Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: a retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization.

INTRODUCTION: The use of chromosome analysis on products of conception from spontaneous abortions is recommended to identify a genetic etiology. However, 20% of products of conception cultures are unsuccessful due to microbial contamination or lack of viable dividing cells. Our laboratory implemented a reflex fluorescent in situ hybridization (FISH) assay to detect numeric chromosome abnormalities for unsuccessful cultures. MATERIALS AND METHODS: All products of conception samples were simultaneously processed for both chromosome analysis and FISH analysis. If the chromosome analysis was unsuccessful, interphase FISH was performed for chromosomes 13, 16, 18, 21, 22, X, and Y. To assess the performance of the FISH assay, a 3-year retrospective comparative analysis of the FISH results versus chromosome results was performed. RESULTS: Of 5555 total specimens, 4189 (75%) represented chorionic villi/fetal tissue and 1366 (25%) represented tissue of unidentified origin. Of the 1189 tissues of unidentified origin with chromosome or FISH results, 1096 (92%) were XX, indicating that the majority of these tissues are likely maternal in origin. Of the 3361 successful chromosome studies on the chorionic villi/fetal tissue specimens, 1734 (52%) samples had a chromosome abnormality. Of the 762 successful FISH studies on chorionic villi/fetal tissue specimens that were unsuccessful by chromosome studies, 181 (25%) had an abnormal result with the targeted FISH panel. Overall, the FISH panel detected approximately 70% of the chromosome abnormalities in products of conception detectable by karyotype. When the FISH panel results were combined with chromosome analysis for the 4189 chorionic villi/fetal tissue specimens, the overall abnormality rate is 47%. CONCLUSIONS: Our reflex FISH assay proved useful for the detection of common chromosome aneuploidies in products of conception samples that failed conventional chromosome analysis. Because of its limited view of the genome, cautious interpretation of FISH results is required for all samples, in particular, trisomy of an acrocentric chromosome, which may represent a Robertsonian translocation. An algorithmic approach to the genetic evaluation of products of conception specimens, with the potential for initial evaluation by a FISH panel, may be warranted.

Development of a dual-color, double fusion FISH assay to detect RPN1/EVI1 gene fusion associated with inv(3), t(3;3), and ins(3;3) in patients with myelodysplasia and acute myeloid leukemia.

Approximately 2-3% of adult patients with acute myeloid leukemia harbor a rearrangement of RPN1 (at 3q21) and EVI1 (at 3q26.2) as inv(3)(q21q26.2), t(3;3)(q21;q26.2), or ins(3;3)(q26.2;q21q26.2). The most recent World Health Organization (WHO) classification has designated AML with inv(3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. We have created a dual color, double fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the RPN1 and EVI1 genes. A blinded investigation was performed using 30 normal bone marrow samples and 51 bone marrow samples from 17 patients with inv(3)(q21q26.2), 11 patients with t(3;3)(q21;q26.2), and one patient with ins(3;3)(q26.2;q21q26.2) previously defined by chromosome analysis. The unblinded results indicated abnormal RPN1/EVI1 fusion results in 30 (97%) of 31 samples from the inv(3)(q21q26.2) group including seven bone marrow samples for which chromosome analysis was unsuccessful or failed to detect an inv(3)(q21q26.2). Abnormal FISH results were detected in 14 (88%) of 16 samples with t(3;3)(q21;q26.2) and in the sole sample with an ins(3;3)(q26.2;q21q26.2). All 30 negative controls were normal and were used to establish a normal cutoff of 0.6% for the typical abnormal D-FISH signal pattern. Overall, this D-FISH assay was more accurate than chromosome analysis and based on the normal cutoff of 0.6%, this assay can be used for minimal residual disease detection and disease monitoring in patients with RPN1/EVI1 fusion.

Isochromosome 12p and polysomy 12 in primary central nervous system germ cell tumors: frequency and association with clinicopathologic features.

