Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders.

We have developed an automated assay to enumerate and characterize circulating multiple myeloma cells (CMMC) from peripheral blood of patients with plasma cell disorders. CMMC show expression of genes characteristic of myeloma and fluorescence in situ hybridisation results on CMMC correlated well with bone marrow results. We enumerated CMMC from over 1000 patient samples including separate cohorts of newly diagnosed multiple myeloma and high/intermediate risk smouldering multiple myeloma (SMM) with clinical follow-up data. In newly diagnosed myeloma patient samples, CMMC counts correlated with other clinical measures of disease burden, including the percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (P < 0·001). Patients with CMMC counts ≥100 at remission showed reduced survival relative to patients with CMMC counts <100. Patients with undetectable CMMC in remission showed further overall survival benefits. In the SMM cohort, there was a trend toward higher CMMC in patients with higher-risk myeloma precursor states. Significantly higher CMMC counts were observed between intermediate/high risk SMM patients that progressed versus those without progression (P = 0·031). CMMC allow a non-invasive means of monitoring tumour biology and may have use as a prognostic test for patients with plasma cell disorders.

Prognostic and predictive value of circulating tumor cells and CXCR4 expression as biomarkers for a CXCR4 peptide antagonist in combination with carboplatin-etoposide in small cell lung cancer: exploratory analysis of a phase II study.

Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.

Mitochondrial Ndufa4l2 Enhances Deposition of Lipids and Expression of Ca9 in the TRACK Model of Early Clear Cell Renal Cell Carcinoma.

Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2.

Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer.

PURPOSE: Development of targeted therapeutic agents in colorectal cancer (CRC) is impeded by the lack of a noninvasive surrogate of drug effect. This pilot study evaluated the ability of immunomagnetic separation to isolate circulating tumor cells (CTCs) and of the fluorescent microscope system and flow cytometry to enumerate and characterize CTCs from patients with metastatic CRC. PATIENTS AND METHODS: Fifty patients with metastatic CRC contributed 50 mL of blood at treatment initiation and disease evaluation timepoints. Fresh tumor specimens were obtained from 17 patients for comparison of circulating and in situ tumor cell characteristics. Epithelial cells were magnetically isolated from whole blood targeting the antiepithelial cell adhesion molecule (EpCAM). Circulating tumor cells were defined as EpCAM isolated, cytokeratin positive, nuclear stain positive, and CD45 negative. Total RNA was isolated from EpCAM-enriched CTCs and multigene reverse-transcriptase polymerase chain reaction analyses were performed. RESULTS: The median number of CTCs detected by flow cytometry was 2/7.5 mL blood. Mean change in cell count was significantly different for patients with tumor progression versus nonprogression (+6.7 vs. +0.2/7.5 mL; P = 0.001). A correlation was noted between mean fluorescence intensity (flow cytometry) of cytokeratin in CTC and matched tumor specimens (r = 0.79, P = 0.06). Nearly 80% (15 of 19) of samples with >or= 2 CTCs expressed >or= 1 epithelial marker gene (CK19, CK20, carcinoembryonic antigen, or epidermal growth factor receptor). CONCLUSION: Isolating and characterizing CTCs from patients with metastatic CRC is feasible. Change in the CTC number might reflect clinical status, and flow cytometric and gene expression data suggest similarity of circulating and in situ tumor cells. Further evaluation of CTCs for pharmacodynamic and clinical monitoring in patients with CRC is warranted.

Circulating tumor cells predict survival in patients with metastatic prostate cancer.

OBJECTIVES: To determine whether circulating tumor cells (CTCs) predict for survival in patients with metastatic prostate cancer (PCa) and to compare its prognostic abilities with other clinical factors. METHODS: Blood samples from 37 patients with metastatic PCa were analyzed for CTCs. CTCs were enriched from 7.5 mL blood using magnetic nanoparticles targeting the epithelial cell adhesion molecule and then fluorescently labeled. The samples were analyzed by multiparameter flow cytometry, and events with appropriate light scatter properties that were nucleic acid dye positive, cytokeratin positive, and CD45 negative were defined as CTCs. RESULTS: The number of CTCs found ranged from 0 to 8586 per 7.5 mL (mean 530 +/- 1887, median 5). A threshold of 5 or more CTCs per 7.5 mL of blood was used to evaluate the ability of CTCs to predict for overall survival. Of the 37 patients, 23 (62%) had 5 or more CTCs, with a median overall survival of 0.70 year compared with more than 4 years for those patients with fewer than 5 CTCs (log-rank P = 0.002, Cox hazards ratio 7.4). In the subset of 26 patients with hormone-refractory PCa, the presence of CTCs was the most significant parameter predictive of survival in univariate and multivariate analyses. CONCLUSIONS: In this pilot study, the presence of 5 or more CTCs in 7.5 mL blood was associated with poor overall survival in patients with metastatic PCa.

Multigene reverse transcription-PCR profiling of circulating tumor cells in hormone-refractory prostate cancer.

BACKGROUND: Circulating tumor cells (CTCs) represent a surrogate source of tissue and conceptually represent a "real-time" biopsy. We previously reported that the number of CTCs mirrors disease progression in hormone-refractory prostate cancer (HRPC). To improve characterization of CTCs we further investigated whether in vitro transcription-based multigene reverse transcription-PCR expression profiles could be obtained from CTCs in HRPC. METHODS: We evaluated the expression of 37 genes with potential utility for epithelial cell characterization from antisense RNA libraries constructed from immunomagnetically enriched CTCs from 7.5-mL blood samples from healthy donors and patients with HRPC. RESULTS: In the control group 13 of 37 genes were not expressed. The most notable of the genes expressed in CTCs of 23 blood specimens drawn from 9 patients with metastatic prostate cancer were prostate-specific antigen (20 of 23; 87%), prostate-specific membrane antigen (17 of 23; 74%), androgen receptor (16 of 23; 70%), human glandular kallikrein 2 (7 of 23; 30%), epidermal growth factor receptor (4 of 23; 17%), and prostate-specific gene with homology to G protein receptor (2 of 23; 9%). The number of CTCs in these samples ranged from 4 to 283 in 7.5 mL of blood (mean, 87; median, 89). Expression of some of the genes was low in the control samples and higher in the patient samples. In all 23 samples, cytokeratin 19, epithelial cell adhesion molecule, or mucin 1 was expressed. Because of background expression in the controls, expression of 13 of the 37 genes, including HER-2, p53, and BCL-2, could not be measured in CTCs. CONCLUSION: Antisense RNA libraries can be constructed from CTCs and gene expression profiles of CTCs obtained from patients with HRPC. This could enhance the characterization of HRPC and facilitate the development of more effective therapies.

Apoptosis of circulating tumor cells in prostate cancer patients.

BACKGROUND: The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. METHODS: In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. RESULTS: In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. CONCLUSIONS: The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients.