Utility of BRAF V600E mutation-specific immunohistochemistry in detecting BRAF V600E-mutated gastrointestinal stromal tumors.

OBJECTIVES: As patients with BRAF V600E mutation respond to BRAF inhibitors, it is important to identify these mutations to stratify patients for the appropriate therapy. In this study, we evaluated the utility of a BRAF V600E allele-specific antibody in gastrointestinal stromal tumors (GISTs). METHODS: BRAF V600E mutation-specific immunohistochemistry (negative, weak, or moderate/strong expression) and BRAF sequencing were performed on 38 consecutive GISTs diagnosed between January 2013 and April 2014. RESULTS: GISTs from a cohort of 25 men and 13 women (mean age, 61 years; range, 39-88 years) were localized to the stomach (18), small bowel (10), colon (three), rectum (two), and pelvis/omentum (five). Strong and diffuse cytoplasmic BRAF expression was noted in two (5%) of 38 cases, while eight (21%) of 38 cases showed weak staining, and 28 (74%) of 38 cases were negative. Both of the strongly positive cases arose in the stomach, occurring in a 42-year-old and a 47-year-old woman, respectively. The lesions measured 0.8 and 1 cm, showed spindle cell morphology, and had no risk of progressive disease by Miettinen criteria. Both cases showed heterozygous BRAF V600E, while no BRAF mutations were detected in cases with weak or negative BRAF expression. CONCLUSIONS: BRAF V600E mutation-specific immunohistochemistry is a highly sensitive and specific method for detecting BRAF-mutated GISTs.

Morphologic and molecular characterization of traditional serrated adenomas of the distal colon and rectum.

Of the serrated polyps, the origin, morphologic features, molecular alterations, and natural history of traditional serrated adenomas (TSAs) are the least understood. Recent studies suggest that these polyps may arise from precursor lesions. The frequencies of KRAS and BRAF mutations vary between these studies, and only 1 small study has measured CpG island methylation using current markers of methylation. Mutations in GNAS, a gene commonly mutated in colorectal villous adenomas, have not been fully evaluated in TSAs. Finally, the expression of annexin A10 (ANXA10), a recently discovered marker of sessile serrated adenomas/polyps, has not been studied in these polyps. To further characterize these polyps, 5 gastrointestinal pathologists reviewed 55 left-sided polyps diagnosed as TSA at a single institution. Pathologists assessed various histologic features including cytoplasmic eosinophilia, ectopic crypt foci, presence of conventional dysplasia, and presence of precursor serrated lesions. KRAS, BRAF, and GNAS mutational analysis was performed, as well as CpG island methylation and ANXA10 immunohistochemistry. Ectopic crypt foci were seen in 62% of TSAs. Precursor lesions were seen in 24% of the study polyps, most of which were hyperplastic polyps. KRAS and BRAF mutations were common and were present in 42% and 48% of polyps, respectively. GNAS mutations occurred in 8% of polyps, often in conjunction with a BRAF mutation. Unlike sessile serrated adenomas/polyps, TSAs rarely had diffuse expression of ANXA10. Importantly, BRAF-mutated TSAs had more widespread methylation of a 5-marker CpG island panel compared with KRAS-mutated polyps. However, ectopic crypt foci, a proposed defining feature of TSA, were not associated with any specific molecular alteration.

STAT6 rabbit monoclonal antibody is a robust diagnostic tool for the distinction of solitary fibrous tumour from its mimics.

