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 (17p11.2) or ORC4 (2q22.3-2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus ( EIF5B , 2q11.2). RESULTS: Results of the HER2 / RAI1 and HER2 / ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold. CONCLUSIONS: Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2 -equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.

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). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. RESULTS: SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. CONCLUSIONS: A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.

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. RESULTS: Three of 43 (7%) endometrial carcinomas harbored FGFR2 exon 7 mutations. All 3 mutations were S252W and occurred in endometrioid (type I) adenocarcinomas. Direct sequencing indicated that 2 of the S252W mutations were heterozygous, whereas 1 was presumably homozygous. No FGFR2 mutations were detected in exon 12. CONCLUSIONS: FGFR2 mutations occur in approximately 7% of adenocarcinomas of the endometrium. Only carcinomas of an endometrioid morphology contain FGFR2 mutations, and in our series all were S252W. FGFR2 exons 7 and 12 unlabeled DNA probes allow for easy screening of endometrial carcinoma for the 2 most common FGFR2 mutations (S252W and N550K). Identification of these mutations may have important implications in directed molecular therapy.

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. METHODS: High-resolution amplicon melting curve analysis and direct sequencing were used to determine the frequency of mutation zygosity in a series of 267 GIST cases with known C-KIT (exons 9, 11, 13 and 17) or PDGFRA (exons 12 and 18) mutations. Mutation zygosity was correlated with clinicopathological characteristics including sex, age, tumor size, tumor location, and C-KIT immunohistochemistry. RESULTS: Forty-two of 267 (15.7%) mutant GISTs were homozygous: 36 in C-KIT exon 11, 1 in C-KIT exon 13, 2 in PDGFRA exon 12, and 3 in PDGFRA exon 18. No correlation was found between mutation zygosity and age, sex, tumor size, or C-KIT expression. Homozygous mutant GISTs from the small intestine were underrepresented (P=0.029) whereas GISTs from metastatic sites such as the liver or pancreas were significantly enriched for mutant homozygosity (P=0.020). CONCLUSIONS: Zygosity of C-KIT or PDGFRA mutations did not correlate with most clinicopathologic features of GISTs including tumor size in our subset. However, homozygous mutant GISTs were associated with metastatic disease.

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. Because the correct diagnosis is the key to successful treatment of this tumor, it is incumbent on the pathologist to be familiar with the various gross and histologic patterns shown by these tumors. GISTs range from small incidental stromal nodules to large cystic and solid tumor masses. GISTs show a variety of microscopic patterns and therefore several other tumors enter the differential diagnosis. Fortunately, with an understanding of GIST histology, and with the proper use of immunohistochemistry and molecular analysis, a correct diagnosis can usually be made. In addition to the correct diagnosis, several key attributes of the tumor need to be determined because they provide the basis for proper clinical management. This article summarizes the gross, microscopic, and molecular findings of GISTs, and discusses the differential diagnosis and key attributes of this interesting group of neoplasms.

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 age range of the patients with composite pheochromocytoma was 15 to 40 years with an equal M/F ratio, including 2 patients with syndromes. None of these composite pheochromocytomas demonstrated N-myc amplification, none recurred, and there were no deaths. Of the classic pheochromocytomas, none demonstrated N-myc amplification, 2 recurred, and there were no deaths. Of the neuroblastomas, 5 (50%) of 10 showed significant N-myc amplification, and there were 4 known recurrences and 5 known deaths. These findings suggest that composite pheochromocytoma may be regarded as a histologic variant of classic pheochromocytoma.

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. MATERIALS AND METHODS: Fifty-two IPMNs were studied for expression of MUC1, MUC2, p16, p21, HER2, cyclin D1, and p53 protein and for mutations in K-ras, HER2, p53, EGFR, and BRAF genes. The cases were evaluated for dysplasia, presence of invasion, and morphology of lining epithelium. RESULTS: Twenty-six IPMNs appeared intestinal (IN). Five were low, 12 moderate, and 9 high grade. K-ras mutations were found in 15, EGFR mutations in 2, and BRAF mutation in 1. Seven cases were pancreaticobiliary (PB) and all showed moderate to high-grade dysplasia. Six K-ras mutations and 2 p53 mutations were found in PB tumors. p53 mutations were in cases with high-grade dysplasia. Nineteen IPMNs demonstrated a gastric foveolar (GF) pattern. The majority of GF cases had low or moderate dysplasia. Sixteen revealed K-ras mutations and 1 case each demonstrated a HER2 or p53 mutation. Five IPMNs revealed invasive adenocarcinoma, including a colloid carcinoma from an IN type epithelium. CONCLUSIONS: IN pattern IPMNs were the most common. Mixed histology was common. K-ras mutations were most common, but did not correlate with dysplasia. p53 mutations were seen in 6% of cases (only in GF and PB subtypes). A HER2 mutation was found in a GF IPMN. EGFR and BRAF mutations were restricted to IN IPMNs. These findings suggest the possibility of alternate pathways for carcinogenesis between epithelial subtypes of IPMNs.

