Reference Size Matching, Whole-Genome Amplification, and Fluorescent Labeling as a Method for Chromosomal Microarray Analysis of Clinically Actionable Copy Number Alterations in Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

Cancer genome copy number alterations (CNAs) assist clinicians in selecting targeted therapeutics. Solid tumor CNAs are most commonly evaluated in formalin-fixed, paraffin-embedded (FFPE) tissue by fluorescence in situ hybridization. Although fluorescence in situ hybridization is a sensitive and specific assay for interrogating preselected genomic regions, it provides no information about coexisting clinically significant copy number changes. Chromosomal microarray analysis is an alternative DNA-based method for interrogating genome-wide CNAs in solid tumors. However, DNA extracted from FFPE tumor tissue produces an essential, yet problematic, sample type. The College of American Pathologists/American Society of Clinical Oncology guidelines for optimal tumor tissue handling, published in 2007 for breast cancer and in 2016 for gastroesophageal adenocarcinomas, are lacking for other solid tumors. Thus, cold ischemia times are seldom monitored in non-breast cancer and non-gastroesophageal adenocarcinomas, and all tumor biospecimens are affected by chemical fixation. Although intended to preserve specimens for long-term storage, formalin fixation causes loss of genetic information through DNA damage. Herein, we describe a reference size matching, whole-genome amplification, and fluorescent labeling method for FFPE-derived DNA designed to improve chromosomal microarray results from suboptimal nucleic acids and salvage highly degraded samples. With this technological advance, whole-genome copy number analysis of tumor DNA can be reliably performed in the clinical laboratory for a wide variety of tissue conditions and tumor types.

Reversal of Refractory Ulcerative Colitis and Severe Chronic Fatigue Syndrome Symptoms Arising from Immune Disturbance in an HLA-DR/DQ Genetically Susceptible Individual with Multiple Biotoxin Exposures.

BACKGROUND: Patients with multisymptom chronic conditions, such as refractory ulcerative colitis (RUC) and chronic fatigue syndrome (CFS), present diagnostic and management challenges for clinicians, as well as the opportunity to recognize and treat emerging disease entities. In the current case we report reversal of co-existing RUC and CFS symptoms arising from biotoxin exposures in a genetically susceptible individual. CASE REPORT: A 25-year-old previously healthy male with new-onset refractory ulcerative colitis (RUC) and chronic fatigue syndrome (CFS) tested negative for autoimmune disease biomarkers. However, urine mycotoxin panel testing was positive for trichothecene group and air filter testing from the patient's water-damaged rental house identified the toxic mold Stachybotrys chartarum. HLA-DR/DQ testing revealed a multisusceptible haplotype for development of chronic inflammation, and serum chronic inflammatory response syndrome (CIRS) biomarker testing was positive for highly elevated TGF-beta and a clinically undetectable level of vasoactive intestinal peptide (VIP). Following elimination of biotoxin exposures, VIP replacement therapy, dental extractions, and implementation of a mind body intervention-relaxation response (MBI-RR) program, the patient's symptoms resolved. He is off medications, back to work, and resuming normal exercise. CONCLUSIONS: This constellation of RUC and CFS symptoms in an HLA-DR/DQ genetically susceptible individual with biotoxin exposures is consistent with the recently described CIRS disease pathophysiology. Chronic immune disturbance (turbatio immuno) can be identified with clinically available CIRS biomarkers and may represent a treatable underlying disease etiology in a subset of genetically susceptible patients with RUC, CFS, and other immune disorders.

Array-based karyotyping in plasma cell neoplasia after plasma cell enrichment increases detection of genomic aberrations.