Recurrent NAB2-STAT6 gene fusions have recently been identified in solitary fibrous tumour by next generation sequencing. Our aim was to examine the sensitivity and specificity of STAT6 immunohistochemistry for solitary fibrous tumour versus other morphologically similar soft tissue tumours. STAT6 expression was evaluated in 54 solitary fibrous tumours of various sites and 99 soft tissue tumours in the histological differential diagnosis. We used a rabbit monoclonal STAT6 antibody (1:100), which has not been reported by others, on formalin fixed, paraffin embedded whole sections and tissue microarray slides. Only nuclear staining of STAT6 was considered positive. Distribution of staining was scored as: 0 (no staining), 1+ (1-25%), 2+ (26-50%), 3+ (>50%). Intensity was scored as weak, moderate or strong. Nuclear STAT6 staining was present in all SFT cases tested (54/54, sensitivity 100%), regardless of histology, anatomical site or CD34 status. The majority of cases showed 3+ and strong staining. All tested cases of cellular angiofibroma (0/9), myofibroblastoma (0/10), spindle cell lipoma (0/10), benign fibrous histiocytoma (0/13), dermatofibrosarcoma protruberans (0/9), low-grade fibromyxoid sarcoma (0/7), schwannoma (0/8), desmoid-type fibromatosis (0/8), monophasic synovial sarcoma (0/11), malignant peripheral nerve sheath tumour (0/7), and mesenchymal chondrosarcoma (0/7) were negative for STAT6 (specificity 100%). Our study further supports the utility of STAT6 immunohistochemistry as an adjunct in the diagnosis of solitary fibrous tumour.

Immunohistochemistry for annexin A10 can distinguish sporadic from Lynch syndrome-associated microsatellite-unstable colorectal carcinoma.

Differentiating sporadic microsatellite-unstable colorectal carcinoma due to MLH1 promoter hypermethylation from Lynch syndrome (LS)-associated tumors due to mutations in mismatch-repair proteins is time consuming, cost intensive, and requires advanced laboratory testing. A mutation in BRAF has been shown to be highly specific for sporadic tumors; however, a significant proportion of sporadic microsatellite-unstable tumors lack BRAF mutations. MLH1 promoter methylation analysis is subsequently used to differentiate LS and sporadic tumors, but both tests require specialized laboratories and are costly. Through previous gene expression profiling of serrated polyps, we identified annexin A10 as a protein highly expressed in sessile serrated adenomas/polyps. As these polyps give rise to the majority of sporadic microsatellite-unstable tumors, we evaluated the ability of annexin A10 expression to discriminate between LS and sporadic tumors. A marked increase in annexin A10 mRNA was observed in sporadic microsatellite-unstable tumors compared with LS tumors (378-fold increase, P<0.001). Using immunohistochemistry, annexin A10 was expressed in 23/53 (43%) BRAF-mutated and 9/22 (41%) BRAF wild-type sporadic tumors. In contrast, only 3/56 (5%) LS tumors were positive for annexin A10 (P<0.0001). One patient had a deleterious MSH2 mutation, and another had a variant of uncertain significance in MSH6. These 2 tumors could be easily distinguished from sporadic tumors using mismatch-repair protein immunohistochemistry. Only 1/28 (4%) LS tumors with loss of MLH1 was positive for annexin A10. This patient did not have a deleterious MLH1 mutation but rather germline promoter hypermethylation of MLH1. On the basis of these results, immunohistochemistry for annexin A10 may be a useful marker to distinguish sporadic from LS-associated microsatellite-unstable colon cancer.

Diagnostic utility of TP53 and cytokeratin 7 immunohistochemistry in idiopathic inflammatory bowel disease-associated neoplasia.

