High detection rate of clinically relevant genomic abnormalities in plasma cells enriched from patients with multiple myeloma

Multiple myeloma is a heterogeneous disease, which is characterized by the occurrence of specific genomic abnormalities that are both of diagnostic and prognostic relevance. Since the detection of these abnormalities through molecular‐genetic techniques is hampered by the overall low percentage of plasma cells present in primary bone marrow aspirates, we assessed the efficacy of these techniques in enriched plasma cell fractions from 61 multiple myeloma patients. Using interphase FISH, genomic abnormalities could be detected in 96% of the enriched samples as compared to 61% in the cultured whole bone marrow samples. We also found that microarray‐based genomic profiling of enriched plasma samples facilitates the detection of additional, possibly clinically relevant, genomic abnormalities. We conclude that the genomic delineation of enriched plasma cells from multiple myeloma patients results in a significantly increased detection rate of clinically relevant genomic abnormalities. In order to facilitate molecular‐genetic data interpretation, we recommend morphological assessment of plasma cell purity after enrichment. © 2012 Wiley Periodicals, Inc.     

Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites

Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern.     

Different patterns of DNA alterations detected by restriction landmark genomic scanning in heterogeneous prostate carcinomas.

Alterations in the genomic DNAs of six heterogeneous prostate carcinomas, as well as that of individual and histologically distinct foci within the tumors, were examined using restriction landmark genomic scanning, a method employing two-dimensional gel analysis of a large number of DNA fragments generated by digestion with highly specific endonucleases. Upon autoradiographic imaging, these fragments appear as spots of varying intensity and location specific for each sample. In our study, comparison of cancer DNAs against normal prostate DNA controls yielded alterations in at least 35 spots. Despite differences in the histological grading of tumors, 3 spots common to all tumor samples showed consistent amplification of intensity and 8 other common spots demonstrated consistent reduction of intensity when compared with control. In addition, spot alterations occurred between histologically identical foci isolated from within single tumors. We suggest that these spot changes detected in DNA profiles generated by restriction landmark genomic scanning reflect aberrations in as yet unidentified oncogenes and tumor suppressor genes and indicate that prostate cancer is not only histologically heterogeneous and multifocal but also genetically multicentric.     

Diagnostic role of circulating free plasma DNA detection in patients with localized prostate cancer

To analyze the potential diagnostic relevance of free plasma DNA (FPDNA), we enrolled 64 patients with localized prostate cancer (CaP). FPDNA was quantified by real-time polymerase chain reaction assessment of the HTERT gene in blood samples from 64 patients with CaP and 45 healthy males. Methylation of the GSTP1 gene was used to confirm the neoplastic origin of FPDNA in selected cases.The mean +/- SD levels of FPDNA were higher in patients with CaP (15.4 +/- 10.9 ng/mL) than in control subjects (5.5 +/- 3.5 ng/mL; P <.001). By using the best cutoff value, the sensitivity of the test was 80%, the specificity was 82%, the area under the receiver operating characteristic curve, 0.881. High FPDNA values were significantly associated with pathologic T3 stage (P = . 035). Methylation of the GSTP1 gene was found in 4 (25%) of 16 FPDNA samples and 15 (94%) of 16 tissue samples.Quantification of FPDNA discriminates between patients with CaP and healthy subjects and correlates with pathologic tumor stage. FPDNA is a candidate biomarker for early diagnosis and monitoring of CaP.     

Comparative analysis of innate immune system function in metastatic breast,colorectal, and prostate cancer patients with circulating tumor cells

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry.     

Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases

TGFβ is a known driver of epithelial-mesenchymal transition (EMT) which is associated with tumor aggressiveness and metastasis. However, EMT has not been fully explored in clinical specimens of castration-resistant prostate cancer (CRPC) metastases. To assess EMT in CRPC, gene expression analysis was performed on 149 visceral and bone metastases from 62 CRPC patients and immunohistochemical analysis was performed on 185 CRPC bone and visceral metastases from 42 CRPC patients. In addition, to assess the potential of metastases to seed further metastases the mitochondrial genome was sequenced at different metastatic sites in one patient. TGFβ was increased in bone versus visceral metastases. While primarily cytoplasmic; nuclear and cytoplasmic Twist were significantly higher in bone than in visceral metastases. Slug and Zeb1 were unchanged, with the exception of nuclear Zeb1 being significantly higher in visceral metastases. Importantly, nuclear Twist, Slug, and Zeb1 were only present in a subset of epithelial cells that had an EMT-like phenotype. Underscoring the relevance of EMT-like cells, mitochondrial sequencing revealed that metastases could seed additional metastases in the same patient. In conclusion, while TGFβ expression and EMT-associated protein expression is present in a considerable number of CRPC visceral and bone metastases, nuclear Twist, Slug, and Zeb1 localization and an EMT-like phenotype (elongated nuclei and cytoplasmic compartment) was only present in a small subset of CRPC bone metastases. Mitochondrial sequencing from different metastases in a CRPC patient provided evidence for the seeding of metastases from previously established metastases, highlighting the biological relevance of EMT-like behavior in CRPC metastases.     

