A novel chromogenic in situ hybridization assay for FGF23 mRNA in phosphaturic mesenchymal tumors.

Phosphaturic mesenchymal tumors of the mixed connective tissue type (PMT) are very rare tumors of bone and soft tissues. Most patients with PMT have long-standing osteomalacia secondary to production of fibroblast growth factor 23 (FGF23), a hormone that inhibits phosphate reuptake within the renal proximal tubule. Previously, we have reported the detection of FGF23 mRNA in PMT by reverse transcription polymerase chain reaction (PCR); however, the low specificity and risk for nontumoral tissue contamination inherent in PCR-based methodology limit its clinical utility. We evaluated RNAscope as a semiquantitative method of in situ FGF23 mRNA detection in the diagnosis of PMT. Twenty-five PMTs (median 52 y, range 5 to 73 y) occurred in patients with tumor-induced osteomalacia (TIO), manifesting as masses (mean 3.9 cm, range 1.4 to 12 cm) in various bones and soft tissues. FGF23 mRNA was positive in 96% (22/23) informative cases of PMT: 16 cases scored 3+; 5 scored as 2+; 1 scored as 1+. Among these cases, FGF23 mRNA was detected in 3 malignant PMTs along with their metastases. Forty control cases included aneurysmal bone cyst (N=4), chondromyxoid fibroma (N=8), high-grade osteosarcomas (N=8), and (nonfamilial) tumoral calcinosis, as well as miscellaneous cartilage-forming tumors or osteoid-forming tumors and soft tissue tumors. All control cases were negative for FGF23 mRNA in the lesional cells. One aneurysmal bone cyst had rare FGF23 mRNA-expressing osteocytes clustered around remodeled bone. One ovarian serous carcinoma in a patient with disseminated disease, elevated serum FGF23, and TIO was negative for FGF23 mRNA in the neoplastic cells. We conclude that RNAscope is a highly sensitive and specific, semiquantitative in situ hybridization method of FGF23 mRNA detection applicable to formalin-fixed, paraffin-embedded tissues. Detection of FGF23 expression is a valuable diagnostic adjunct, especially in patients with occult TIO. Compared with reverse transcription PCR, this method preserves tissue morphology and reduces "false positives" related to detection of endogenous FGF23 mRNA expression by osteocytes.

Islet-1 is a sensitive but not entirely specific marker for pancreatic neuroendocrine neoplasms and their metastases.

Islet-1 (Isl1) is a transcription factor involved in the embryogenesis of islets of Langerhans. Immunohistochemically, Isl1 is a sensitive lineage-specific marker for pancreatic neuroendocrine neoplasms (NENs) and their metastases. Its specificity has not been documented, nor have large numbers of NENs from other parts of the gut or other organs been studied. We examined Isl1 expression in 203 primary NENs (gastroenteropancreatic, lung, breast, and ovarian neoplasms) and 40 hepatic NEN metastases (enteropancreatic and lung neoplasms) from known primaries. The correlation between Isl1 and CDX2 expression was studied using a tissue microarray containing 46 pancreatic NENs. Immunostaining for Isl1 and CDX2 was also performed in selected NENs from other sites. Isl1 was positive in 90% of pancreatic, 89% of duodenal, 100% of rectal, 38% of colonic, 14% of appendiceal, and 6% of ileal primaries. Isl1 was negative in all other NENs. Among metastatic neoplasms, 76% of pancreatic and 2 of 2 rectal NEN metastases were Isl1 positive, whereas all other tested metastases were negative. The overall sensitivity and specificity of Isl1 in identifying primary pancreatic NENs was 88% and 80%, respectively. Thirty-six of 46 pancreatic NENs in the tissue microarray were Isl1 positive; 4 were Isl1 negative but CDX2 positive. Our findings confirm that Isl1 is a sensitive marker of pancreatic origin in cases of metastatic NEN. However, Isl1 does not distinguish pancreatic NEN from duodenal and colorectal NEN, even when used in association with CDX2.

Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: IHC score algorithm for FISH.

INTRODUCTION: Accurate, cost-effective methods for testing anaplastic lymphoma kinase gene rearrangement (ALK+) are needed to select patients with non-small cell lung carcinoma for ALK-inhibitor therapy. Fluorescent in situ hybridization (FISH) is used to detect ALK+, but it is expensive and not routinely available. We explored the potential of an immunohistochemistry (IHC) scoring system as an affordable, accessible approach. METHODS: One hundred one samples were obtained from an enriched cohort of never-smokers with adenocarcinoma from the Mayo Clinic Lung Cancer Cohort. IHC was performed using the ALK1 monoclonal antibody with ADVANCE detection system (Dako) and FISH with dual-color, break-apart probe (Abbott Molecular) on formalin-fixed, paraffin-embedded tissue. RESULTS: Cases were assessed as IHC score 0 (no staining; n = 69), 1+ (faint cytoplasmic staining, n = 21), 2+ (moderate, smooth cytoplasmic staining; n = 3), or 3+ (intense, granular cytoplasmic staining in ≥10% of tumor cells; n = 8). All IHC 3+ cases were FISH+, whereas 1 of 3 IHC 2+ and 1 of 21 IHC 1+ cases were FISH+. All 69 IHC 0 cases were FISH-. Considering FISH a gold-standard reference in this study, sensitivity and specificity of IHC were 90 and 97.8%, respectively, when 2+ and 3+ were regarded as IHC positive and 0 and 1+ as IHC negative. CONCLUSIONS: IHC scoring correlates with FISH and may be a useful algorithm in testing ALK+ by FISH in non-small cell lung carcinoma, similar to human epidermal growth factor-2 testing in breast cancer. Further study is needed to validate this approach.

