (moderate staining at ×100 magnification and circumferential staining at ×400 magnification) to 4+ (intense diffuse and strong staining at ×100 magnification and circumferential staining at ×400 magnification). Only membranous staining was considered significant and non-specific nuclear or cytoplasmic uptake was disregarded. Based on the findings of Korner et al.8Korner M. Waser B. Schonbrunn A. et al.Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumours suitable for in vitro somatostatin receptor targeting.Am J Surg Pathol. 2012; 36: 242-252Google Scholar who correlated SSTR2A expression with the gold standard of in vitro 125I-[Tyr3]-octreotide autoradiography, for the purposes of binary analysis we classified cases that scored 0 or 1 as negative, and those that scored >1 as positive for SSTR expression. Statistical analysis was performed using SPSS Version 21.0 (Statistical Package for the Social Sciences, USA). Differences between groups were studied using the Kruskal–Wallis H test, and differences in proportions were studied using the Fisher exact test. p values less than 0.05 were considered statistically significant. There were 299 pituitary adenomas with assessable material in the TMA slides. These comprised 25 somatotroph adenomas, 23 lactotroph adenomas, 17 thyrotroph adenomas, 18 corticotroph adenomas, 13 gonadotroph adenomas, 78 null cell adenomas, and 125 plurihormonal adenomas (46 with GH expression and 79 without). The results of SSTR2A and SSTR5 IHC are presented in Table 1. Briefly, the majority of somatotroph adenomas were positive for SSTR2A (64%). The somatotroph adenomas also had the highest proportion of SSTR5 positivity (32%). Positive staining for both SSTR2A and SSTR5 was found in five of the 18 (28%) corticotroph adenomas. Within the plurihormonal group, the proportion of both SSTR2A and SSTR5 positivity was significantly higher for those tumours that expressed GH compared to those that were GH negative (p < 0.001, Fisher's exact test). SSTR2A and SSTR5 expression was less common in other tumours. Five of the 23 (22%) lactotroph adenomas were positive for SSTR2A, although none demonstrated 4+ staining. Only one (4%) lactotroph adenoma was positive for SSTR5. Three of the 17 (18%) thyrotroph adenomas were SSTR2A positive, however all were negative for SSTR5. Of the 78 null cell adenomas, 17 (22%) were positive for SSTR2A and five (6%) were positive for SSTR5. Of the 13 gonadotroph adenomas, two (15%) were positive for SSTR2A and one was positive for SSTR5 (6%). A Kruskal–Wallis H test showed that there was a statistically significant difference in SSTR2A and SSTR5 expression between the different adenoma types (H = 32.565, df = 6, p < 0.001 and H = 26.769, df = 6, p < 0.001, respectively).Table 1Expression of SSTR2A and SSTR5 in the pituitary adenomas subclassified by immunohistochemical evidence of hormone productionAdenoma type (n = 299)SSTR2ASSTR5NegativePositiveNegativePositiveSomatotroph (n = 25)9 (36%)16 (64%)17 (68%)8 (32%)Lactotroph (n = 23)18 (78%)5 (22%)22 (96%)1 (4%)Thyrotroph (n = 17)14 (82%)3 (18%)17 (100%)0Corticotroph (n = 18)13 (72%)5 (28%)13 (72%)5 (28%)Gonadotroph (n = 13)11 (85%)2 (15%)12 (92%)1 (8%)Null cell (n = 78)61 (78%)17 (22%)73 (94%)5 (6%)Plurihormonal with GH (n = 46)10 (22%)36 (78%)27 (59%)19 (41%)Plurihormonal without GH (n = 79)58 (73%)21 (27%)69 (87%)10 (13%) Open table in a new tab Limitations of our study include the fact that IHC was performed on TMA sections rather than whole sections so that focal staining may have been missed and also that we classified tumours on the basis of hormone production as determined by IHC rather than clinical evidence of hormone secretion which is considered the gold standard for pituitary adenoma classification. Despite this, our main finding that GH producing adenomas are the most likely to be SSTR2A and SSTR5 positive by IHC is in keeping with previous studies and the clinical impression that somatotroph adenomas are most likely to respond to somatostatin agonists.