Toll-like receptor (TLR) expression of immune system cells from metastatic breast cancer patients with circulating tumor cells

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p < 0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population.     

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.     

Advances in liquid biopsy using circulating tumor cells and circulating cell-free tumor DNA for detection and monitoring of breast cancer

Clinical and Experimental Medicine - Overview the progress of liquid biopsy using circulating tumor cells (CTCs) and circulating cell-free tumor DNA (cfDNA) to detect and monitor breast cancer....     

Assessment of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors

The objective of this study was to assess the incidence of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors (less than 0.5 cc) after radical prostatectomy (RP). Blood samples were collected from 64 RP patients to assess the incidence of CTCs following RP. The specimens were processed by whole‐mount section. Clinicopathological data (e.g. patient age, race, specimen weight, tumor volume, grade, stage and surgical margin status) and follow‐up PSA data were compared to CTC status. Of the 64 RP patients, nine had ‘low‐volume prostate cancer’. Seven of these patients had detectable levels of CTCs. In two of the seven patients with detectable CTCs, PSA elevation was also observed. Isolation and detection of circulating epithelial cells is possible in low‐volume prostate cancer patients. In the setting of low‐volume prostate cancer, CTCs may be associated with the presence of detectable PSA levels. However, the detection of CTCs did not predict PSA failure.     

DNA copy number alterations and expression of relevant genes in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is defined by a lack of expression of estrogen, progesterone, and HER2 receptors, and genetically most of them fall into the basal subgroup of breast cancer. The important issue of TNBC is poorer clinical outcome and absence of effective targeted therapy. In this study, we sought to identify DNA copy number alterations and expression of relevant genes characteristic of TNBC to discover potential therapeutic targets. Frozen tissues from 114 breast cancers were analyzed using high-resolution array comparative genomic hybridization. The classification into subtype was determined by estrogen and progesterone receptor expression, and by the presence or absence of gain on the ERBB2 containing clone. The ACE algorithm was used for calling gain and loss of clones. Twenty-eight cases (25%) were classified as TNBC. Recurrent gains (> or =25%) unique to TNBC were 9p24-p21, 10p15-p13, 12p13, 13q31-q34, 18q12, 18q21-q23, and 21q22. Two published gene expression array data sets comparing basal subtype versus other subtype breast cancers were used for searching candidate genes. Of the genes upregulated in the basal subtype, 45 of 686 genes in one data set and 59 of 1,428 in the second data set were found to be located in the gained regions. Of these candidate genes, gain of NFIB (9p24.1) was specific for TNBC in a validation set by real-time PCR. In conclusion, we have identified recurrently gained regions characteristic of TNBC, and found that NFIB copy number and expression is increased in TNBC across the data sets. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Development and clinical validation of a circulating tumor DNA test for the identification of clinically actionable mutations in nonsmall cell lung cancer

Molecular analysis of potentially actionable mutations has become routine practice in oncological pathology. However, testing a wide range of oncogenes and mutations can be technically challenging because of limitations associated with tumor biopsy. Circulating tumor DNA (ctDNA) is a potential tool for the noninvasive profiling of tumors. In this study, we developed a next‐generation sequencing (NGS)‐based test for the detection of clinically relevant mutations in ctDNA and evaluated the feasibility of using this ctDNA NGS‐based assay as an alternative to tissue genotyping. Tissue and matched blood samples were obtained from 72 patients with advanced nonsmall cell lung cancer (NSCLC). NGS‐based testing was performed using plasma cell‐free DNA (cfDNA) samples of all 72 patients as well as tumor DNA samples of 46 patients. Of the remaining 26 patients, tDNA was tested by amplification refractory mutation system PCR (ARMS‐PCR) because of insufficient tissue sample or quality for NGS. Of the 46 patients who had tDNA and cfDNA NGS performed, we found 20 patients were concordant between tDNA and ctDNA alterations and 21 sample pairs were discordant because of additional alterations found in tDNA. Considering all clinically relevant alterations, the concordance rate between tDNA and ctDNA alterations was 54.9% with a sensitivity of 53.2% and a specificity of 75.0%. Our findings demonstrate that targeted NGS using cfDNA is a feasible approach for rapid and accurate identification of actionable mutations in patients with advanced NSCLC, and may provide a safe and robust alternative approach to tissue biopsy.     