Germ cell tumors arising within the central nervous system are rare neoplasms that typically occur along midline structures in children and young adults. Although isochromosome 12p is established as a frequent chromosomal abnormality in testicular germ cell tumors, studies examining isochromosome 12p in primary central nervous system germ cell tumors are limited. Herein, we studied 24 primary central nervous system germ cell tumors from 23 patients using fluorescence in situ hybridization to determine the frequency of isochromosome 12p in these neoplasms. Of the 24 primary central nervous system germ cell tumors, fluorescence in situ hybridization detected isochromosome 12p in 6 (25%) tumors, whereas 11 (46%) tumors showed polysomy (multiple copies) of chromosome 12. One case with isochromosome 12p also showed increased 12p independent of isochromosome 12p formation. The remaining 7 tumors yielded a normal result by fluorescence in situ hybridization. Clinical follow-up of this patient cohort indicated 8 patients (32%) developed a recurrence, although no association was demonstrated between the presence or absence of chromosomal 12 abnormalities and tumor relapse. We confirm that isochromosome 12p is less frequent in primary central nervous system germ cell tumors relative to testicular germ cell tumors, and although our numbers are limited, the presence or absence of isochromosome 12p does not appear to impact tumor recurrence. Similarly, although polysomy 12 was identified in nearly half of our central nervous system germ cell tumors, no prognostic significance was attributed to this abnormality. These results suggest that fluorescence in situ hybridization studies for isochromosome 12p or polysomy 12 may have limited use in the evaluation of these rare neoplasms.

Evaluation of a commercially available focused aCGH platform for the detection of constitutional chromosome anomalies.

Microarray-based comparative genomic hybridization (aCGH) allows for simultaneous high-resolution analysis of multiple genomic loci. Recently, focused aCGH platforms have emerged allowing for analysis of numerous clinically relevant chromosome loci. The purpose of our study was to evaluate the Spectral Genomics Constitutional Chip 1.0 (CC) for use in the clinical laboratory. The CC consisted of 429 BAC clones for 41 known genetic deletion/duplication syndromes, subtelomeric regions, and chromosomal backbone clones. We conducted a blinded study of 48 samples including 46 patients (one sample was run in triplicate) with previously determined constitutional chromosome anomalies and two negative controls. Patient samples included 31 microdeletions, four duplications, three derivative chromosomes, three trisomies, and five sex chromosome aneuploidies. Our results show that the CC identified the expected gains and/or losses in 46 of 48 samples. The two negative controls were considered to be normal and the three replicates of the same patient sample were concordant. Two samples yielded false-negative results; however, repeat analysis produced acceptable results for one of them. One sample ultimately had an insufficient amount of DNA precluding aCGH analysis. While promising, the results suggest that further studies are needed to reduce protocol variability and to establish standard analysis and interpretation criteria. Further, this study verifies the importance of extensive validation studies prior to clinical implementation of new clinically available methodologies.

Automated Duet spot counting system and manual technologist scoring using dual-fusion fluorescence in situ hybridization (D-FISH) strategy: comparison and application to FISH minimal residual disease testing in patients with chronic myeloid leukemia.

The automated BioView Duet system was compared with manual technologist scoring (MTS) using a BCR/ABL dual-fusion FISH (D-FISH) probe strategy for chronic myeloid leukemia (CML) specimens. In the first study, 500 nuclei were evaluated for 10 distinct signal patterns in various abnormal cell percentages from each of 89 specimens. The Duet system correctly identified all 27 normal specimens and the abnormal signal pattern of all 63 abnormal specimens. The percentage of abnormal nuclei detected was also concordant, with an average difference between MTS and the Duet system of only 2.7%. However, achievement of accurate quantitative results required reclassification by a technologist for nearly 50% of nuclei per specimen. Next, the Duet system was used to evaluate BCR/ABL D-FISH for FISH minimal residual disease (MRD) detection in CML patients. Up to 6,000 nuclei were evaluated for four signal pattern categories for each of 60 CML MRD samples. Excluding four abnormal specimens with insufficient samples, the Duet system correctly identified all of the abnormal specimens and identified four additional abnormal specimens previously diagnosed as normal by MTS. The technologist time required for evaluation and reclassification of the Duet system data for the FISH MRD samples averaged only 1 minute per case, saving significant technologist effort. We conclude that the Duet system appears to be more sensitive and cost-effective than MTS for CML FISH MRD testing.