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar Of note, other studies have reported a significantly higher rate of somatostatin receptor expression in somatotroph adenomas of up to 93% for SSTR2A compared to our rate of 64% and up to 83% for SSTR5 compared to our rate of 32%.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar This discrepancy may be due to our use of TMA rather than whole sections and because we classified tumours based on IHC evidence of hormone production rather than on clinical evidence of hormone excess. That is, in addition to sampling artefact, it is possible that some of our GH IHC positive but SSTR2A and SSTR5 IHC negative adenomas were clinically non-functioning. This is supported by the findings of Pawlikowski et al.,9Pawlikowski M. Pisarek H. Kunert-Radek J. et al.Immunohistochemical detection of somatostatin receptor subtypes in “clinically non-functioning” pituitary adenomas.Endocr Pathol. 2003; 14: 231-238Google Scholar who demonstrated that clinically non-functioning pituitary adenomas in fact represent a heterogeneous group, the majority of which express pituitary hormones and somatostatin receptor proteins. Although some older studies using less specific and sensitive antibodies are conflicting, recent studies using clone UMB-4 have suggested an approximately 20% incidence of SSTR5 expression in corticotroph adenomas, very similar to the 28% (5/18) incidence we identified.1Chinezu L. Vasiljevic A. Jouanneau E. et al.Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumours using immunohistochemical detection with new specific monoclonal antibodies.Hum Pathol. 2014; 45: 71-77Google Scholar, 10Lupp A. Hunder A. Petrich A. et al.Reassessment of sst2 somatostatin receptor expression in human normal and neoplastic tissue using the novel rabbit monoclonal antibody UMB-4.Neuroendocrinology. 2011; 94: 255-264Google Scholar In this context, it is particularly interesting that in a recent clinical study the SSTR5 agonist pasireotide demonstrated a 20% response rate in pituitary Cushing's disease.11Colao A. Petersenn S. Newell-Price J. et al.A 12-month phase 3 study of pasireotide in Cushing’s disease.N Engl J Med. 2012; 366: 914-924Google Scholar Clinical data including whether or not patients were treated with, or responded to, somatostatin analogues was not available for our study participants, which we acknowledge as an important limitation. Whilst we do not have data on pasireotide treatment in this cohort, this similar incidence of IHC positivity and treatment-response supports the impression that SSTR5 expression by IHC may correlate with treatment response to pasireotide.8Korner M. Waser B. Schonbrunn A. et al.Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumours suitable for in vitro somatostatin receptor targeting.Am J Surg Pathol. 2012; 36: 242-252Google Scholar In conclusion, we report the results of IHC for SSTR2A and SSTR5 in a large cohort of pituitary adenomas. Of note the subtypes of adenomas which are reported to be most likely to respond to SSTR2A and SSTR5 agonists are also the subtypes of adenomas which we found most likely to demonstrate positive staining for these receptors by IHC. Further dedicated studies will be required to definitively confirm or refute the inference that IHC for the somatostatin receptors may be useful to predict response to somatostatin analogue therapy, particularly if major management decisions are to be based on an immunohistochemical staining. Nonetheless, our study adds some weight to the impression that in future IHC for somatostatin receptors may have a role to tailor treatment in routine clinical practice. This project was supported by the Sydney Vital, Translational Cancer Research Centre, through a Cancer Institute NSW competitive grant. The views expressed herein are those of the authors and are not necessarily those of the Cancer Institute NSW (CINSW). The authors state that there are no conflicts of interest to disclose.