Long-standing inflammatory bowel disease is associated with increased risk of developing colorectal adenocarcinoma. Significant intra- and inter-observers' variability exists in histologic interpretation of dysplasia in surveillance biopsies. In this study, we evaluated the utility of a panel of immunohistochemical markers in diagnosing inflammatory bowel disease-associated neoplasia. We reviewed 39 colectomy specimens with inflammatory bowel disease-associated neoplasia. In these 39 cases, we identified 172 foci of interest (5 normal, 58 negative for dysplasia, 15 indefinite for dysplasia, 59 low-grade dysplasia, 18 high-grade dysplasia, and 17 invasive adenocarcinoma). They were subjected to immunohistochemistry for TP53 and CK7. Logistic regression was used to evaluate their association with the presence of dysplasia. Receiver operating characteristic curves were used to determine the optimal cutoffs and assess the diagnostic performance of TP53 and CK7. Both TP53 nuclear staining and CK7 immunoreactivity gradually increased in the progression of inflammatory bowel disease-associated neoplasia (P<0.0001). CK7 immunoreactivity increased along with the increase of inflammation severity (P=0.0002) as well as reactive changes (P=0.04) in the colonic mucosa. But TP53 nuclear staining was independent of either feature. When both TP53>8% and CK7>30% as identified from logistic regression and receiver operating characteristic curves were used to diagnose dysplasia, the specificity achieved as high as 95%. When either TP53>8% or CK7>30% was used to diagnose dysplasia, the sensitivity achieved was 82%. Our results suggested that a combination of CK7 and TP53 immunohistochemistry may be helpful in diagnosing inflammatory bowel disease-associated dysplasia in difficult cases.

Gene expression profiling of serrated polyps identifies annexin A10 as a marker of a sessile serrated adenoma/polyp.

Sessile serrated adenomas/polyps (SSA/Ps) are precursors of colon cancer, particularly those that exhibit microsatellite instability. Distinguishing SSA/Ps from the related, but innocuous, microvesicular hyperplastic polyp (MVHP) can be challenging. In this study seven gastrointestinal pathologists reviewed 109 serrated polyps and identified 60 polyps with histological consensus. Microarray analysis was performed on six distal consensus MVHPs < 9 mm, six proximal consensus SSA/Ps > 9 mm, and six normal colon biopsies (three proximal, three distal). Comparative gene expression analysis confirmed the close relationship between SSA/Ps and MVHPs as there was overlapping expression of many genes. However, the gene expression profile in SSA/Ps had stronger and more numerous associations with cancer-related genes compared with MVHPs. Three genes (TFF2, FABP6, and ANXA10) were identified as candidates whose expression can differentiate SSA/Ps from MVHPs, and the differences in expression were confirmed by quantitative RT-PCR. As ANXA10 showed the most promise in differentiating these polyps, the expression of ANXA10 was evaluated by immunohistochemistry in consensus SSA/Ps (n = 26), MVHPs (n = 21), and normal colon (n = 9). Immunohistochemical expression of ANXA10 was not identified in separate samples of normal colon or in the normal colonic epithelium adjacent to the serrated polyps. Consistent with the microarray and quantitative RT-PCR experiments, immunohistochemical expression of ANXA10 was markedly increased in SSA/Ps compared to MVHPs (p < 0.0001). An ANXA10 score ≥ 3 has a sensitivity of 73% and a specificity of 95% in the diagnosis of an SSA/P. In conclusion, we show that SSA/Ps and MVHPs have significant overlap in gene expression, but also important differences, particularly in cancer-related pathways. Expression of ANXA10 may be a potential marker of the serrated pathway to colon cancer.

Endometriosis involving the mucosa of the intestinal tract: a clinicopathologic study of 15 cases.

Endometriosis involving the mucosa of the intestines is rare, but may lead to diagnostic pitfalls. We reviewed 15 cases (seven biopsies and eight resections) from 14 patients. The patients' mean age is 48 years (31-66 years). Presenting symptoms included lower gastrointestinal bleeding, pelvic pain, rectal urgency, abdominal mass, and bowel obstruction. In the majority of cases, the lesion was located in the rectum (73%) with the remainder in the sigmoid colon (20%) and ileum (7%). The most common indication for biopsy was a polypoid lesion seen endoscopically (eight cases). For patients who underwent resections, the most common clinical impression was colonic carcinoma (75%), due to mass lesions and stricture as the most common macroscopic findings. Histologically, one case had stromal endometriosis only, but the remaining 14 cases had both endometrial glands and stroma. Epithelial metaplasia was present in all cases, mostly tubal metaplasia (ciliated epithelium). Hybrid glands and replacement of the surface epithelium by endometrial epithelium were also seen. Crypt architectural distortion, cryptitis, and crypt abscesses were seen in some cases, mimicking chronic active colitis or enteritis. A panel of immunohistochemical stains (CK7, CK20, CDX2, and ER) was found to be useful in biopsies with suspected endometriosis demonstrating unusual histology or only containing endometrioid stroma tissue. Vascular involvement by endometriosis was identified in one case. Endometrial hyperplasia (n=2) and cancer (n=1) were also seen in the ectopic tissue. All patients were alive at follow-up (3-216 months, mean 67 months).

