Mansoor S. Mohammed

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.

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.

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.

Molecular cytogenetics as a clinical test for prognostic and predictive biomarkers in newly diagnosed ovarian cancer

BackgroundThere is a clinical need for routinely available genomic biomarker testing in newly diagnosed ovarian cancer. In the current study we performed molecular cytogenetics using a validated array based comparative genomic hybridization (array CGH) assay to screen for the presence of predictive and prognostic biomarkers in archival diagnostic tissue from ovarian cancer patients. We hypothesized that biomarkers of high-risk disease would be detectable in tumor samples from patients with treatment refractory, advanced disease, and would be detected less frequently in tumor samples from patients with more favorable outcomes. In addition, we predicted that the use of a genome-wide copy number analysis (CNA) testing platform would enable us to identify novel potentially targetable chromosomal alterations of therapeutic significance in a percentage of cases.MethodsFormalin-fixed paraffin-embedded tissue (FFPE) tumor bank specimens were retrieved from the initial surgical resection for 18 ovarian cancer patients. Molecular cytogenetics was performed by array CGH for the detection of somatic chromosomal alterations associated with high-risk disease including amplifications of the CCNE1 and HER2 genes. Genomic risk stratification results were correlated with available clinical data. CGH data from each patient’s tumor genome was also surveyed for the presence of potentially targetable aberrations. Relevant therapeutic agents and open studies for investigational drugs were reported for each patient.ResultsHigh-risk genomic alterations were identified in 12/18 (67%) of cases and all patients with high-risk markers had advanced, treatment refractory disease. Three tumors with minimal genomic changes had no high-risk markers and were from patients with Stage I/II disease that had been completely resected and under surveillance for recurrence. Eleven patients (61%) had at least one potentially targetable genomic alteration including CCNE1, HER2, KRAS gene amplifications, and somatic BRCA1 and/or BRCA2 gene deletions. Bi-allelic PTEN gene deletion was detected in one patient’s tumor.ConclusionsClinical genomic profiling of ovarian tumors by array CGH augments pathologic grade and stage to help stratify newly diagnosed ovarian cancer into high and low-risk disease. This personalized genomic information can also help guide treatment planning and disease monitoring by identifying novel potentially targetable genomic alterations that can be used by clinicians to choose rational directed therapies for patients with chemo-resistant disease.