Background: The diagnosis of epithelioid haemangioendothelioma (EHE) can be challenging, particularly on small biopsies. A specific recurrent chromosomal translocation t(1;3)(p36.23;q25.1) resulting in expression of the WWTR1-CAMTA1 fusion oncogene has been reported in 80–90% of EHE. Recently immunohistochemistry (IHC) using a novel rabbit polyclonal antibody (NBP1-93620: Novus Biologicals, CO, USA) has been reported as a sensitive and specific marker of EHE.1,2

In the routine clinical setting there is often an indication to test colorectal carcinoma (CRC) for the presence of somatic BRAFV600E mutation. BRAFV600E mutations are found in approximately two-thirds of CRCs demonstrating dual loss of PMS2 and MLH1 expression by immunohistochemistry (IHC) attributable to somatic hypermethylation of the MLH1 promoter locus, but in less than 1% with dual loss of PMS2/MLH1 expression due to Lynch syndrome.1Toon C.W. Walsh M.J. Chou A. et al.BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome.Am J Surg Pathol. 2013; 37: 1592-1602Crossref PubMed Scopus (111) Google Scholar, 2Capper D. Voigt A. Bozukova G. et al.BRAF V600E-specific immunohistochemistry for the exclusion of Lynch syndrome in MSI-H colorectal cancer.Int J Cancer. 2013; 133: 1624-1630Crossref PubMed Scopus (94) Google Scholar, 3Parsons M.T. Buchanan D.D. Thompson B. Young J.P. Spurdle A.B. Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification.J Med Genet. 2012; 49: 151-157Crossref PubMed Scopus (210) Google Scholar Therefore the presence of a BRAFV600E mutation in a dual PMS2/MLH1 negative CRC virtually excludes the diagnosis of Lynch syndrome – an association commonly used in population based screening programmes.4Jin M. Hampel H. Zhou X. Schunemann L. Yearsley M. Frankel W.L. BRAF V600E mutation analysis simplifies the testing algorithm for Lynch syndrome.Am J Clin Pathol. 2013; 140: 177-183Crossref PubMed Scopus (37) Google Scholar Furthermore BRAFV600E mutated microsatellite stable CRCs have a worse prognosis,5Toon C.W. Chou A. DeSilva K. et al.BRAFV600E immunohistochemistry in conjunction with mismatch repair status predicts survival in patients with colorectal cancer.Mod Pathol. 2014; 27: 644-650Crossref PubMed Scopus (47) Google Scholar and BRAFV600E mutated CRCs appear to be resistant to anti-EGFR targeted therapies with cetuzimab and panitumumab.6Nicolantonio F.D. Martini M. Molinari F. et al.Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer.J Clin Oncol. 2008; 26: 5705-5712Crossref PubMed Scopus (1438) Google Scholar That is, a strong argument could be made to test all CRCs for the presence of the BRAFV600E mutation, but only if such testing can be delivered reliably and in a cost effective manner. Molecular testing, which is usually considered the gold standard for identification of the BRAFV600E mutation, is relatively expensive and not available in all diagnostic laboratories. Mutation specific immunohistochemistry (IHC) has been proposed as a cheap and rapidly deployable method to detect BRAFV600E mutation in routine clinical care.1Toon C.W. Walsh M.J. Chou A. et al.BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome.Am J Surg Pathol. 2013; 37: 1592-1602Crossref PubMed Scopus (111) Google Scholar However, not all groups have found mutation specific IHC for BRAFV600E to be reliable in CRC. A fair reading of literature indicates that while some have found BRAFV600E IHC to be highly sensitive (98.2–100%) and specific (98.1–98.7%) in CRC,2Capper D. Voigt A. Bozukova G. et al.BRAF V600E-specific immunohistochemistry for the exclusion of Lynch syndrome in MSI-H colorectal cancer.Int J Cancer. 2013; 133: 1624-1630Crossref PubMed Scopus (94) Google Scholar, 7Day F. Muranyi A. Singh S. et al.A mutant BRAF V600E-specific immunohistochemical assay: correlation with molecular mutation status and clinical outcome in colorectal cancer.Target Oncol. 