The association between KRAS and histopathological growth patterns and the impact on resection margins around vasculature and bile ducts in colorectal liver metastases.

The liver is the most frequent and often the only site of distant disease in colorectal cancer and, of all treatment protocols currently in use, resection is the most likely to result in long-term cure. Within the liver, tumour proximity to major vasculature and biliary structures poses a resection challenge, requiring a balance of achieving negative margins while preserving adequate vascular circulation and biliary drainage. The focus on parenchymal sparing resections are important but just as important may be the 'biological' behaviour of the tumour. In colorectal liver metastases (CRLM), biomarkers such as the Kirsten rat sarcoma oncogene homologue (KRAS) gene and histological growth patterns (HGPs) further improve the prognostication after resection. However, to date, the association between the KRAS status and HGPs in CRLM and their impact on resection margins around major vasculature or biliary structures in terms of overall survival and recurrence rates are unclear. The aim of this review was to explore the available evidence for the association between KRAS and HGPs in CRLM and attempt to define their impact on resection margins near major structures.

The prognostic significance of lymphovascular invasion in cutaneous squamous cell carcinoma.

BACKGROUND: The majority of cutaneous squamous cell carcinomas (cSCC) have a favourable prognosis. However, a subset of cases follow an aggressive disease course with progression to metastasis and death. Several histopathological parameters are associated with poor outcomes, but lymphovascular invasion (LVI) has not been well studied. OBJECTIVE: To assess the prognostic significance of LVI in cSCC and determine associations between LVI and cSCC. METHODS: A retrospective review of 486 consecutive cases of cSCC over a 5-year period from a single centre was stratified by the presence or absence of LVI. Logistic regression and multivariate survival analysis were used to determine associations of LVI and prognostic significance of LVI, respectively. FINDINGS: LVI was present in 41 cases (9.2%). LVI was significantly associated with increasing depth of invasion, microanatomical tumour location (subcutis vs. dermis), and tumour dimensions (P < 0.05). Univariate survival analysis revealed significantly lower 2-year overall survival rates for patients with LVI (37.1%) compared with those without (66.6%) (95% CI = 60.6-73.3, P < 0.001). LVI was also found to be an independent marker of poor disease-specific survival (HR = 0.232 (95% CI = 0.090-0.600), P = 0.003), poor overall survival (HR 0.338 (95% CI = 0.184-0.623), P < 0.001) and poor disease-free survival (HR 0.461 (95% CI = 0.230-0.923), P = 0.029) through multivariate analysis. CONCLUSIONS: This study confirms that LVI is an independent poor prognosticator in cSCC, with significantly worse survival indices at 2 years. Future systems of risk stratification for cSCC should incorporate LVI.

Granular cell tumour of the pancreas: a case report and systematic review.

PURPOSE: Granular cell tumours (GCTs) of the pancreas are mostly benign and exceptionally rare, with no unique identifying radiological features. Following a case discussion of a patient with GCT, a comprehensive review of available literature was conducted to identify the common diagnostic features associated with GCT. METHODS: Following a case report identified in our institution, a systematic review was conducted by two authors in accordance with Preferred Reporting Items for Systematic review and Meta-Analysis protocols (PRISMA) guidelines. Databases MEDLINE, EMBASE, Scopus, World of Science, and grey literature were searched on August 2021. Inclusion criteria were histopathology diagnosed granular cell tumour of the pancreas. RESULTS: A 37-year-old male presented with 1 month of abdominal pain and an MRI demonstrating a dilated main pancreatic duct, distal parenchymal atrophy, but no focal lesion. Repeat MRI at 6 months re-demonstrated similar findings and subsequent endoscopic ultrasound was suspicious for main duct IPMN. Following multidisciplinary team discussion, a spleen-preserving distal pancreatectomy was performed. Histopathology demonstrated granular cell tumour with cells diffusely positive for S100 and no malignant transformation. 11 case reports were identified in the literature with diagnosis confirmed on tissue histopathology based on positive immunohistochemical staining for S-100 protein. Eight patients presented with gastrointestinal symptoms with abdominal pain the main presenting complaint (50%). 10 patients underwent CT with portal venous contrast and all underwent endoscopic examination. Imaging findings were similar in five studies for EUS which demonstrated a hypoechoic lesion with homogenous appearance. On non-contrast CT GCT was iso-enhancing, and with portal venous contrast demonstrated hypo-enhancement that gradually enhanced on late phases. Pre-operative diagnosis of pancreatic carcinoma was described in six cases based on imaging and biopsy, resulting in progression to surgical resection. Nine patients were managed surgically and no complications identified on follow-up (6-52 months). CONCLUSION: The currently proposed management pathway includes EUS with biopsy and CT, and surgical resection recommended due to malignancy risk. Improved sample collection with EUS-FNA and microscopic assessment utilising S-100 immunohistochemistry may improve pre-operative diagnosis. Limitations include rare numbers in reported literature and short follow-up not allowing an assessment of GCT's natural history and malignancy risk. Additional cases would expand the current dataset of GCTs of the pancreas, so that surgical resection may be avoided in the future.