The role of desmoglein-3 in the diagnosis of squamous cell carcinoma of the lung.

Results from several microarray-based studies have led to the identification of up-regulated expression levels of the DSG3 gene in pulmonary squamous cell carcinomas (SQCCs). The purpose of this study was to determine the role of DSG3 expression in the diagnosis of SQCCs of the lung and to compare DSG3 with p63, CK5, and CK6, as markers of squamous cell differentiation. Expression of DSG3 mRNA was evaluated in bulk laser capture microdissection-derived microarray data and by quantitative reverse transcription PCR on both SQCCs and adenocarcinomas. Expression levels of p63, CK5, and CK6 were evaluated in microarray data from the same set. An immunohistochemical study using antibodies directed against DSG3, p63, and CK5/6 was also performed. DSG3 was over-expressed in SQCCs but had very limited expression in both adenocarcinomas and non-neoplastic lungs. The microarray data showed that DSG3 had a sensitivity and specificity of 88% and 98%, respectively, in detecting SQCC versus adenocarcinoma. In comparison, sensitivity and specificity was 92% and 82% for p63, and 85% and 96% for CK5, respectively. The correlation coefficient between the microarray and immunohistochemical data for these genes was greater than or equal to 0.9. Using immunohistochemistry, sensitivity and specificity of DSG3 for lung cancers were 98% and 99%, respectively. Therefore, DSG3 can be a useful ancillary marker to separate SQCC from other subtypes of lung cancer.

GLUT-1 expression in mesenchymal tumors: an immunohistochemical study of 247 soft tissue and bone neoplasms.

GLUT-1, an erythrocyte-type glucose transporter protein expressed in juvenile hemangiomas, has recently been shown to be a sensitive marker of perineurial cells and their tumors in a small number of cases. However, GLUT-1 expression has not been systematically examined in other mesenchymal neoplasms. Prompted by a recent report of GLUT-1 expression in epithelioid sarcoma, a tumor not generally felt to show perineurial differentiation, we examined GLUT-1 expression in a wide variety of mesenchymal tumors. Sections from 247 mesenchymal tumors of a variety of histologic subtypes were retrieved from our archives and immunostained for GLUT-1 using heat-induced epitope retrieval and the DAKO ADVANCE detection system (DAKO, Carpinteria, CA). Scoring was as follows: negative (<5% of cells), 1+ (5%-25% of cells), 2+ (25%-50% of cells), and 3+ (>50% of cells). All benign nerve sheath tumors showed a peripheral rim of positive normal perineurial cells, with 2 neurofibromas and 3 schwannomas showing more extensive staining. Three of 4 perineuriomas showed strong GLUT-1 expression. All juvenile hemangiomas were GLUT-1 positive. GLUT-1 expression was also seen in a wide variety of benign and malignant mesenchymal tumors. However, GLUT-1 expression was absent in nonjuvenile hemangioma endothelial tumors and in almost all low-grade lesions that enter the histologic differential diagnosis of perineurial tumors, including low-grade fibromyxoid sarcoma, dermatofibrosarcoma protuberans, and myxofibrosarcoma. We conclude that GLUT-1 expression in mesenchymal tumors is by no means specific for perineurial differentiation, but may instead represent upregulation of this protein within hypoxic zones, secondary to upstream activation of proteins such as hypoxia-inducible factor 1-alpha.

MGMT immunohistochemical expression and promoter methylation in human glioblastoma.

O6-methylguanine-DNA methyltransferase (MGMT) expression has been recently proposed as a useful prognostic and/or predictive marker in glioblastoma patients receiving adjuvant therapy after the surgery. We studied samples from 50 patients with histologically confirmed GBM to evaluate MGMT expression by immunohistochemistry and its relation to promoter methylation status. Genomic DNA was extracted from scrapings of formalin-fixed, paraffin-embedded tissue corresponding to hematoxylin and eosin sections. Using the mouse monoclonal antibody MT3.1, MGMT expression was assessed and scored in tumor cells: (1=negative or limited to <10% positive tumor cells, 2=10% to 50%, 3=>50%). Methylation-specific polymerase chain reaction was performed after bisulfite treatment. Assessment of MGMT expression in neoplastic tissue required careful scrutiny because of its expression in a variety of non-neoplastic cells. MGMT expression was present in tumor cells with a score of 1, 2, and 3, respectively in 36 (72%), 13 (26%), and 1 (2%) cases. Methylation-specific polymerase chain reaction yielded interpretable results in 39 cases (78%). MGMT promoter methylation was detected in 15 cases (38.5%), whereas 24 (61.5%) were unmethylated. Among the methylated samples, 14 (of 15) had a score of 1, and 1 had a score of 3 by immunohistochemistry. Of the 24 unmethylated samples, 18 had a score of 1, and 6 of 2. There was no significant correlation between MGMT expression and methylation, and no significant survival difference was observed between patients whose tumors were negative versus positive for MGMT protein by immunohistochemistry. This study underscores some of the difficulties in applying immunohistochemistry to assess MGMT expression.