Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status.

Objectives: Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application.

Methods: Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 ( HER2 , 17q12) and either RAI1 (17p11.

Genomic Copy Number Analysis of HER2-Equivocal Breast Cancers.

Objectives: Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup.

Methods: Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array).

Utilization of unlabeled probes for the detection of fibroblast growth factor receptor 2 exons 7 and 12 mutations in endometrial carcinoma.

Background: Endometrial adenocarcinomas are associated with a variety of molecular abnormalities including microsatellite instability, Kirsten rat sarcoma viral oncogene homolog mutations, and phosphatase and tensin homolog inactivation. Recently, mutations in fibroblast growth factor receptor 2 (FGFR2) have been described but their frequency and clinicopathologic characteristics are incompletely known.

Methods: To determine the frequency of mutations in FGFR2 exons 7 and 12, 43 adenocarcinomas of the endometrium were studied by high-resolution melting analysis utilizing unlabeled probes and sequencing.

C-KIT and PDGFRA zygosity in gastrointestinal stromal tumors: Correlation with tumor site, tumor size, exon, and CD117 immunohistochemistry.

Background: Gastrointestinal stromal tumors (GISTs) often harbor activating mutations in the receptor tyrosine kinases C-KIT or platelet derived growth factor receptor-alpha (PDGFRA). Gain-of-function mutations in these 2 genes, which result in constitutive signaling, are presumed to be dominant and therefore are usually heterozygous. However, homozygous C-KIT mutations have been reported in GISTs, although at varying frequencies in different subsets.

Gastrointestinal Stromal Tumors: A Guide to the Diagnosis.

Gastrointestinal stromal tumors (GISTs) have emerged from being a poorly understood and therapeutically refractory sarcoma to a tumor whose biology has not only provided insight into a mechanism of oncogenesis but has also led to a rational basis for therapy. Most GISTs are characterized by KIT protein (CD117) expression and constitutive activating mutations in either the c-kit or platelet-derived growth factor receptor α genes. This information can now be obtained from routine formalin-fixed and paraffin-embedded tissue.

Composite pheochromocytoma: a clinicopathologic and molecular comparison with ordinary pheochromocytoma and neuroblastoma.

Composite pheochromocytoma is a rare adrenal tumor composed of ordinary pheochromocytoma and other components, most frequently neuroblastic elements. Little is known about its biologic potential, therefore creating a clinical dilemma on diagnosis. This study investigates the clinical characteristics and N-myc amplification status of 4 cases of composite pheochromocytoma and compares them with selected cases of ordinary pheochromocytoma and neuroblastoma.

The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer.

Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy.

Lack of BRAF activating mutations in prostate adenocarcinoma: a study of 93 cases.

Oncogenic activating mutations in the cytosolic serine/threonine kinase, BRAF, have been reported in a variety of neoplasms. BRAF relays signals from membrane-bound RAS downstream through the MAP/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) signaling pathway. The presence of BRAF activating mutations suggests the importance of the MAP/ERK kinase pathway for tumor growth and points to possible therapeutic interventions.

Monoplex LightCycler polymerase chain reaction quantitation of the HER2 gene for quality assurance of HER2/neu testing by immunohistochemistry and fluorescence in situ hybridization.

Background: Assessment of HER2 by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is a standard practice for breast carcinomas. Testing is associated with a 20% disagreement between laboratories. The College of American Pathologists (CAP) guidelines for HER2 testing include validation of HER2 test methods by achieving 95% concordance with another validated method.

Histologic, immunohistochemical, and molecular classification of 52 IPMNs of the pancreas.

Background: Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas account for approximately 5% of pancreatic neoplasms. Prognosis is superior to that of pancreatic invasive ductal carcinoma. IPMNs reveal a variety of epithelial linings expressing different mucin staining patterns and may progress along different oncogenic pathways.

A gastrointestinal stromal tumor of the stomach morphologically resembling a neurofibroma: demonstration of a novel platelet-derived growth factor receptor alpha exon 18 mutation.

Gastrointestinal stromal tumors are increasingly being recognized because of their characteristic expression of KIT (CD 117). Most KIT-positive gastrointestinal stromal tumors have activating mutations in the c-kit gene. A subgroup of gastrointestinal stromal tumors are negative for KIT expression, and in these tumors, activating mutations in platelet-derived growth factor receptor alpha are common.

Translocations and amplifications of chromosome 12 in liposarcoma demonstrated by the LSI CHOP breakapart rearrangement probe.

Context: Liposarcomas display a number of molecular abnormalities involving chromosome 12. Myxoid and round cell liposarcomas are characterized by t(12;16)(q13; p11) or t(12;22)(q13;q12) translocations. Amplifications occur within the 12q13-15 region of atypical lipomatous tumors and well-differentiated liposarcomas but not lipomas.

Tyrosine kinase activating mutations in human malignancies: implications for diagnostic pathology.

Constitutively activated tyrosine kinases play an important role in human malignancies. Their constant downstream signaling leads to cell proliferation and the inhibition of anti-apoptotic mechanisms. New cancer therapeutics have been designed to specifically target the activated kinases in human cancers and in some instances treatment with these agents leads to tumor regression.

Fluorescence in situ hybridization determination of aneusomy: criteria and technical considerations.

Objective: To investigate a series of tissues to determine if proliferation rate can affect chromosome counts by fluorescence in situ hybridization (FISH).