Expression of ERG protein, a prostate cancer specific marker, in high grade prostatic intraepithelial neoplasia (HGPIN): lack of utility to stratify cancer risks associated with HGPIN.

UNLABELLED: What's known on the subject? and What does the study add? High grade prostatic intraepithelial neoplasia is a pre-malignant lesion to prostate cancer and is associated with 20%-25% risk of prostate cancer in subsequent repeat biopsies. ERG is a highly prostate-cancer-specific marker. Expression of ERG is rare in isolated high grade prostatic intraepithelial neoplasia diagnosed in prostate biopsy and is not associated with cancer risk in subsequent repeat biopsies. OBJECTIVES: • To evaluate how often ERG, a highly prostate-cancer-specific marker, is expressed in isolated high grade prostatic intraepithelial neoplasia (HGPIN) by immunohistochemistry. • To study whether a positive ERG immunostain in HGPIN correlates with prostate cancer (PCa) detection in subsequent repeat biopsies. PATIENTS AND METHODS: • Patients with initial HGPIN in biopsies and at least one follow-up prostate biopsy were included. • Biopsies with HGPIN were immunostained for ERG. • The ERG staining results were then correlated with the PCa risk in subsequent biopsies. RESULTS: • The mean age of 94 patients was 63 years (range 48-78). A mean of 1.8 (range 1-5) repeat biopsy sessions were carried out at a mean interval of 27.4 months (range 1.5-140). The repeat biopsies showed PCa and non-cancer lesions (benign, HGPIN, atypical glands suspicious for cancer) in 36 patients (38%) and 58 patients (62%) respectively. • ERG immunostain was positive in five (5.3%) biopsies with HGPIN, in which PCa was found in two (40%) subsequent biopsies. Of 89 biopsies with negative ERG staining, PCa was found in 34 (38%) repeat biopsies. The cancer detection rate was not different between ERG positive and negative cases (P= 0.299). CONCLUSIONS: • This is the first study to investigate the ERG protein expression in prostate biopsy containing HGPIN only and its use to stratify the cancer risk associated with HGPIN. We found that ERG expression is distinctly uncommon in isolated HGPIN (5.3%). • Positive ERG expression is not associated with increased cancer detection in subsequent repeat biopsies. The use of ERG immunostain in the evaluation and cancer risk stratification of HGPIN is of limited value.

Histomorphologic and molecular features of pouch and peripouch adenocarcinoma: a comparison with ulcerative colitis-associated adenocarcinoma.