Utility of ALK-1 protein expression and ALK rearrangements in distinguishing inflammatory myofibroblastic tumor from malignant spindle cell lesions of the urinary bladder.

Inflammatory myofibroblastic tumor of the urinary bladder is an unusual spindle cell neoplasm that displays cytologic atypia, infiltrative growth and mitotic activity mimicking malignant tumors, such as leiomyosarcoma, rhabdomyosarcoma and sarcomatoid carcinoma. The objective of this study was to determine if anaplastic lymphoma kinase (ALK-1) protein expression detected by immunohistochemistry and ALK rearrangements detected by fluorescence in situ hybridization (FISH) were useful in distinguishing inflammatory myofibroblastic tumor from malignant spindle cell tumors of the urinary bladder. In inflammatory myofibroblastic tumor, ALK-1 expression was identified in 13 of 21 cases (62%) and ALK rearrangements in 14 of 21 cases (67%). All cases of inflammatory myofibroblastic tumor demonstrating ALK-1 expression, carried ALK rearrangements. One case negative for ALK-1 expression exhibited ALK rearrangement. ALK rearrangements were more common in women (P=0.0032). Leiomyosarcoma, sarcomatoid carcinoma, embryonal rhabdomyosarcoma and reactive myofibroblastic proliferations were negative for ALK-1 protein and ALK rearrangements. Immunohistochemistry using markers of muscle, epithelial, neural, and follicular dendritic cell differentiation showed overlap between inflammatory myofibroblastic tumor with and without ALK gene rearrangements, and between inflammatory myofibroblastic tumor and spindle cell malignancies. However, coexpression of cytokeratin and muscle-specific antigens was unique to inflammatory myofibroblastic tumor, observed in approximately half the tumors. This study indicates that detection of ALK protein and ALK gene rearrangements are useful in distinguishing inflammatory myofibroblastic tumor from spindle cell malignancies in the urinary bladder. Additionally, our findings suggest that ALK rearrangement is the primary mechanism for ALK activation and that inflammatory myofibroblastic tumor likely represents a heterogeneous group of spindle cell proliferations with the majority associated with ALK translocations, and the remaining associated with other etiologies.

Preclinical validation of fluorescence in situ hybridization assays for clinical practice.

PURPOSE: Validation of fluorescence in situ hybridization assays is required before using them in clinical practice. Yet, there are few published examples that describe the validation process, leading to inconsistent and sometimes inadequate validation practices. The purpose of this article is to describe a broadly applicable preclinical validation process. METHODS: Validation is performed using four consecutive experiments. The Familiarization experiment tests probe performance on metaphase cells to measure analytic sensitivity and specificity for normal blood specimens. The Pilot Study tests a variety of normal and abnormal specimens, using the intended tissue type, to set a preliminary normal cutoff and establish the analytic sensitivity. The Clinical Evaluation experiment tests these parameters in a series of normal and abnormal specimens to simulate clinical practice, establish the normal cutoff and abnormal reference ranges, and finalize the standard operating procedure. The Precision experiment measures the reproducibility of the new assay over 10 consecutive working days. To illustrate documentation and analysis of data with this process, the results for a new assay to detect fusion of IGH and BCL3 associated with t(14;19)(q32;q13.3) in lymphoproliferative disorders are provided in this report. RESULTS: These four experiments determine the analytic sensitivity and specificity, normal values, precision, and reportable reference ranges for validation of the new test. CONCLUSION: This report describes a method for preclinical validation of fluorescence in situ hybridization studies of metaphase cells and interphase nuclei using commercial or home brew probes.

Interphase FISH to detect PBX1/E2A fusion resulting from the der(19)t(1;19)(q23;p13.3) or t(1;19)(q23;p13.3) in paediatric patients with acute lymphoblastic leukaemia.

