Non-canonical transcriptional start sites in E. coli O157:H7 EDL933 are regulated and appear in surprisingly high numbers.

Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.

The impact of Down syndrome-specific non-malignant hematopoietic regeneration in the bone marrow on the detection of leukemic measurable residual disease.

BACKGROUND: Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS. METHODS: A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the "difference from normal (ΔN)" technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions. RESULTS: 93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34+ CD56+ population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34+ HLA-DRheterogeneous population was observed among 92% of patients at a lower frequency (0.04%-0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34+ CD56+ HLA-DRheterogeneous expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells. CONCLUSIONS: Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.

Three Novel Antisense Overlapping Genes in E. coli O157:H7 EDL933.

The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping candidate genes are equipped with typical structural elements required for transcription and translation, i.e., promoters, transcription start sites, as well as terminators, all of which were experimentally verified. Translationally arrested mutants, unable to produce the overlapping encoded protein, were found to have a growth disadvantage when grown competitively against the wild type. Thus, the phenotypes found imply biological functionality of the genes at the level of proteins produced. The addition of 3 more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes emphasizes the underestimated coding capacity of bacterial genomes. IMPORTANCE The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated, since such genes are not allowed in genome annotations. However, ribosome profiling catches mRNA in the moment of being template for protein production. Using this technique and subsequent experiments, we verified 3 novel overlapping genes encoded in antisense of known genes. This adds more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes.

Integrated analysis of genotype and phenotype reveals clonal evolution and cytogenetically driven disruption of myeloid cell maturation in myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogenous collection of clonal bone marrow diseases characterized by cytopenias, abnormal karyotypes, molecular abnormalities, and dysplasia by flow cytometry and/or morphology. The progression of MDS to severe cytopenias and/or overt leukemia is associated with the accumulation of additional cytogenetic abnormalities, suggesting clonal evolution. The impact of these accumulated abnormalities on myeloid maturation and the severity of the disease is poorly understood. METHODS: Bone marrow specimens from 16 patients with cytogenetic abnormalities were flow cytometrically sorted into three myeloid populations: progenitors, immature myeloid cells, and mature myeloid cells. Fluorescence in situ hybridization analysis was performed on each to determine the distribution of chromosomal abnormalities during myeloid maturation. RESULTS: Our findings revealed three distinct distributions of cytogenetic abnormalities across myeloid maturation, each of which corresponded to specific cytogenetic abnormalities. Group 1 had continuous distribution across all maturational stages and contained patients with a single cytogenetic aberration associated with good-to-intermediate prognosis; Group 2 had accumulation of abnormalities in immature cells and contained patients with high-risk monosomy 7; and Group 3 had abnormalities defining the founding clone equally distributed across maturational stages while subclonal abnormalities were enriched in progenitor cells and contained patients with multiple, non-monosomy 7, abnormalities with evidence of clonal evolution. CONCLUSIONS: Our findings demonstrate that low-risk abnormalities (e.g., del(20q) and trisomy 8) occurring in the founding clone display a markedly different disease etiology, with respect to myeloid maturation, than monosomy 7 or abnormalities acquired in subclones, which result in a disruption of myeloid cell maturation in MDS.

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC).

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.

Evidence for BCR/ABL1-positive T-cell acute lymphoblastic leukemia arising in an early lymphoid progenitor cell.

BCR-ABL1-positive leukemias have historically been classified as either chronic myelogenous leukemia or Ph+ acute lymphoblastic leukemia. Recent analyses suggest there may be a wider range of subtypes. We report a patient with BCR-ABL1 fusion positive T-cell ALL with a previously undescribed cell distribution of the fusion gene. The examination of sorted cells by fluorescence in situ hybridization showed the BCR-ABL1 fusion in the malignant T cells and a subpopulation of the nonmalignant B cells, but not nonmalignant T cells or myeloid or CD34+ progenitor cells providing evidence that the fusion may have occurred in an early lymphoid progenitor.

The novel EHEC gene asa overlaps the TEGT transporter gene in antisense and is regulated by NaCl and growth phase.