Peri-ureteric mass an unusual case of immunoglobin G4-related disease (IgG4-RD).

Immunoglobulin G4-related disease (IgG4-RD) of the ureter is a rarely reported disease, often mimicking urothelial carcinoma. This paper describes a case of an otherwise healthy patient with a lesion involving the ureter revealed on Computed tomography (CT), avid on fludeoxyglucose positron emission tomography (FDG PET), that prior to surgery was suspicious for urothelial carcinoma, until intra-op frozen section revealed otherwise. Diagnosis of ureteral IgG4-RD should be considered as a differential diagnosis, with serum IgG4 levels obtained.

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.

Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A as a Potential Glioblastoma Biomarker with Prognostic Significance.

Glioblastoma, WHO-grade IV glioma, carries a dismal prognosis owing to its infiltrative growth and limited treatment options. Glioblastoma-derived extracellular vesicles (EVs; 30-1000 nm membranous particles) influence the microenvironment to mediate tumor aggressiveness and carry oncogenic cargo across the blood-brain barrier into the circulation. As such, EVs are biomarker reservoirs with enormous potential for assessing glioblastoma tumors in situ. Neurosurgical aspirates are rich sources of EVs, isolated directly from glioma microenvironments. EV proteomes enriched from glioblastoma (n = 15) and glioma grade II-III (n = 7) aspirates are compared and 298 differentially-abundant proteins (p-value < 0.00496) are identified using quantitative LC-MS/MS. Along with previously reported glioblastoma-associated biomarkers, levels of all eight subunits of the key molecular chaperone, T-complex protein 1 Ring complex (TRiC), are higher in glioblastoma-EVs, including CCT2, CCT3, CCT5, CCT6A, CCT7, and TCP1 (p < 0.00496). Analogous increases in TRiC transcript levels and DNA copy numbers are detected in silico; CCT6A has the greatest induction of expression and amplification in glioblastoma and shows a negative association with survival (p = 0.006). CCT6A is co-localized with EGFR at 7p11.2, with a strong tendency for co-amplification (p < 0.001). Immunohistochemistry corroborates the CCT6A proteomics measurements and indicated a potential link between EGFR and CCT6A tissue expression. Putative EV-biomarkers described here should be further assessed in peripheral blood.

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.

NRASQ61R Mutation-specific Immunohistochemistry is Highly Specific for Either NRASQ61R or KRASQ61R Mutation in Colorectal Carcinoma.

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 mutations in melanoma, we assessed the specificity of NRAS 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 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.

Hypermutation In Pancreatic Cancer.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

Loss of INI1 expression in colorectal carcinoma is associated with high tumor grade, poor survival, BRAFV600E mutation, and mismatch repair deficiency.

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.

Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma.