Study Design: We studied 9 non-neoplastic tissues and a trisomy 7 and tetrasomy 13 cell line by FISH. For each sample, 100 cells were analyzed for chromosome 7 and 13 number and MIB-1 expression.

Mantle cell lymphoma presenting with unusual morphology in an adolescent female: a case report and review of the literature.

We report the case of an 18-year-old female initially diagnosed with CD5-negative diffuse large B-cell lymphoma in an inguinal lymph node in 1999 who subsequently relapsed with classic-morphology mantle cell lymphoma with involvement of the bone marrow, gastrointestinal tract, and spleen in 2004. Both the 1999 and 2004 lesions were retrospectively positive for Cyclin D1 by immunohistochemistry and positive for t(11:14)(q13;q32) by fluorescence in situ hybridization, and both lesions had identical B-cell receptor gene rearrangements by polymerase chain reaction. This case of a CD5-negative large cell or pleomorphic blastoid variant of mantle cell lymphoma arising in an 18-year-old represents a very early incidence for this type of lymphoma, which is usually not seen in younger patients.

High-resolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors.

High-resolution melting amplicon analysis (HRMAA) was used to detect c-kit and platelet-derived growth factor receptor alpha (PDGFRA) activating mutations in 96 gastrointestinal stromal tumors (GISTs). HRMAA detected mutations in 87 GISTs (91%). Of the 87 cases, 69 (79%) contained c-kit mutations and 18 (21%), PDGFRA mutations.

Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is used to determine amplification status of the Her-2/neu gene in specimens of newly diagnosed breast carcinoma. The Vysis kit for FISH analysis stipulates that the tissue be formalin-fixed and paraffin-embedded. Concerns regarding carcinogenicity of formalin and environmental effects of formalin waste have led to the development of formalin replacement products.

Molecular diagnostic testing as an adjunct to morphologic evaluation of pancreatic ductal system brushings: potential augmentation for diagnostic sensitivity.

Malignancies arising from the pancreatic and biliary ductal systems present the gastroenterologist and pathologist with diagnostic challenges. Tumors of the pancreatic and/or biliary ductal system may present as either duct strictures or mass lesions. When lesions present as strictures without associated demonstrable masses, brushing cytology may represent the only reasonable diagnostic technique aside from open biopsy.

Tumor cell nuclei extraction from paraffin-embedded lymphoid tissue for fluorescence in situ hybridization.

In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-embedded lymphoma samples before performing FISH testing to detect the lymphoma-specific trans-location t(11;14) that defines mantle cell lymphoma. Well-characterized surgical pathology cases of mantle cell lymphoma were identified from pathology archives. Thin sections were cut from the paraffin-embedded tissue blocks.

Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis.

A subgroup of testicular seminomas has been reported to contain activating mutations in KIT, the transmembrane tyrosine kinase receptor encoded by the c-kit gene. Most mutations are in exon 17, although exon 11-activating mutations have recently been described. For patients refractory to standard therapeutic protocols for seminoma, the presence of c-kit-activating mutations in some of these neoplasms might suggest an alternative therapy with KIT targeting drugs.

Detection of epidermal growth factor receptor and human epidermal growth factor receptor 2 activating mutations in lung adenocarcinoma by high-resolution melting amplicon analysis: correlation with gene copy number, protein expression, and hormone receptor expression.

Activating mutations in the epidermal growth factor receptor (EGFR) (7p12.3-p12.1) and in the human epidermal growth factor receptor 2 (HER2) (17q21.

Evaluation of Her-2/neu gene status in osteosarcoma by fluorescence in situ hybridization and multiplex and monoplex polymerase chain reactions.

Context: Previous reports suggest that the human epidermal growth factor 2 (HER-2/neu) receptor may be overexpressed in osteosarcoma.

Objective: To determine whether osteosarcomas have amplifications of the HER-2/neu gene.

Design: We studied a series of osteosarcomas by fluorescence in situ hybridization (FISH) and by 2 real-time polymerase chain reaction assays that measure the amount of HER-2/neu DNA relative to a control gene.

BRAF and c-kit gene copy number in mutation-positive malignant melanoma.

Activating mutations in BRAF or c-kit have been reported in malignant melanoma. Because the activating mutations are dominant, it has been assumed that they are heterozygous in the affected tumors. To test this, we have carefully examined the DNA sequencing electropherograms on 43 BRAF mutation-positive and 3 c-kit mutation-positive malignant melanomas to determine the ratio of the normal to mutant allele.

Detection of EGFR- and HER2-activating mutations in squamous cell carcinoma involving the head and neck.

Recent reports have indicated that mutations in the epidermal growth factor receptor-1 (EGFR) occur in about 7% of patients with squamous cell carcinoma of the head and neck. It is known that many patients with non-small-cell lung cancer who respond to the EGFR inhibitors gefitinib and erlotinib have tumors with EGFR-activating mutations. This might suggest that patients with head and neck squamous carcinoma, who also have tumors with EGFR-activating mutations, might represent a patient population who could benefit from gefitinib or erlotinib therapy.

Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis.

Activating mutations in the BRAF kinase have been reported in a large number of cases of malignant melanoma. This suggests that therapy with specific RAF kinase inhibitors may find use in treating this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of malignant melanoma for the presence or absence of BRAF mutations.