2015; 10: 99-109Crossref PubMed Scopus (40) Google Scholar, 8Nakaji Y. Oki E. Nakanishi R. et al.Prognostic value of BRAF V600E mutation and microsatellite instability in Japanese patients with sporadic colorectal cancer.J Cancer Res Clin Oncol. 2017; 143: 151-160Crossref PubMed Scopus (32) Google Scholar others have found it to be relatively inaccurate and reported sensitivities as low as 75% sometimes with particularly low specificities of 35–68%.9Lasota J. Kowalik A. Wasag B. et al.Detection of the BRAFV600E mutation in colon carcinoma – critical evaluation of the immunohistochemical approach.Am J Surg Pathol. 2014; 38: 1235-1241Crossref PubMed Scopus (51) Google Scholar, 10Adackapara C.A. Sholl L.M. Barletta J.A. Hornick J.L. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma.Histopathology. 2013; 63: 187-193Crossref PubMed Scopus (72) Google Scholar, 11Loes I.M. Immervoll H. Angelsen J.H. et al.Performance comparison of three BRAF V600E detection methods in malignant melanoma and colorectal cancer specimens.Tumour Biol. 2015; 36: 1003-1013Crossref PubMed Scopus (19) Google Scholar, 12Estrella J.S. Tetzlaff M.T. Bassett R.L. et al.Assessment of BRAFV600E status in colorectal carcinoma: tissue-specific discordances between immunohistochemistry and sequencing.Mol Cancer Ther. 2015; 14: 2887-2895Crossref PubMed Scopus (34) Google Scholar As proponents and early adopters of BRAFV600E mutation specific IHC in CRC1Toon C.W. Walsh M.J. Chou A. et al.BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome.Am J Surg Pathol. 2013; 37: 1592-1602Crossref PubMed Scopus (111) Google Scholar we have performed reflex BRAFV600E mutation specific IHC in all CRCs since August 2012. However, we were recently taken aback to encounter a patient with Lynch syndrome who presented with a PMS2/MLH1 negative CRC which was initially interpreted to be BRAFV600E IHC positive. Further investigation of this case determined that this discrepancy was due to false positive interpretation of the BRAFV600E IHC at the time of primary reporting. Therefore, we felt obliged to conduct a retrospective audit of the sensitivity and specificity of BRAFV600E mutation specific IHC as deployed at out institution since 2012 as part of our Lynch syndrome screening program. We now share our findings in the hope that the results will inform the debate on the accuracy of BRAFV600E mutation specific IHC in CRC in the routine clinical setting. We searched the surgical pathology database of the Royal North Shore Hospital, Sydney Australia, for all PMS2/MLH1 dual negative CRCs from the introduction of reflex BRAFV600E mutation specific IHC on 22 August 2012 until 31 May 2016. Detailed methods for performing and interpreting the BRAFV600E IHC have been previously reported1Toon C.W. Walsh M.J. Chou A. et al.BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome.Am J Surg Pathol. 2013; 37: 1592-1602Crossref PubMed Scopus (111) Google Scholar, 5Toon C.W. Chou A. DeSilva K. et al.BRAFV600E immunohistochemistry in conjunction with mismatch repair status predicts survival in patients with colorectal cancer.Mod Pathol. 2014; 27: 644-650Crossref PubMed Scopus (47) Google Scholar and throughout the study period we used the now commercially available clone VE1 (Spring Bioscience, USA). Over the study period the results of BRAFV600E IHC had been interpreted by 10 different pathologists. The original BRAFV600E IHC stained slides were retrieved from the files and rescored (blinded to the previous interpretation) by a pathologist with particular interest and expertise in mutation specific IHC. All PMS2/MLH1 negative cases were then macrodissected and underwent molecular testing on the same block used for IHC using a multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry detection assay (Agena MassArray).1Toon C.W. Walsh M.J. Chou A. et al.BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome.Am J Surg Pathol. 