Barbara K. Zehentner

Evaluation of a panel of tumor markers for molecular detection of circulating cancer cells in women with suspected breast cancer

Purpose: We examined the feasibility of using molecular characterization of circulating tumor cells as a method for early detection of breast cancer. Research Design: Women without a prior history of cancer who had a breast abnormality detected on imaging followed by a breast biopsy were enrolled in this study. Density gradient centrifugation and immunomagnetic capture were used to enrich for epithelial cells from ∼20 mL of blood. Real-time reverse transcription-PCR was used to quantitate the expression levels of the highly breast-specific genes, mammaglobin, γ-aminobutyric acid type A receptor π subunit (GABA Aπ), B305D-C, and B726P in the epithelial cell–enriched samples. Results: The assay was technically feasible in 154 of 199 accrued patients. From their clinical assessment, 100 patients had benign breast disease, 10 patients had ductal carcinoma in situ, and 44 patients had invasive breast cancer. We constructed a diagnostic test that classified patients with mammaglobin levels of at least 32.2 copies/pg β-actin (units) in their circulating epithelial cells as positive for invasive breast cancer. This resulted in a sensitivity and specificity of 63.3% and 75.0%, respectively. A diagnostic test that classified patients as positive for invasive breast cancer when either mammaglobin levels were >46.3 units or B305D-C levels were >11.6 units increased the sensitivity and specificity to 70.5% and 81.0%, respectively. In the latter test, 12 of the 14 node-positive breast cancer patients were correctly identified. Including GABA Aπ and B726P in the test did not increase its diagnostic potential. Conclusions: These results suggest that molecular characterization of circulating epithelial cells using mammaglobin and B305D-C offers potential for early detection of invasive breast cancer.

Transcriptional complementarity in breast cancer: application to detection of circulating tumor cells.

AbstractBackground: We used a combination of genetic subtraction, silicon DNA micro-array analysis, and quantitative PCR to identify tissue-and tumor-specific genes as diagnostic targets for breast cancer. Methods and Results: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor—specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. Conclusion: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

Detection of α-Methylacyl-Coenzyme-A Racemase Transcripts in Blood and Urine Samples of Prostate Cancer Patients

AbstractBackground: α-Methylacyl-coenzyme-A racemase (AMACR) has been shown to be a highly specific marker for prostate cancer cells, even in the earliest stages of malignant progression. It is expressed at much higher levels than prostate-specific antigen (PSA) in malignant tissues, and is not expressed at appreciable levels in normal prostatic epithelium. In this study, we demonstrate the quantitative detection of AMACR transcripts in peripheral blood of prostate cancer patients using real-time RT-PCR. In addition, we have undertaken a pilot study to demonstrate the potential application of this technique for the detection of prostate tumor cells in urine samples from patients with prostate cancer. Methods: A real-time RT-PCR assay was developed for detection of the expression of AMACR in prostate cancer patients. Blood samples from 163 patients were tested at various stages of disease progression, with or without therapy. Blood specimens from patients with benign prostate disorders and other types of cancer were also evaluated. Results: In 28 of 58 samples from patients with known metastatic disease who were undergoing treatment, an AMACR expression signal above the cut-off value was detected, consistent with the presence of circulating tumor cells. In 39 of 88 patients with presumptive organ-confined disease, there was evidence of low levels of circulating tumor cells. Comparison of AMACR RT-PCR with known serum PSA values indicated that a combination of these parameters significantly increased the sensitivity for detection of progressive disease. In a pilot study analyzing urine samples from seven prostate cancer patients, elevated AMACR expression levels were detected in the urine sediments of four of six stage-T1 prostate cancer patients and in the one patient with stage-T2 prostate cancer. Conclusion: The data presented in this study indicates that AMACR real-time RT-PCR may aid in the detection and staging of prostate cancer.

Phenotypic Abnormalities Strongly Reflect Genotype in Patients with Unexplained Cytopenias

In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology.

Detection of Genomic Abnormalities in Multiple Myeloma The Application of FISH Analysis in Combination With Various Plasma Cell Enrichment Techniques

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.

Detection of circulating tumor cells in peripheral blood of breast cancer patients during or after therapy using a multigene real-time RT-PCR assay.

AbstractAim: To evaluate the utility of a multigene real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay to detect circulating tumor cells in peripheral blood specimens of breast cancer patients during or after treatment. Method: Using this assay, peripheral blood samples were analyzed for expression levels of mammaglobin and three complementary transcribed breast cancer-specific genes: B305D, γ-aminobutyrate type A receptor π subunit (GABA π; GABRP), and B726P. We examined 172 blood specimens from 82 breast cancer patients during or after therapy for the presence of circulating tumor cells using the multigene real-time RT-PCR assay. Results: In 63.4% of the blood samples, a positive signal for mammaglobin and/or three breast cancer-associated gene transcripts was detected. Of breast cancer patients, 75.6% had at least one positive blood sample. Blood specimens from 51 of 53 healthy female volunteers tested negative in the assay whereas two samples had a low expression signal. In addition, three patients were monitored for more than a year during their adjuvant therapy treatment. Conclusion: This assay could be a valuable tool for monitoring breast cancer patients during and after therapy.

Assessment of erythroid dysplasia by “Difference from normal” in routine clinical flow cytometry workup

While multidimensional flow cytometry (MDF) has great utility in diagnostic workups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia.

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.

Detection of Clonal Evolution in Hematopoietic Malignancies by Combining Comparative Genomic Hybridization and Single Nucleotide Polymorphism Arrays

BACKGROUND Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. METHODS This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. RESULTS Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the samples clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. CONCLUSIONS Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated.

A minority of concurrent monoclonal lymphocytes and plasmacytic cells sharing light chains are genetically related in putative lymphoplasmacytic lymphoma

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenströms/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.