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. Recently, BRAF mutations were reported to characterize a series of prostate adenocarcinomas. In this study, we used DNA melting analysis with high-resolution technology to screen a series of 93 prostate carcinomas for BRAF mutations. None were found. This suggests that BRAF mutations may not play an important role in the oncogenesis or therapy of prostate adenocarcinoma.

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. Our laboratory requires IHC 3+ FISH nonamplified specimens to undergo retesting by polymerase chain reaction (PCR). A random sample of IHC 2+ cases are routinely tested by PCR. We found this practice useful for resolving discrepancies in HER2 testing. METHODS: At clinician request, seventy-nine 3+ and one hundred forty-eight 2+ cases were tested by FISH. In 22 cases, IHC was 3+ but FISH was nonamplified. These 22 cases underwent HER2 LightCycler monoplex polymerase chain reaction (MPCR) testing. Seventeen 2+ nonamplified cases were tested by MPCR. RESULTS: Twenty-one 3+, FISH nonamplified cases were found to be MPCR nonamplified. One IHC 3+, FISH nonamplified case was MPCR amplified. Seventeen 2+, FISH nonamplified cases were MPCR nonamplified. In all but one case, FISH and MPCR were concordant. DISCUSSION: American Society of Clinical Oncology/CAP guidelines propose validation of testing procedures by showing 95% concordance with a validated test for positive and negative assays. Specific actions are not recommended to resolve discordances between tests. Our laboratory uses 3 different modalities for HER2 testing. We have found that our 2 methods for testing gene amplification status show a higher degree of concordance between themselves than either did with IHC. Review of the 3+ IHC nonamplified cases showed them to have a dark, granular circumferential staining pattern.

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. Most platelet-derived growth factor receptor alpha mutation-positive gastrointestinal stromal tumors show an epithelioid histology and are located in the stomach. Herein, we describe an unusual gastric stromal tumor. The tumor was negative for KIT expression and the morphology did not show an epithelioid pattern but rather was composed of bland spindle cells reminiscent of a neurofibroma. Molecular analysis revealed a somatic mutation in platelet-derived growth factor receptor alpha exon 18 (D842F). Aside from demonstrating a new platelet-derived growth factor receptor alpha mutation, this case illustrates the usefulness of molecular testing as a diagnostic tool and clearly indicates the wide range of morphology that can be observed in gastrointestinal stromal tumors.

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. OBJECTIVE: To investigate the performance characteristics of the LSI CHOP Breakapart Rearrangement Probe for the diagnosis of myxoid/round cell liposarcomas and atypical lipomas/well-differentiated liposarcomas. DESIGN: We investigated a series of lipomatous neoplasms (5 lipomas, 5 well-differentiated liposarcomas, 22 myxoid/round cell liposarcomas, 2 liposarcomas not otherwise specified, and 2 dedifferentiated liposarcomas) and normal myometrium for abnormalities in the q13-15 region of chromosome 12. Cases were studied for the presence or absence of t(12;16)(q13;p11) or t(12;22)(q13;q12) translocations by the LSI CHOP Breakapart Rearrangement Probe. These probes contain a sequence encompassing the SAS and CDK4 genes. Four or more copies of this sequence were considered to represent amplification of these genes. RESULTS: Rearrangement of the CHOP gene was seen in all evaluable myxoid liposarcomas. Rearrangements were seen in 1 dedifferentiated liposarcoma but not in normal myometrium or lipomas. Probe signal amplification was seen in all 5 well-differentiated liposarcomas and 1 myxoid liposarcoma. No signal amplification was seen in lipomas or myometrium. CONCLUSIONS: Demonstration of translocations t(12; 16)(q13;p11) and t(12;22)(q13;q12) by the LSI CHOP Breakapart Rearrangement Probe appears to correlate with round cell/myxoid liposarcoma. The probe also demonstrated amplification of the 12q13-15 region in well-differentiated liposarcomas, making it useful for the diagnosis of these neoplasms. In a significant percentage of cases, high background fluorescence or poor probe staining made interpretation difficult.

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. With the use of new molecular techniques, it is now possible in routine diagnostic work to characterize human malignancies with respect to the presence or absence of activated tyrosine kinases. This may have important predictive and prognostic implications.

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. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations.

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. One c-kit mutation-positive case contained an exon 9 mutation, ins FY at codon 503, that has not been previously described. One PDGFRA mutation-positive case contained mutation D842V del 843, also not previously described. Of 18 PDGFRA mutation-positive cases, 3 (17%) were strongly positive for kit expression as measured by CD117 immunohistochemical analysis. Of 69 c-kit mutation-positive cases, 66 (96%) showed strong kit immunohistochemical expression, but 3 (4%) showed negative to weak CD117 expression. Of 96 cases, 9 (9%) were wild type for c-kit and PDGFRA. Of the wild-type cases, 8 still showed strong immunohistochemical kit expression, whereas 1 showed weak kit expression. GISTs with PDGFRA mutations were found in the stomach, omentum, and peritoneum but not the small intestine. GISTs with c-kit exon 9 mutations were found primarily in the small intestine. HRMAA is a sensitive technique that can be used to rapidly identify c-kit and PDGFRA activating mutations in GISTs.

