Ryan S. Robetorye

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 Oncologys 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.

Copy number abnormalities, MYC activity, and the genetic fingerprint of normal B cells mechanistically define the microRNA profile of diffuse large B-cell lymphoma.

MicroRNA (miRNA) deregulation contributes to cancer pathogenesis. However, analysis of miRNAs in diffuse large B-cell lymphoma (DLBCL) has been hindered by a focus on cell lines, limited number of miRNAs examined, and lack of copy number data. To address these restrictions, we investigated genomewide miRNA expression and copy number data in 86 DLBCLs. Permutation analysis showed that 63 miRNAs were recurrently disrupted in DLBCL, including highly expressed oncomirs not previously linked to chromosomal abnormalities. Further, using training and validation tumor groups, we defined a collection of miRNAs that robustly segregates DLBCLs into 3 subsets, which are independent of the cell-of-origin classification, extent of T-cell infiltrate, and tumor site. Instead, these unique miRNA-driven DLBCL subgroups showed markedly different MYC transcriptional activity, which explained the dominance of miRNAs regulated by MYC in their expression signatures. In addition, analysis of miRNA expression patterns of normal B cells and integration of copy number and expression data showed that genomic abnormalities and the genetic fingerprint of nonmalignant cells also contribute to the miRNA profile of DLBCL. In conclusion, we created a comprehensive map of the miRNA genome in DLBCL and, in the process, have uncovered and mechanistically elucidated the basis for additional molecular heterogeneity in this tumor.

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.

Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia

Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia

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.

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.

Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction

The real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) has become the method of choice for the quantification of specific mRNAs. This method is fast, extremely sensitive, and accurate, requires only very small amounts of input RNA, and is relatively simple to perform. These characteristics have made it the method of choice for minimal residual disease monitoring such as in chronic myelogenous leukemia (CML). CML comprises approximately 20% of all leukemias and is characterized by a balanced (9;22) chromosomal translocation that results in the formation of a chimeric gene comprised of the BCR (breakpoint cluster region) gene and the ABL oncogene (BCR-ABL fusion gene). The chimeric gene encodes a fusion protein with constitutively increased tyrosine kinase activity, resulting in growth factor-independent proliferation. This kinase is the target for current CML therapy, and BCR-ABL fusion gene levels are monitored to determine the effectiveness of this therapy. This chapter uses BCR-ABL transcript detection to illustrate an example for the RQ-PCR and describes a RQ-PCR method to detect the most common form of the BCR-ABL fusion transcript in CML, known as p210 BCR-ABL.

Integrity of the CBL gene in mature B-cell malignancies

To the editor: Identification of biomarkers for response to novel therapeutic agents is an essential step for the success of tailored treatments in cancer. Preliminary data from clinical trials indicate that inhibitors of the spleen tyrosine kinase (SYK) may be efficacious in subsets of diffuse

Epstein–Barr Virus (EBV) Load Determination Using Real-Time Quantitative Polymerase Chain Reaction

Epstein-Barr virus (EBV) infects virtually the entire human population and infection persists throughout the lifetime of its host. EBV has been associated with the development of a wide variety of neoplasms, including lymphoma, carcinoma, and sarcoma. In addition, EBV-associated lymphoproliferative disorders are particularly prevalent in immunosuppressed individuals, including AIDS patients, transplant recipients, and patients with congenital immunodeficiencies. In recent years, EBV viral load assessment has been extensively implemented in clinical practice for the diagnosis and monitoring of EBV-associated malignancies and lymphoproliferative disorders. The real-time quantitative polymerase chain reaction (RQ-PCR) has become the method of choice for quantification of specific EBV nucleic acid sequences. This method is fast, extremely sensitive, and accurate, requires only very small amounts of input nucleic acid, and is relatively simple to perform. These characteristics have made it the method of choice for EBV viral load determination. This chapter describes the use of a laboratory-developed RQ-PCR EBV viral load assay for the detection of EBV DNA in cell-free plasma and cerebrospinal fluid samples.

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.