Assessment of the role of circulating breast cancer cells in tumor formation and metastatic potential using in vivo flow cytometry

The identification of breast cancer patients who will ultimately progress to metastatic disease is of significant clinical importance. The quantification and assessment of circulating tumor cells (CTCs) has been proposed as one strategy to monitor treatment effectiveness and disease prognosis. However, CTCs have been an elusive population of cells to study because of their small number and difficulties associated with isolation protocols. In vivo flow cytometry (IVFC) can overcome these limitations and provide insights in the role these cells play during primary and metastatic tumor growth. In this study, we used two-color IVFC to examine, for up to ten weeks following orthotopic implantation, changes in the number of circulating human breast cells expressing GFP and a population of circulating hematopoietic cells with strong autofluorescence. We found that the number of detected CTCs in combination with the number of red autofluorescent cells (650 to 690 nm) during the first seven days following implantation was predictive in development of tumor formation and metastasis eight weeks later. These results suggest that the combined detection of these two cell populations could offer a novel approach in the monitoring and prognosis of breast cancer progression, which in turn could aid significantly in their effective treatment.     

Tracking recurrent quantitative genomic alterations in colorectal cancer: allelic losses in chromosome 4 correlate with tumor aggressiveness

Allelic imbalances are common events in cancer cells. Quantitative alterations in specific chromosomal loci have been linked to activation (gain) or inactivation (loss) of genes with a proven impact on tumor cell biology. The aim of this study was to detect new chromosomal regions recurrently altered in colorectal tumorigenesis and with a potential effect on patient's outcome. We have analyzed a series of human colorectal tumor biopsy specimens by using the DNA fingerprinting technique arbitrarily primed PCR. This approach provided information on 95 different loci randomly selected and distributed through out the cell's genome. Eight sequences displayed recurrent alterations associated with diminished patient survival. Four of them (showing allelic losses) were located in chromosome 4, one sequence in chromosome 2, and one sequence in chromosome 17. The chromosomal origin of the two remaining sequences could not be determined. Fine mapping of chromosome 4 bands suggested that there are at least two regions in chromosome 4 (4p14-16 and 4q21-28) susceptible to containing tumor suppressor genes the loss of which may affect tumor aggressiveness.     

Activin A circulating levels in patients with bone metastasis from breast or prostate cancer

Recent studies have highlighted that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, may be involved in the regulation of osteoblastic activity and in osteoclast differentiation. Therefore, we have investigated the clinical significance of its circulating levels in patients with bone metastasis. Activin A serum concentrations were determined, by a commercially available enzyme-linked immunosorbent assay kit, in 72 patients with breast cancer (BC) or prostatic cancer (PC) with (BM+) or without (BM−) bone metastases, in 15 female patients with age-related osteoporosis (OP), in 20 patients with benign prostatic hypertrophy (BPH) and in 48 registered healthy blood donors (HS) of both sex (25 female and 23 male). Activin A serum concentrations were significantly increased in BC or PC patients as compared to OP (P < 0.0001) or BPH (P = 0.045), respectively, or to sex matched HS (P < 0.0001). Additionally, these levels resulted more elevated in PC patients as compared to BC patients (P = 0.032). Interestingly, Activin A was significantly higher in BM+ patients than in BM− patients (BC, P = 0.047; PC, P = 0.016). In BC patients, a significant correlation was observed only between Activin A and number of bone metastases (P = 0.0065) while, in PC patients, Activin A levels were strongly correlated with the Gleason score (P = 0.011) or PSA levels (P = 0.0001) and, to a lessen extent, with the number of bone metastases (P = 0.056). Receiver operating characteristic curve (ROC) analysis showed a fair diagnostic accuracy of Activin A to discriminate between BM+ and BM− patients (BC: AUC = 0.71 ± 0.09, P = 0.03; PC: AUC = 0.73 ± 0.081, P = 0.005). These findings indicate that Activin A may be implicated in the pathogenesis of bone metastasis. Therefore, this cytokine may be considered a novel potential target for a more selective therapeutic approach in the treatment of skeletal metastasis and may be also useful as additional biochemical marker of metastatic bone disease.