The discovery of genomic abnormalities present in monoclonal plasma cells has diagnostic, prognostic, and disease-monitoring implications in plasma cell neoplasms (PCNs). However, technical and disease-related limitations hamper the detection of these abnormalities using cytogenetic analysis or fluorescence in situ hybridization (FISH). In this study, 28 bone marrow specimens with known PCNs were examined for the presence of genomic abnormalities using microarray analysis after plasma cell enrichment. Cytogenetic analysis was performed on 15 of 28 samples, revealing disease-related genomic aberrations in only 3 (20%) of 15 cases. FISH analysis was performed on enriched plasma cells and detected aberrations in 84.6% of specimens while array comparative genomic hybridization (aCGH) detected abnormalities in 89.3% of cases. Furthermore, aCGH revealed additional abnormalities in 24 cases compared with FISH alone. We conclude that aCGH after plasma cell enrichment, in combination with FISH, is a valuable approach for routine clinical use in achieving a more complete genetic characterization of patients with PCN.

Inversion and deletion of 16q22 defined by array CGH, FISH, and RT-PCR in a patient with AML.

Acute myelomonocytic leukemia with eosinophilia is commonly associated with pericentric inversions of chromosome 16, involving the core binding factor beta gene (CBFB) on 16q22 and the myosin heavy chain gene (MYH11) on 16p13. The inv(16)(p13q22) results in a fusion gene comprising the 5'CBFB gene and the 3'MYH11 gene on the short arm of chromosome 16. The fusion gene interferes with the normal transcription of the CBFA/CBFB heterodimer and disrupts myeloid differentiation. The inv(16) is associated with a good prognosis. The inv(16) with deletion of the 3'CBFB region of the gene is a very rare occurrence. Although the number of cases is small, inv(16) with a deleted 3'CBFB seems to be associated with a poorer prognosis than that generally associated with inv(16). Our patient was a 30-year-old man with newly diagnosed acute myeloid leukemia who was found to have a CBFB-MYH11 fusion by reverse transcriptase-polymerase chain reaction. The high blast count and lack of differentiation were not typical for this entity and suggested clonal progression. The initial karyotype by conventional cytogenetic analysis, in all metaphases examined, was 46,XY,del(7)(q32),del(16)(q22). Fluorescence in situ hybridization analysis with a dual-color, break-apart probe corresponding to the CBFB gene locus (Abbott, Des Plaines, IL) showed a derivative chromosome 16 resulting from an inversion of the CBFB gene with a deletion of the 3'CBFB probe region. Oligonucleotide array comparative genetic hybridization analysis was performed on this patient's diagnostic bone marrow DNA referenced to a normal male control DNA by using the DNAarray Heme Profile (CombiMatrix Diagnostics, Irvine, CA) microarray. This analysis showed a 1.2 Mb loss of 16q22.1, which did not include loss of the 3'CBFB gene locus, but rather sequences distal to this locus. The DNAarray Heme Profile results illustrate the importance of microarray in the correct identification of abnormalities that will affect prognosis.

The vanguard has arrived in the clinical laboratory: array-based karyotyping for prognostic markers in chronic lymphocytic leukemia.

This Commentary provides a state of the art for array-based karyotyping in cancer diagnostics.

Clinical validation of an array CGH test for HER2 status in breast cancer reveals that polysomy 17 is a rare event.

The HER2 gene is an important prognostic and therapeutic marker in newly diagnosed breast cancer. Currently, HER2 status is most frequently determined by immunohistochemical detection of HER2 protein expression on the cellular membrane surface or by fluorescence in situ hybridization analysis of HER2 gene copy number in fixed tissue using locus-specific probes for the HER2 gene and chromosome 17 centromere. However, these methods are problematic because of issues with intra- and inter-laboratory reproducibility and preanalytic variables, such as fixation time. In addition, the commonly used HER2/chromosome 17 ratio presumes that chromosome 17 polysomy is present when the centromere is amplified, even though analysis of the rest of the chromosome is not included in the assay. In this study, 97 frozen samples of invasive lobular and invasive ductal carcinoma, with known immunohistochemistry and fluorescence in situ hybridization results for HER2, were analyzed by comparative genomic hybridization to a commercially available bacterial artificial chromosome whole-genome array containing 99 probes targeted to chromosome 17 and the HER2/TOP2 amplicon. Results were 97% concordant for HER2 status, meeting the College of American Pathologists/American Society of Clinical Oncology's validation requirements for HER2 testing. Surprisingly, not a single case of complete polysomy 17 was detected even though multiple breast cancer cases showed clear polysomies of other chromosomes. We conclude that array comparative genomic hybridization is an accurate and objective DNA-based alternative for clinical evaluation of HER2 gene copy number, and that polysomy 17 is a rare event in breast cancer.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene.