Approximately 6% of paediatric patients with precursor B-cell acute lymphoblastic leukaemia (B-ALL) harbour a rearrangement involving the gene regions of PBX1 (1q23) and E2A (19p13.3) which is visualized cytogenetically either as a der(19)t(1;19)(q23;p13.3) or the less common balanced t(1;19)(q23;p13.3). Unfortunately, no commercial dual-colour, double fusion fluorescence in situ hybridization (D-FISH) strategies are available to detect this recurrent anomaly. Therefore, we have created a D-FISH assay to detect these translocations and monitor minimal residual disease. This probe set was created using four bacterial artificial chromosomes (BACs) corresponding to the PBX1 gene region at 1q23 and four BACs corresponding to the E2A gene region at 19p13.3. We analysed 30 negative bone marrow controls and 20 diagnostic and post-treatment specimens from 13 paediatric B-ALL patients with a cytogenetically defined 1;19 translocation. Once unblinded, the results demonstrated that our D-FISH method effectively identified all diagnostic samples as abnormal and identified disease in four post-treatment samples that were previously considered to be normal by conventional cytogenetic analysis. The development of this FISH strategy for the detection of der(19)t(1;19)(q23;p13.3) and t(1;19)(q23;p13.3) proved to be an effective technique, allowing both the detection of disease in diagnostic samples and in post-treatment samples.

Molecular diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization.

Recent studies have suggested that formalin-fixed paraffin-embedded (FFPE) tissues can be used for molecular analyses by fluorescence in situ hybridization (FISH) and RT-PCR. We analyzed 18 cases of ES/PNET for the t(11;22)(q24;q12) and t(21;22)(q22;q12) fusion transcripts by RT-PCR and analyzed for EWS translocation by interphase FISH with a dual color fusion probe to compare these two approaches directly. RT-PCR detected 13 (72%) EWS-FLI-1 fusions (type I=10, type II=3) and 2 (11%) EWS-ERG fusions. Three cases could not be evaluated because the housekeeping gene phosphoglycerate kinase (internal mRNA control) was not amplified. FISH was diagnostic in 15 of 18 cases (83%). There were three discordant cases between RT-PCR and FISH (concordance of 83%). Using a combination of RT-PCR and FISH, the results were complementary. One advantage of RT-PCR analysis was that subtypes of EWS translocation could be determined specifically (type I, type II and ERG). These findings indicate that because of the difficulties and limitations associated with the molecular analysis of FFPE tissues, a combination of RT-PCR and FISH may be a better approach to enhance the sensitivity and accuracy of detecting ES/PNET translocations in FFPE tissues with suboptimally preserved nucleic acids.

CHIC2 deletion, a surrogate for FIP1L1-PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy.

Imatinib mesylate is effective in the treatment of hematologic malignancies that are characterized by either abl- or PDGFR beta- activating mutations. The drug is also active in a subset of patients with eosinophilic disorders and systemic mast cell disease (SMCD). Recently, a novel tyrosine kinase that is generated from fusion of the Fip1-like 1 (FIP1L1) and PDGFR alpha (PDGFRA) genes has been identified as a therapeutic target for imatinib mesylate in hypereosinophilic syndrome (HES). We used fluorescence in situ hybridization (FISH) to detect deletion of the CHIC2 locus at 4q12 as a surrogate for the FIP1L1-PDGFRA fusion. CHIC2 deletion was observed in bone marrow cells for 3 of 5 patients with SMCD associated with eosinophilia. Deletion of this locus and expression of the FIP1L1-platelet-derived growth factor receptor alpha (PDGFRA) fusion was also documented in enriched eosinophils, neutrophils, or mononuclear cells by both FISH and reverse transcriptase-polymerase chain reaction (RT-PCR) for one patient. While all 3 patients with the FIP1L1-PDGFRA rearrangement achieved a sustained complete response with imatinib mesylate therapy, the other two, both carrying the c-kit Asp816 to Val (Asp816Val) mutation, did not. These observations suggest that the FIP1L1-PDGFRA rearrangement occurs in an early hematopoietic progenitor and suggests that the molecular pathogenesis for a subset of SMCD patients is similar to that of HES. Screening for the FIP1L1-PDGFRA rearrangement and Asp816Val mutation will advance rational therapy decisions in SMCD.