The occurrence of adenocarcinoma after ileal pouch-anal anastomosis for ulcerative colitis (UC) is an infrequent but potentially lethal complication. Neither histomorphologic nor molecular features of pouch adenocarcinoma after ileal pouch-anal anastomosis have been fully investigated. We report the largest series of 12 pouch and peripouch adenocarcinomas and compared them with 58 randomly selected UC-associated adenocarcinomas. The mean age of patients with pouch/peripouch adenocarcinoma was 55.2 years (SD 14.8), which was not significantly different from that of controls (P=0.52). Pouch/peripouch adenocarcinoma and UC-associated adenocarcinoma had a comparable frequency of tumor-infiltrating lymphocytes, lack of dirty necrosis, mucin differentiation, signet ring cell differentiation, heterogeneity, and well differentiation (P>0.05 for all). Pouch/peripouch adenocarcinoma was more likely to show Crohn-like reaction compared with UC-associated adenocarcinoma (P=0.047). Loss of at least 1 mismatch repair protein was noted in 9% of pouch/peripouch adenocarcinomas and 9.6% of UC-related adenocarcinomas (P=1.0). There was no significant difference in the frequency of p53 overexpression (36.4% vs. 61.1%, P=0.184) or nuclear immunoreactivity for ß-catenin (9% vs. 7.4%, P=0.99) in pouch/peripouch versus UC-associated adenocarcinomas, respectively. Pouch/peripouch and UC-associated adenocarcinoma had a comparable positive rate for CK7 (54.5% vs. 55.5%, P=0.99), CK20 (100% vs. 98.1%, P=0.99), and CDX2 (72.8% vs. 72.2%, P=0.99) by immunohistochemistry. In summary, pouch and peripouch adenocarcinoma can occur in patients without colorectal neoplasia and in those with idiopathic inflammatory bowel disease, can be potentially lethal, and has histomorphologic and molecular features similar to those of UC-associated adenocarcinoma.

Solitary fibrous tumor: is there a molecular relationship with cellular angiofibroma, spindle cell lipoma, and mammary-type myofibroblastoma?

Solitary fibrous tumor (SFT) is a mesenchymal tumor characterized by ovoid cells, branching blood vessels, stromal hyalinization, and CD34 immunoreactivity. Studies have shown loss of 13q in a group of morphologically similar entities, including cellular angiofibroma, mammary-type myofibroblastoma, and spindle cell lipoma. The histologic and immunophenotypic overlap between SFT and the latter group of tumors suggests that these tumors may be genetically linked. We tested a group of 40 SFTs to assess for loss of RB1 (13q14) by fluorescence in situ hybridization (FISH). All 38 SFTs with evaluable signals failed to show loss of RB1 (13q14) by FISH. All cases of cellular angiofibroma (1/1), spindle cell lipoma (6/6), and mammary-type myofibroblastoma (4/4), which were used as a control group, showed monoallelic or biallelic loss of RB1. The absence of RB1 loss in SFTs suggests that they are not related to cellular angiofibroma, mammary-type myofibroblastoma, or spindle cell lipoma.

The expanded histologic spectrum of myxoid liposarcoma with an emphasis on newly described patterns: implications for diagnosis on small biopsy specimens.

The variety of histologic patterns in myxoid liposarcoma is underappreciated. The diversity of these patterns can lead to diagnostic difficulty. We examined the morphologic spectrum of myxoid liposarcoma by cataloguing and describing different patterns identified in biopsy and resection specimens of 46 primary, recurrent, and metastatic myxoid liposarcomas. The patterns identified in the 46 cases included traditional myxoid (43 [93%]), traditional round cell (17 [37%]), pseudoacinar (24 [52%]), lipoblast-rich (13 [28%]), island (11 [24%]), lipomatous (10 [22%]), stromal hyalinization (7 [15%]), cord-like (5 [11%]), nested (3 [7%]), chondroid metaplasia (2 [4%]), and hemangiopericytoma (HPC)-like (1 [2%]). Island and nested patterns had not previously been described. The diagnosis of myxoid liposarcoma was confirmed by fluorescence in situ hybridization studies for DDIT3 (also known as CHOP) rearrangement. The morphologic spectrum of myxoid liposarcoma spans well beyond its typical appearance of spindle cells in a myxoid stroma with a prominent vascular pattern. Awareness of the variety of histologic patterns is critical to avoid misdiagnosis, especially in small biopsy specimens.

ERG gene rearrangement status in prostate cancer detected by immunohistochemistry.

