Serum cell-free DNA concentration as a possible prognostic marker in newly diagnosed diffuse large B-cell lymphoma.

Cell-free DNA (cfDNA) is a fragment of DNA circulating in the blood, and its concentration is often elevated in cancer patients. To investigate the relationships between serum cfDNA concentration and clinical characteristics, including prognosis, we measured serum cfDNA concentration in 114 newly diagnosed lymphoma patients. The cfDNA concentrations in diffuse large B cell lymphoma (DLBCL) (62.5 ng/mL) and follicular lymphoma patients (51.6 ng/mL) were significantly elevated compared to healthy individuals (7.5 ng/mL, P < 0.001). In DLBCL, patients with elevated serum cfDNA (> 38.9 ng/mL) at diagnosis had significantly shorter time-to-progression compared to those without (P = 0.033). The addition of cfDNA concentration to the international prognostic index showed improved predictive power for time-to-progression. Moreover, cfDNA added significant prognostic value to other inflammatory markers such as B symptoms and sIL2R. There was a trend towards shorter progression-free survival and overall survival in patients with elevated cfDNA. Furthermore, B symptoms (P = 0.038), bulky masses (P = 0.031), non-GCB subtype (P = 0.012), and serum sIL-2R levels > 2,000 U/mL (P = 0.012) were associated with higher cfDNA levels. Our study showed that serum cfDNA concentration at diagnosis was associated with certain clinicopathological characteristics, and may be predictive of survival outcomes in DLBCL patients.

Detection of HER2 Amplification in Circulating Tumor Cells of HER2-Negative Gastric Cancer Patients.

A key to the successful use of targeted cancer therapy is the ability to preselect patients who are likely to benefit from the treatment according to molecular markers. Assessment for predicting therapy response is mostly done using tumor biopsies. However, these might not truly represent all of the patient's malignant cells because of tumor heterogeneity and/or clonal evolution during disease progression. One potential strategy that can complement primary tumor biopsy is the analysis of circulating tumor cells (CTCs). In this study, we analyzed CTCs of patients with gastric cancer (GC) to find those who were likely to benefit from trastuzumab therapies. We developed an imaging-based method that enabled CTC identification simultaneously with evaluation of HER2 gene amplification (the 3D-IF-FISH method). Then we performed a study enrolling 101 GC patients in whom we analyzed CTCs by both 3D-IF-FISH and an FDA-approved CellSearch system. As compared with the CellSearch system, 3D-IF-FISH methods identified a higher number of patients whose primary tumors were HER2- but who had HER2+ CTCs, suggesting that the 3D-IF-FISH method is effective in preselecting patients for trastuzumab therapies. To demonstrate this, we performed an exploratory clinical study to evaluate the clinical benefits of trastuzumab treatment for advanced GC patients (n = 15) whose primary tumors were HER2-, but whose CTCs showed HER2 amplification. An interim evaluation after the first stage showed that these preselected patients had response rates comparable to those reported in the trastuzumab-plus-chemotherapy arm of the ToGA study. The present study offers a new, non-invasive strategy to select patients who are likely to benefit from trastuzumab-based therapies, despite their primary biopsy being HER2-negative. (UMIN ID: UMIN000008622).

A novel detection strategy for living circulating tumor cells using 5-aminolevulinic acid.

Most circulating tumor cell (CTC) detection methods have technical limitations, allowing the detection of only cells expressing epithelial antigens, and they cannot identify if the CTCs are alive or dead. Herein, we constructed a novel CTC detection system comprised of filter separation and 5-aminolevulinic acid (5-ALA)-based labeling, termed "Fs-ALA". Blood specimens (7.5 mL) were subjected to this method. Cells enriched on the filter were incubated with 5-ALA and Hoechst 33342 as positive markers for CTCs. Images of the whole filter surface were obtained using a fluorescence microscope. No 5-ALA positive cells were detected in healthy blood specimens. The Fs-ALA method was capable of detecting not only EpCAM-positive, but also EpCAM-negative tumor cells. In the Fs-ALA method, one or more CTCs were detected in samples from 13 of 18 (72.2%) colorectal cancer patients. The Fs-ALA method had a significantly higher CTC detection rate than CellSearch™ in colorectal cancer patients (P <0.05), and only the former was capable of identifying live cells. This method is highly efficient for detecting CTC populations having undergone phenotypic changes, such as epithelial-mesenchymal transition.

Circulating tumor cell (CTC) count and epithelial growth factor receptor expression on CTCs as biomarkers for cetuximab efficacy in advanced colorectal cancer.

BACKGROUND/AIM: The purpose of this study was to establish whether CTC count and epidermal growth factor receptor (EGFR) expression in CTCs predicted outcome in patients with advance colorectal cancer (ACC) receiving cetuximab as third-line treatment. PATIENTS AND METHODS: Between October 2008 and March 2011, 63 patients with KRAS wild-type ACC were treated with cetuximab-containing chemotherapy at the Cancer Institute Hospital. We measured the CTC count and EGFR expression on CTCs using the CellSearch System (Veridex LLC, NJ, USA). RESULTS: Nineteen patients (30%) with a high number of CTCs had a significantly lower overall survival compared with 44 patients with a low number of CTCs. No significant difference was observed in progression-free survival between the two groups. Out of the 33 patients positive for CTCs (one or more CTC), seven patients (21%) were positive for EGFR expression. No statistically significant difference was observed in clinical outcome between EGFR-positive and EGFR-negative patients. CONCLUSION: A high CTC count predicted reduced overall survival in patients with ACC treated with cetuximab-combination chemotherapy as third-line treatment. These results suggest that the assessment of CTCs might provide with important prognostic information for such patients.

Retroviral integration site analysis and the fate of transduced clones in an MDR1 gene therapy protocol targeting metastatic breast cancer.

A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.

Pilot study of MDR1 gene transfer into hematopoietic stem cells and chemoprotection in metastatic breast cancer patients.

A major problem in high-dose chemotherapy with autologous hematopoietic stem cell transplantation is insufficient function of reconstituted bone marrow that limits the efficacy of post-transplantation chemotherapy. Because transduction of hematopoietic stem cells with the multidrug resistance 1 (MDR1) gene might circumvent this problem, we conducted a pilot study of MDR1 gene therapy against metastatic breast cancer. Peripheral blood stem cells were harvested, and one-third of the cells were transduced with MDR1 retrovirus. After the reconstitution of bone marrow function, the patients received high-dose chemotherapy with transplantation of both MDR1-transduced and unprocessed peripheral blood stem cells. The patients then received docetaxel chemotherapy. Two patients received transplantation of the MDR1-transduced cells in 2001. Peripheral blood MDR1-transduced leukocytes were 3-5% of the total cells after transplantation, but decreased gradually. During docetaxel chemotherapy, an increase in the rate of MDR1-transduced leukocytes (up to 10%) was observed. Comparison of docetaxel-induced granulocytopenia in the two patients suggested a bone marrow-protective effect of the MDR1-transduced cells. No serious side-effect was observed, and the patients were in complete remission for more than 3 years. The MDR1-transduced cells gradually decreased and disappeared almost entirely by the end of 2004. Results of linear amplification-mediated polymerase chain reaction of the MDR1-transduced leukocytes suggested no sign of abnormal amplification of the transduced cells. A third patient received transplantation of the MDR1-transduced cells in 2004. These results suggest the feasibility of our MDR1 gene therapy against metastatic breast cancer, and follow-up is ongoing.