Toshiyuki Tanahashi

Is conversion therapy possible in stage IV gastric cancer: the proposal of new biological categories of classification

Conversion therapy for gastric cancer (GC) has been the subject of much recent attention. It is defined as a surgical treatment aiming at an R0 resection after chemotherapy for tumors that were originally unresectable or marginally resectable for technical and/or oncological reasons. However, the indications for resection remain to be clarified. In the present review, we focus on the biology and heterogeneous characteristics of stage IV GC and propose new categories of classification. Stage IV GC patients can be divided based on the absence (categories 1 and 2) or presence (categories 3 and 4) of macroscopically detectable peritoneal dissemination, which has a different biological outcome compared to hematological metastasis. Category 1 is defined oncologically as stage IV but the metastasis is technically resectable. Category 2 includes a marginally resectable metastasis or patients for whom the operation would not necessarily be the best choice. Category 3 includes a potentially unresectable metastasis of peritoneal dissemination that is only macroscopically detectable. Category 4 includes noncurable metastasis with peritoneal and other organ metastasis. The indications for conversion therapy might include the patients from category 2, some patients from category 3 and a very small number of patients from category 4. The longer survival can be expected for patients corresponding to categories 1, 2 and, to a lesser extent, 3, while the treatment of other patients focuses on “care.” The provision of conversion therapy for stage IV GC patients might be one of the main roles of surgical oncologists in the near future.

Laparoscopic technique and safety experience with barbed suture closure for pelvic cavity after abdominoperineal resection.

BackgroundBetween April 2005 and December 2012, we performed laparoscopic colorectal resection with regional lymph node dissection on 273 cases of colorectal cancer patients. However, Laparoscopic rectal cancer surgery requires a high degree of skill. Any surgeon who is going to embark on these difficult resections should have at a minimum laparoscopic suturing skills in order to be able to close the peritoneal defect.MethodsIn laparoscopic surgery for rectal cancer, the intracorporeal suture technique required to close the pelvic cavity is very difficult. Barbed sutures have recently been proposed to facilitate laparoscopic suturing. Two patients with rectal cancer who underwent laparoscopic abdominoperineal resection (APR) with intracorporeal closure of the pelvic cavity from September to October 2012 were enrolled in this study.ResultsWe present our initial experience of two consecutive cases of intracorporeal closure of the pelvic cavity by totally laparoscopic APR. After clinical follow-up, the two patients have no complaints and have shown no signs of recurrence.ConclusionsWe hypothesized that barbed sutures could potentially improve the efficiency of intracorporeal closure of the pelvic cavity after laparoscopic APR. Further, we expect that use of the V-Loc™ will reduce intra-operative stress on the endoscopic surgeon.

Extracellular signal-regulated kinase and Akt activation play a critical role in the process of hepatocyte growth factor-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) has recently been studied to elucidate mechanisms of the liver metastatic process. We investigated EMT in the process of liver metastasis and the effects of chemotherapy on EMT cells as therapeutic strategy for colorectal liver metastasis. We used the CT26 murine colorectal carcinoma cell line to create an in vivo mouse liver metastasis model. Liver tumors were stained immuno-histochemically. Expression of proteins associated with TGF-β/Smad and hepatocyte growth factor (HGF)/c-Met pathways were investigated by western blotting. Cells with c-Met mRNA knockdown by siRNA techniques showed clearly reduced liver metastases compared with regular cells at 21 days. TGF-β and HGF induced EMT expression, but signal transduction was quite different. TGF-β induced ERK, but not Akt phosphory-lation. HGF mediated both ERK and Akt phosphorylation. Akt inhibitor blocked Akt phosphorylation but did not affect TGF-β-induced activation of ERK, Snail and Slug. U-0126 did not reduce Snail activity by TGF-β at a concentration to block ERK phosphorylation. However, Akt inhibitor and U-0126 completely inhibited HGF-induced Slug activation. 5-FU mediated cell death in the EMT process induced by TGF-β more effectively than HGF. ERK/Akt signaling, but not the Smad pathway, may be one of the main processes in HGF-induced EMT, despite the Smad pathway, but not ERK/Akt, being critical for TGF-β-induced EMT. The MAPK/Akt pathway is indispensable in HGF/c-Met signaling. The ERK/Akt pathway particularly may be critical in the HGF-induced EMT process. However, long-term use of chemotherapeutic agents may induce drug resistance and distant metastases through EMT-related signaling pathway activation.

Decreased FANCJ caused by 5FU contributes to the increased sensitivity to oxaliplatin in gastric cancer cells