Only a few overlapping gene pairs are known in the best-analyzed bacterial model organism Escherichia coli. Automatic annotation programs usually annotate only one out of six reading frames at a locus, allowing only small overlaps between protein-coding sequences. However, both RNAseq and RIBOseq show signals corresponding to non-trivially overlapping reading frames in antisense to annotated genes, which may constitute protein-coding genes. The transcription and translation of the novel 264 nt gene asa, which overlaps in antisense to a putative TEGT (Testis-Enhanced Gene Transfer) transporter gene is detected in pathogenic E. coli, but not in two apathogenic E. coli strains. The gene in E. coli O157:H7 (EHEC) was further analyzed. An overexpression phenotype was identified in two stress conditions, i.e. excess in salt or arginine. For this, EHEC overexpressing asa was grown competitively against EHEC with a translationally arrested asa mutant gene. RT-qPCR revealed conditional expression dependent on growth phase, sodium chloride, and arginine. Two potential promoters were computationally identified and experimentally verified by reporter gene expression and determination of the transcription start site. The protein Asa was verified by Western blot. Close homologues of asa have not been found in protein databases, but bioinformatic analyses showed that it may be membrane associated, having a largely disordered structure.

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.

Atypical Chronic Myeloid Leukemia in Two Pediatric Patients.

Atypical chronic myeloid leukemia, BCR-ABL1-negative, (aCML) is a rare myeloid neoplasm. Recent adult data suggest the leukemic cells in a subset of patients are dependent on JAK/STAT signaling and harbor CSF3R-activating mutations. We hypothesized that, similar to adult patients, the presence of CSF3R-activating mutations would be clinically relevant in pediatric myeloid neoplasms as patients would be sensitive to the JAK inhibitor, ruxolitinib. We report two cases of morphologically similar pediatric aCML, BCR-ABL1-negative based on WHO 2008 criteria. One patient had CSF3R-activating mutation (T618I) and demonstrated a robust response to ruxolitinib, which was used to bridge to a successful stem cell transplant. The other patient did not have a CSF3R-activating mutation and succumbed to refractory disease <6 months from diagnosis. This report documents CSF3R-T618I in pediatric aCML and demonstrates the efficacy of ruxolitinib in a pediatric malignancy. As the third documented case successfully treating aCML with ruxolitinib, this case highlights the importance of prompt CSF3R sequencing analysis for myeloproliferative and myelodysplastic/myeloproliferative neoplasms.

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry workup.

INTRODUCTION: 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 (NH4 Cl) 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. METHOD: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2(V617F) positive myeloproliferative neoplasms (MPN), and 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. RESULTS: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%), and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. DISCUSSION: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS using a lysis protocol practical for routine implementation in clinical flow cytometric workup. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically.

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 sample's 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.

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry work-up.

Introduction: While multidimensional flow cytometry (MDF) has great utility in diagnostic work-ups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4 Cl) 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. Method: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2V617F positive myeloproliferative neoplasms (MPN), 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. Results: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%) and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. Discussion: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS utilizing a lysis protocol practical for routine implementation in clinical flow cytometric work-up. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. © 2014 Clinical Cytometry Society.

Intraclonal heterogeneity in concomitant monoclonal lymphocyte and plasma cell populations: combining flow cytometric cell sorting with molecular monoclonality profiling.

Flow cytometric cell sorting combined with molecular gene rearrangement analysis can assist in further characterizing simultaneously occurring, phenotypically distinct, monoclonal B-lymphoid and monoclonal plasma cell populations that express immunoglobulin of the same light chain. We previously established monoclonality profiles for lymphoid and plasma cell populations of lymphoplasmacytic lymphoma (LPL) bone marrow aspirates by using flow cytometric cell sorting and subsequent monoclonal gene rearrangement analysis. Our findings demonstrated that related genetic processes are less likely than unrelated genetic processes. Here, we demonstrated the utility of cell sorting combined with gene rearrangement (both immunoglobulin IgH and IgK) and IgVH sequence analysis as well as plasma cell targeted fluorescence in situ hybridization analysis in clinical cases of presumed Waldenström macroglobulinemia/LPL in which multiple distinct B-cell and plasma cell populations were identified. Combining cell sorting with subsequent molecular analysis can provide proof of identical monoclonal genotype for Waldenström macroglobulinemia/LPL and nonidentical distinct lymphoid and plasma cell populations in the clinical setting. Understanding how many clonal processes (molecular profiles) are present can help guide patient monitoring throughout treatment and potentially identify patients with worse outcomes.

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 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.

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 Waldenström's/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.

Phenotypic abnormalities strongly reflect genotype in patients with unexplained cytopenias.

BACKGROUND: 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. METHODS: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality. RESULTS: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥ 2. Trisomy 8, del(20 q), and minus Y had flow scores ≥ 2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (> 20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells. CONCLUSIONS: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities.

Detection of alpha-methylacyl-coenzyme-A racemase transcripts in blood and urine samples of prostate cancer patients.

BACKGROUND: Alpha-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.

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

AIM: 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, gamma-aminobutyrate type A receptor pi subunit (GABA pi; 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.