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2, and PRAME genes. Quantitative real-time PCR revealed that ZNF280A, ZNF280B, and PRAME mRNA expression was significantly lower in the 22q11 deletion cases compared to non-deleted cases.

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.

BACKGROUND: Whole-genome analysis by array-based comparative genomic hybridization (array CGH) is an emerging technique for the detection of recurrent unbalanced chromosomal aberrations in chronic lymphocytic leukemia (CLL). These chromosomal changes can be highly predictive of clinical course and are evaluated at present using classical cytogenetics and interphase fluorescence in situ hybridization. However, the significant limitations of these assays have resulted in efforts to move array CGH from use as a discovery tool in the research laboratory into the clinical laboratory as an alternative method for the evaluation of genomic prognostic markers in patients with CLL. OBJECTIVE: The HemeScan(™) array was developed as a clinical tool to provide prognostic marker identification with simultaneous diagnostic monitoring of the entire genome in CLL and other hematological malignancies. METHODS: The authors review representative data from clinical testing of HemeScan for genomic aberration identification in CLL and present suggestions for the integration of array CGH genome scanning into the clinical laboratory. RESULTS/CONCLUSION: The HemeScan assay for CLL precludes the need for G-banded cytognetics by simultaneously revealing prognostic marker status and the level of genomic complexity in > 85% of cases.

Comparative genomic hybridization arrays in clinical pathology: progress and challenges.

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.

Use of DNA sequencing analysis to confirm fungemia due to Trichosporon dermatis in a pediatric patient.

This is the first reported case of human disease caused by Tricosporon dermatis, an organism recently transferred to the genus Trichosporon from Cryptococcus and now confirmed to be a human pathogen.

Molecular characterization of a patient with central nervous system dysmyelination and cryptic unbalanced translocation between chromosomes 4q and 18q.

We report on a 12-year-old boy who presented with delayed development and CNS dysmyelination. Genetic studies showed a normal 46,XY karyotype by routine cytogenetic analysis, and 46,XY.ish del(18)(q23)(D18Z1+, MBP-) by FISH using a locus-specific probe for the MBP gene (18q23). Though the patient appeared to have normal chromosome 18s by repeated high resolution banding analysis, his clinical features were suggestive of a deletion of 18q. These included hearing loss secondary to stenosis of the external auditory canals, abnormal facial features, and foot deformities. FISH studies with genomic probes from 18q22.3 to 18qter confirmed a cryptic deletion which encompassed the MBP gene. In an attempt to further characterize the deletion, whole genome screening was conducted using array based comparative genomic hybridization (array CGH) analysis. The array CGH data not only confirmed a cryptic deletion in the 18q22.3 to 18qter region of approximately 7 Mb, it also showed a previously undetected 3.7 Mb gain of 4q material. FISH studies demonstrated that the gained 4q material was translocated distal to the 18qter deletion breakpoint. The 18q deletion contains, in addition to MBP, other known genes including CYB5, ZNF236, GALR1, and NFATC1, while the gained 4q material includes the genes FACL1 and 2, KLKB1, F11 and MTNR1A. The use of these combined methodologies has resulted in the first reported case in which array CGH has been used to characterize a congenital chromosomal abnormality, highlighting the need for innovative molecular cytogenetic techniques in the diagnosis of patients with idiopathic neurological abnormalities.