Gynecologic and primary peritoneal serous carcinoma may be difficult to distinguish from abdominal mesotheliomas clinically, morphologically, and immunohistochemically. BAP1 double-hit inactivation and subsequent loss of protein expression have been reported in more than half of all abdominal mesotheliomas. We therefore sought to investigate the expression of BAP1 in serous carcinoma and explore its potential utility as a marker in the differential diagnosis with mesothelioma. We searched the computerized database of the Department of Anatomical Pathology, Royal North Shore Hospital, Australia, for all cases of gynecologic and peritoneal serous carcinomas and mesotheliomas diagnosed between 1998 and 2014. Immunohistochemistry for BAP1 was then performed on tissue microarray sections. Cases with completely absent nuclear staining in the presence of a positive internal control in nonneoplastic cells were considered negative. If staining was equivocal (eg, absent nuclear staining but no internal control), staining was repeated on whole sections. Loss of BAP1 expression was found in only 1 of 395 (0.3%) serous carcinomas but in 6 of 9 (67%) abdominal mesotheliomas (P < .001) and 131 of 277 (47%) thoracic mesotheliomas (P < .001). We conclude that BAP1 loss occurs extremely infrequently in gynecologic and peritoneal serous adenocarcinomas, whereas it is very common in mesotheliomas including abdominal mesothelioma. Therefore, although positive staining for BAP1 cannot be used to exclude a diagnosis of mesothelioma, loss of BAP1 expression can be used to very strongly support a pathological diagnosis of abdominal mesothelioma over serous carcinoma.

Loss of expression of BAP1 is very rare in non-small cell lung carcinoma.

Germline mutations of the BAP1 gene have been implicated in a cancer predisposition syndrome which includes mesothelioma, uveal melanoma, cutaneous melanocytic lesions, renal cell carcinoma, and possibly other malignancies. Double hit inactivation of BAP1 with subsequent loss of expression of the BAP1 protein also occurs in approximately 50% of mesotheliomas. The link between BAP1 mutation and lung cancer is yet to be fully explored. We sought to assess BAP1 expression in a large cohort of lung cancers undergoing surgery with curative intent. We searched the Anatomical Pathology database of our institution for lung cancer patients undergoing surgery with curative intent between 2000 and 2010. Immunohistochemistry for BAP1 was then performed in tissue microarray format. Our cohort included 257 lung cancer patients, of which 155 (60%) were adenocarcinomas and 72 (28%) were squamous cell carcinomas, with no other subtype comprising more than 3%. BAP1 loss of expression was found in only one lung cancer. We conclude that BAP1 mutation occurs very infrequently (0.4%) in non-small cell lung cancer. Given that the pathological differential diagnosis between lung carcinoma and mesothelioma may sometimes be difficult, this finding increases the specificity of loss of expression for BAP1 for the diagnosis of mesothelioma.

Mutation specific immunohistochemistry is highly specific for the presence of calreticulin mutations in myeloproliferative neoplasms.

The identification of somatic calreticulin (CALR) mutations can be used to confirm the diagnosis of a myeloproliferative disorder in Philadelphia chromosome-negative, JAK2 and MPL wild type patients with thrombocytosis. All pathogenic CALR mutations result in an identical C-terminal protein and therefore may be identifiable by immunohistochemistry. We sought to test the sensitivity and specificity of mutation specific immunohistochemistry for pathogenic CALR mutations using a commercially available mouse monoclonal antibody (clone CAL2). Immunohistochemistry for mutant calreticulin was performed on the most recent bone marrow trephine from a cohort of patients enriched for CALR mutations and compared to mutation testing performed by polymerase chain reaction (PCR) amplification followed by fragment length analysis. Twenty-nine patients underwent both immunohistochemistry and molecular testing. Eleven patients had CALR mutation, and immunohistochemistry was positive in nine (82%). One discrepant case appeared to represent genuine false negative immunohistochemistry. The other may be attributable to a 12 year delay between the bone marrow trephine and the specimen which underwent molecular testing, particularly because a liver biopsy performed at the same time as molecular testing demonstrated positive staining in megakaryocytes in extramedullary haematopoiesis. All 18 cases which lacked CALR mutation demonstrated negative staining. In this population enriched for CALR mutations, the specificity was 100%; sensitivity 82-91%, positive predictive value 100% and negative predictive value 90-95%. We conclude that mutation specific immunohistochemistry is highly specific for the presence of CALR mutations. Whilst it may not identify all mutations, it may be very valuable in routine clinical care.