TMPRSS2-ERG, the most common gene fusion in prostate cancer, is associated with expression of a truncated protein product of the oncogene ERG. A novel anti-ERG monoclonal antibody has been recently characterized. We investigated the correlation between ERG rearrangement assessed by fluorescence in situ hybridization (FISH) and ERG expression detected by immunohistochemistry in a large cohort of patients treated with radical prostatectomy for clinically localized prostate cancer. Thirteen tissue microarrays comprising 305 tumors and a subset of 112 samples of nonneoplastic prostatic tissue were assessed for ERG rearrangement status by FISH and for ERG expression by immunohistochemistry. Accuracy of ERG detection by immunohistochemistry in predicting ERG status as assessed by FISH (criterion standard) was calculated in terms of sensitivity, specificity, positive and negative predictive values. Of 305 tumor foci, 103 (34%) showed ERG rearrangement by FISH. ERG was detected by immunohistochemistry in 100 (33%) cases, 99 of which were FISH positive. ERG detection by immunohistochemistry demonstrated a sensitivity and specificity of 96% and 99%, respectively, with positive and negative predictive values of 99% and 98%, respectively. None of the 112 samples of nonneoplastic prostatic tissue was rearranged by FISH or showed any ERG expression. In conclusion, ERG detection by immunohistochemistry in prostate cancer was highly predictive of ERG rearrangement as assessed by FISH in a large cohort of prostatectomy patients. Given the high yield and the easier task of performing immunohistochemistry vs. FISH, ERG assessment by immunohistochemistry may be useful for characterizing ERG status in prostate cancer.

The diagnostic utility of novel immunohistochemical marker ERG in the workup of prostate biopsies with "atypical glands suspicious for cancer".

A diagnosis of "atypical glands suspicious for cancer" (ATYP) in prostate needle biopsy is associated with a 40% to 50% risk of finding prostate carcinoma (PCa) in subsequent biopsies. Many studies have attempted to identify clinical, histologic, or molecular characteristics of ATYP that correlated with the risk of PCa in follow-up biopsies. TMPRSS2:ERG gene rearrangement is the most common chromosomal alteration and is highly specific for PCa. Recently, 2 studies reported that positive immunohistochemical (IHC) stains with an ERG antibody highly correlated with the TMPRSS2:ERG gene rearrangement status. We evaluated the use of this antibody as an IHC marker on prostate biopsies with an initial ATYP diagnosis to determine whether positive ERG IHC was associated with increased PCa detection in subsequent biopsies, which therefore might be useful for stratifying ATYP prostate biopsies. ERG IHC was performed on 103 biopsies with initial ATYP diagnosis. Positive ERG IHC staining was detected in 16 of the 103 cases (15.5%) of the ATYP prostate biopsies. Of these 16 ERG-positive cases, the atypical glands were positive for ERG in 9 cases. In the remaining 7 cases, positive ERG staining was found in glands other than ATYP glands, including high-grade prostatic intraepithelial neoplasia and morphologically benign glands. ERG IHC was negative in other benign prostate lesions, including simple atrophy, partial atrophy, proliferative inflammatory atrophy, basal cell hyperplasia, postatrophic hyperplasia, and squamous metaplasia. In subsequent follow-up biopsies, PCa was detected in 7 of the 16 (43.8%) ERG-positive cases and in 42 of the 87 (48.3%) ERG-negative cases (P=0.952 by χ test). In biopsies with ERG-positive ATYP glands, cancer was found in 5 of 9 (55.6%) cases in subsequent biopsies. This is the first study to investigate the use of ERG IHC in difficult prostate biopsies. ERG IHC was positive in a small percentage (15.5%) of the ATYP prostate biopsies, and positive ERG staining did not correlate with the increased cancer detection in subsequent prostate biopsies. Therefore, ERG IHC is not useful for stratifying ATYP prostate biopsies to identify patients who have increased risk for PCa in repeat biopsies. Furthermore, positive ERG staining is not entirely specific for PCa and can occasionally be found in high-grade prostatic intraepithelial neoplasia and benign glands that are not associated with PCa in prostate biopsies.