2013; 37: 1592-1602Crossref PubMed Scopus (111) Google Scholar This platform has been specifically validated for BRAFV600E, V600R, V600K, and V600M mutations and the laboratory holds full IANZ ISO15189 accreditation for this assay. For cases in which both the IHC results and MALDI-TOF PCR were concordant, IHC was considered accurate and no further testing was undertaken. Any cases in which there were any discrepancies between the initial IHC, the rescored IHC or the MALDI-TOF PCR underwent Sanger sequencing on macrodissected tumour tissue to resolve the discordance. That is, the gold standard for cases in which IHC and MALDI-TOF PCR were discrepant was Sanger sequencing. The results are summarised in the flow chart in Fig. 1. In the study period there were 972 colorectal adenocarcinomas of which 227 (23.4%) demonstrated negative staining for PMS2 and MLH1 and formed the primary study cohort. Of these cases 200 (88.1%) were considered BRAFV600E mutated based on initial interpretation of IHC by the primary reporting pathologist. Upon blinded review for this study, 10 of these cases were considered to be false positive interpretation of the IHC. All 227 PMS2/MLH1 dual negative CRCs underwent MALDI-TOF PCR and of these 182 (80.1%) were found to harbour a BRAFV600E mutation. MALDI-TOF PCR was discordant with the initial IHC in 20 cases (19 IHC positive, MALDI-TOF PCR negative; 1 IHC negative and MALDI-TOF PCR positive). These 20 discrepant cases then underwent Sanger sequencing. When Sanger sequencing was considered the gold standard in these 20 cases, the initial interpretation of the IHC was 'incorrect' but the rescoring of the same slide by an expert was 'correct' in 10 cases (50% of discrepant cases). Six cases had incorrect IHC as both initially scored and rescored but 'correct' MALDI-TOF PCR. Four cases (20%) had initially correct IHC and review IHC correct but incorrect MALDI-TOF results (Table 1).Table 1Dual PMS2/MLH1 negative colorectal carcinomas with discrepant BRAF immunohistochemistry and/or MALDI-TOF PCR testingCase no.AgeGenderBRAFV600E IHC as initially reportedBRAFV600E IHC result expert reviewBRAFV600E MALDI-TOF PCRBRAF sanger sequencingConsensus BRAF molecular test resultCases in which MALDI-TOF PCR is 'wrong' and IHC 'correct'474FPositivePositiveNegativePositivePositive588FPositivePositiveNegativePositivePositive1378FPositivePositiveNegativePositivePositive1961MPositivePositiveNegativePositivePositiveCases in which IHC (both initial and review) is 'wrong' and MALDI-TOF PCR 'correct'170FNegativeNegativePositivePositivePositive370FPositivePositiveNegativeNegativeNegative956MPositivePositiveNegativeNegativeNegative1181FPositivePositiveNegativeNegativeNegative1280FPositivePositiveNegativeNegativeNegative2079FPositivePositiveNegativeNegativeNegativeCases in which initial IHC is 'wrong' and review IHC is 'correct'260MPositiveNegativeNegativeNegativeNegative684FPositiveNegativeNegativeNegativeNegative779FPositiveNegativeNegativeNegativeNegative882FPositiveNegativeNegativeNegativeNegative1087MPositiveNegativeNegativeNegativeNegative1462FPositiveNegativeNegativeNegativeNegative1545MPositiveNegativeNegativeNegativeNegative1681FPositiveNegativeNegativeNegativeNegative1778FPositiveNegativeNegativeNegativeNegative1868MPositiveNegativeNegativeNegativeNegativeF, female; M, male.IHC = BRAF specific immunohistochemistry. Open table in a new tab F, female; M, male. IHC = BRAF specific immunohistochemistry. That is, in the routine clinical setting IHC demonstrates similar accuracy to MALDI-TOF PCR (97.4% versus 98.3% accurate), but only if scored with care by a pathologist with experience in its interpretation and awareness of the possibility of false positive staining, otherwise the accuracy falls to 93%. Review of the 10 cases the initial reporting pathologists interpreted as positive but were thought to be negative upon rescoring and confirmed negative by molecular testing, provides important lessons on the artifacts which may lead to false positive interpretation. The primary reason for the error was