Mismatch repair deficiency as a prognostic factor in mucinous colorectal cancer.

There is some uncertainty about pathological grading of mucinous colorectal adenocarcinoma, defined as colorectal cancer demonstrating at least 50% mucinous differentiation. Under the WHO 2000 classification mucinous colorectal cancer was considered high grade. However under the current WHO 2010 classification microsatellite unstable/mismatch repair-deficient (MSI/MMRd) mucinous colorectal cancer is considered low grade, whereas microsatellite stable/mismatch repair proficient (MSS/MMRp) tumours are high grade. However there is little empirical evidence for this approach. We therefore compared the long term survival of patients with MSI/MMRd vs MSS/MMRp mucinous colorectal cancer in a large unselected cohort of patients undergoing surgery at our institution from 1998 to 2011. There were 2608 patients in the cohort, of which 264 (10.1%) were mucinous. 95 (36%) of the mucinous tumours were microsatellite unstable. The all-cause 5-year survival of mucinous MSI/MMRd colorectal cancer was similar to that of non-mucinous low-grade colorectal cancer (73 vs 67%, P=0.368), and significantly better than that of both non-mucinous high-grade (73 vs 53%, P<0.001) and mucinous MSS/MMRp colorectal cancer (73 vs 57%, P=0.023). The 5-year survival of mucinous MSS/MMRp colorectal cancer was slightly better than that of non-mucinous high-grade patients (57 vs 53%, P=0.027), but significantly worse than that of non-mucinous low-grade colorectal cancer (57 vs 67%, P=0.018). In multivariate Cox regression analysis, conventional histological grade based on glandular differentiation maintained prognostic significance (P=0.003) whereas MSI/MMRd status just failed to be statistically significant (P=0.062). Our findings support the WHO 2010 approach that as a group mucinous MSS/MMRp colorectal cancers are biologically aggressive. However, grading based exclusively on MSI/MMR status may be overly simplistic as conventional grading based on the degree of glandular differentiation still holds greater prognostic significance in multivariate analysis.

Loss of BAP1 Expression Occurs Frequently in Intrahepatic Cholangiocarcinoma.

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor gene. Double hit BAP1 inactivation has been reported in a range of tumor types, including intrahepatic cholangiocarcinoma (ICC), sometimes in association with germline mutation.We performed immunohistochemistry for BAP1 on a well-characterized cohort of 211 ICC patients undergoing surgical resection with curative intent at 3 institutions based in 3 different countries. The median age at diagnosis was 65 years (range, 36.5-86) and 108 (51%) were men. Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in nonneoplastic cells) occurred in 55 ICCs (26%). BAP1 loss predicted a strong trend toward improved median survival of 40.80 months (95% CI, 28.14-53.46) versus 24.87 months (95% CI, 18.73-31.01), P = 0.059). In a multivariate model including age, sex, BAP1 status, tumor stage, tumor grade, lymphovascular invasion, and tumor size, female sex was associated with improved survival (hazard ratio [HR] 0.54; 95% CI, 0.34-0.85), while advanced tumor stage and lymphovascular invasion (HR 1.89; 95% CI, 1.09-3.28) correlated with decreased survival. In a multivariate analysis, high grade tumors were associated with BAP1 loss (odds ratio [OR] 3.32; 95% CI, 1.29-8.55), while lymphatic invasion was inversely associated with BAP1 loss (OR 0.36; 95% CI, 0.13-0.99).In conclusion, we observed a trend toward improved prognosis in ICC associated with absent expression of BAP1 and an association of BAP1 loss with higher histological grade and absent lymphatic invasion. Female sex was associated with improved survival while advanced tumor stage and lymphatic invasion were associated with decreased survival.