Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents.

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.

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.

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 approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti­EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus.

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.

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.

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.