misinterpretation of non-specific staining not limited to the cytoplasm of the neoplastic cells but also staining their nucleus and other background elements including benign epithelium and smooth muscle with similar intensity (illustrated in Fig. 2). From a practical point of view, we recommend that extreme caution be taken if there is evidence of such non-specific background staining and, if uncertainty persists, such cases can be considered indeterminate for BRAFV600E mutation by IHC. In addition to errors in interpretation, other factors which may be associated with poor antibody performance include differences in fixation, antibody concentration and antibody retrieval solutions.9Lasota J. Kowalik A. Wasag B. et al.Detection of the BRAFV600E mutation in colon carcinoma – critical evaluation of the immunohistochemical approach.Am J Surg Pathol. 2014; 38: 1235-1241Crossref PubMed Scopus (51) Google Scholar, 10Adackapara C.A. Sholl L.M. Barletta J.A. Hornick J.L. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma.Histopathology. 2013; 63: 187-193Crossref PubMed Scopus (72) Google Scholar, 11Loes I.M. Immervoll H. Angelsen J.H. et al.Performance comparison of three BRAF V600E detection methods in malignant melanoma and colorectal cancer specimens.Tumour Biol. 2015; 36: 1003-1013Crossref PubMed Scopus (19) Google Scholar, 12Estrella J.S. Tetzlaff M.T. Bassett R.L. et al.Assessment of BRAFV600E status in colorectal carcinoma: tissue-specific discordances between immunohistochemistry and sequencing.Mol Cancer Ther. 2015; 14: 2887-2895Crossref PubMed Scopus (34) Google Scholar Anecdotally, in our hands we have found that the antibody performs more reliably when staining is performed on one commercially available staining platform in comparison to another. In conclusion, as early implementers of mutation specific IHC for BRAFV600E in CRC, our experience demonstrates that it can be highly accurate when scored by pathologists with experience in its interpretation and awareness of the pitfalls of non-specific staining. The authors state that there are no conflicts of interest to disclose.

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.

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.

Anti–epidermal growth factor receptor–targeted therapy is only indicated in RAS wild-type colorectal carcinomas (CRCs). It is recommended that both NRAS and KRAS mutation testing to be performed before a CRC is considered RAS wild-type. Given that mutation-specific immunohistochemistry (IHC) has been shown to be sensitive and specific for the detection of NRAS Q61R mutations in melanoma, we assessed the specificity of NRASQ61R mutation-specific IHC in CRC. IHC was performed on tissue microarrays containing 2823 consecutive CRC undergoing surgery with curative intent using a novel mutation-specific antibody to the protein produced by the NRAS Q61R mutation (clone SP174). Tissue microarrays were assessed by 2 observers and all IHC-positive or equivocal cases were repeated on whole sections to confirm the result. Positive cases then underwent molecular testing by matrix-assisted laser desorption/ionization-time of flight polymerase chain reaction. In total, 22 of 2823 (0.8%) CRCs demonstrated confirmed positive staining with complete interobserver concordance. RAS mutations were confirmed in all IHC-positive CRCs. In total, 11 cases harbored the NRASQ61R mutation. Surprisingly, 11 cases demonstrated the KRASQ61R mutation. We conclude that mutation-specific IHC with this currently available NRASQ61R antibody is highly specific for the presence of either NRASQ61R or KRASQ61R mutations in CRC. We caution that we did not assess the sensitivity of IHC and that this antibody does not detect other RAS mutations. Therefore, negative staining does not exclude a clinically significant RAS mutation. However, positive staining confirms the presence of an NRASQ61R or KRASQ61R mutation without the need for further molecular testing.