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. An increasing number of breast biopsy specimens are being fixed in these substitutes. We tested 6 non-formalin-based fixatives to determine their impact on FISH testing for Her-2/neu gene amplification status by comparison with formalin-fixed control specimens from the same neoplasm. Specimens fixed in Pen-Fix, Prefer, Histochoice, UniFix, and GTF were associated with absent or technically compromised staining in at least one of the 3 neoplasms tested for each fixative when compared to the formalin-fixed control. O-Fix did not seem to compromise staining quality in 3 paired specimens tested.

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. Diagnostic sensitivities for brushing cytology have ranged from 18 to 90%. Positive diagnoses of malignancy are of great clinical value but a negative result is of relatively little clinical aid when the radiographic or clinical findings are suspicious for a malignancy.A variety of techniques have been used in an attempt to improve diagnostic sensitivity for brushing cytology. These have included immunohistochemistry and various molecular diagnostic techniques. Using the high resolution melting curve technique, we performed mutational analysis on 20 bile duct brushing specimens for mutations in p53, K-ras, BRAF, and EGFR genes. Eleven specimens had corresponding surgical specimens, which were similarly analyzed. Our series included twelve adenocarcinomas, one islet cell tumor, one case of dysplasia, and six benign cases. K-ras mutations were found in cytology specimens of 3 out of 12 malignancies. No EGFR or B-raf mutations were detected and only a single p53 mutation in an adenocarcinoma was detected in the corresponding cytology specimen. No mutations were detected in benign lesions or in the dysplasia. Only 8% of specimens from adenocarcinomas had p53 mutations and only 33% of cases had K-ras mutations. Mutational analysis did not appear to improve the cytologic detection of adenocarcinoma by bile duct brushings.

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. Centrometric enumeration probe (CEP) 7 counts were correlated with proliferation index. RESULTS: Average CEP 7 number showed a relationship to proliferation index, with higher CEP 7 averages associated with higher proliferation indices. Specimens of brain tissue demonstrated average CEP 7 counts between 1.64 and 1.75. Tissues with high proliferation indices (23-66%) demonstrated CEP 7 counts between 2.14 and 2.31. The average CEP 7 count for the trisomy 7 cell line was 2.61. The average LSI 13 count for the tetrasomy 13 cell line was 3.65. CONCLUSION: Chromosome 7 FISH counts demonstrated overlap between diploid tissues with high proliferation and triploid chromosome 7 tissues. This overlap was seen when 95% CI limits were used. The trisomic 7 and tetrasomic 13 cell lines demonstrated average CEP 7 and CEP 13 levels below 3 and 4, respectively. Definitions used for determination of polysomy should take into account tissue proliferation and section thicknesses.

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. We used the novel mutation scanning technique, high-resolution melting amplicon analysis, to screen a series of 22 testicular seminomas for c-kit-activating mutations. Four cases (18%) had exon 17-activating mutations and these included D816Y, D816V, Y823N and one case that contained both D816E and D820H. A single case (5%) had an exon 11-activating mutation. Interestingly, the exon 11-activating mutation was L576P, the same mutation that characterizes the rare c-kit mutation-positive cases of malignant melanoma. Fluorescence in situ hybridization (FISH) for c-kit suggested that most seminomas are probably polysomic for c-kit and there was not a significant difference in c-kit FISH characteristics between the mutation-positive and mutation-negative cases. The use of high-resolution melting amplicon analysis as a screening technique will allow for the rapid identification of patients with testicular seminomas whose tumors contain c-kit-activating mutations. This could benefit patients whose tumors are refractory to standard therapeutic protocols.

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. One section was stained using hematoxylin and eosin and an area composed exclusively of malignant cells was identified and marked on the slide. The corresponding area of the tissue block corresponding to this region underwent needle core biopsy, and the tissue was processed to isolate tumor cell nuclei and deposited onto a glass slide. The paired sample preparations underwent routine FISH testing for detection of the t(11;14)(q13;q32) chromosomal trans-location. DNA probe hybridization quality was compared between the tissue and isolated nuclei. Individual tumor cell nuclei were successfully extracted from each of the tissue blocks. The t(11;14) trans-location was detected by FISH in all of the samples diagnosed as mantle cell lymphoma. The hybridization signals found in the nuclei of extracted tumor cells were bright, planar, and easily identified. Detection of signal was superior to that on whole tissue samples, where signals often overlapped or were truncated. This technique produces intact nuclei for analysis, preserves the tissue block for additional studies, and allows sampling of a specific area of the tissue block. This approach may be particularly useful when the amount of diagnostic tissue is limited.