BackgroundOxaliplatin is effective against many types of cancer, and the combination of 5-fluorouracil (5FU) and oxaliplatin is synergistically effective against gastric cancer, as well as colon cancer. The FANCJ protein is one of the Fanconi anemia (FA) gene products, and its interaction with the tumor suppressor BRCA1 is required for DNA double-strand break (DSB) repair. FANCJ also functions in interstrand crosslinks (ICLs) repair by linking to mismatch repair protein complex MLH1-PMS2 (MutLα). While oxaliplatin causes ICLs, 5FU is considered to cause DSBs. Therefore, we investigated the importance of FANCJ in the synergistic effects of oxaliplatin and 5FU in MKN45 gastric cancer cells and the derived 5FU-resistant cell line, MKN45/F2R.MethodsMKN1, TMK1, MKN45, and MKN45/F2R (5FU-resistant) gastric cancer cells were treated with 5FU and/or oxaliplatin. The signaling pathway was evaluated by a western blotting analysis and reverse transcription polymerase chain reaction (RT-PCR). Drug resistance was evaluated by the 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay.ResultsIn MKN45 cells, the combination of 5FU and oxaliplatin had synergistic effects. DSBs appeared when the cells were treated with 5FU. FANCJ was down-regulated, and BRCA1 was induced in a dose- and time-dependent manner. MKN45 cells showed increased sensitivity to oxaliplatin when FANCJ was knocked down by short interfering (si) RNA. However, these findings were not observed in MKN45/F2R 5FU-resistant cells.ConclusionThese results strongly suggest that the decrease in FANCJ caused by 5FU treatment leads to an increase in sensitivity to oxaliplatin, thus indicating that the FANCJ protein plays an important role in the synergism of the combination of 5FU and oxaliplatin.

Actionable gene-based classification toward precision medicine in gastric cancer

BackgroundIntertumoral heterogeneity represents a significant hurdle to identifying optimized targeted therapies in gastric cancer (GC). To realize precision medicine for GC patients, an actionable gene alteration-based molecular classification that directly associates GCs with targeted therapies is needed.MethodsA total of 207 Japanese patients with GC were included in this study. Formalin-fixed, paraffin-embedded (FFPE) tumor tissues were obtained from surgical or biopsy specimens and were subjected to DNA extraction. We generated comprehensive genomic profiling data using a 435-gene panel including 69 actionable genes paired with US Food and Drug Administration-approved targeted therapies, and the evaluation of Epstein-Barr virus (EBV) infection and microsatellite instability (MSI) status.ResultsComprehensive genomic sequencing detected at least one alteration of 435 cancer-related genes in 194 GCs (93.7%) and of 69 actionable genes in 141 GCs (68.1%). We classified the 207 GCs into four The Cancer Genome Atlas (TCGA) subtypes using the genomic profiling data; EBV (N = 9), MSI (N = 17), chromosomal instability (N = 119), and genomically stable subtype (N = 62). Actionable gene alterations were not specific and were widely observed throughout all TCGA subtypes. To discover a novel classification which more precisely selects candidates for targeted therapies, 207 GCs were classified using hypermutated phenotype and the mutation profile of 69 actionable genes. We identified a hypermutated group (N = 32), while the others (N = 175) were sub-divided into six clusters including five with actionable gene alterations: ERBB2 (N = 25), CDKN2A, and CDKN2B (N = 10), KRAS (N = 10), BRCA2 (N = 9), and ATM cluster (N = 12). The clinical utility of this classification was demonstrated by a case of unresectable GC with a remarkable response to anti-HER2 therapy in the ERBB2 cluster.ConclusionsThis actionable gene-based classification creates a framework for further studies for realizing precision medicine in GC.

Combination therapy with zoledronic acid and cetuximab effectively suppresses growth of colorectal cancer cells regardless of KRAS status

Targeted molecular therapy is an effective anticancer strategy. Anti‐EGFR monoclonal antibodies such as cetuximab (CTX) have been approved for the treatment of various malignancies, including colorectal cancer (CRC) with wild‐type KRAS. However, their efficacy in patients with KRAS mutations has not been established. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. CRC cell lines SW48 (wild‐type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX and a combination of both drugs. Cytotoxicity was measured using the MTT assay. Changes in the levels of intracellular signaling proteins were evaluated using western blot analysis. Finally, we evaluated the efficacy of the combination treatment in an in vivo xenograft model. We observed that ZOL apparently inhibited growth in both cell lines, whereas CTX showed little effect. ZOL also increased the levels of unprenylated RAS. Combined ZOL and CTX treatment was synergistic in both cell lines and was associated with inhibition of the RAS‐MAPK and AKT‐mTOR signaling pathways. Furthermore, the combination treatment was more effective in suppressing the growth of xenografts derived from both SW48 and LS174T cells; this effect was associated with increased apoptosis. These results demonstrate that ZOL inhibits the growth of colon cancer cells regardless of KRAS status, and combination therapy using ZOL and CTX enhances this growth suppression. These findings suggest a novel strategy for the treatment of CRC independent of KRAS mutational status.

Phase II trial of neoadjuvant chemotherapy with docetaxel, nedaplatin, and S1 for advanced esophageal squamous cell carcinoma

Although standard chemotherapy for esophageal cancer patients is fluorouracil and cisplatin, the prognosis is still unsatisfactory. A new therapeutic regimen combining docetaxel, cisplatin, and 5‐fluorouracil was recently developed to improve both local and distant tumor control. We developed a new regimen of docetaxel, nedaplatin, and S1 (DGS) and previously reported the recommended dose in a phase I dose‐escalation study. We then undertook a phase II study of DGS for advanced esophageal squamous cell carcinoma. Patients with clinical stage IB/II/III disease were eligible. Patients received two courses of chemotherapy: docetaxel 35 mg/m2 with nedaplatin 40 mg/m2 on day 8, 80 mg/m2 S1 on days 1–14, and 2 weeks off. After completion of chemotherapy, patients underwent esophagectomy. The primary endpoint was the completion rate of protocol treatment (completion of two courses of preoperative chemotherapy and R0 surgery [no residual tumor]). We enrolled 32 patients. The completion rate of protocol treatment was 96.9%. During chemotherapy, the most common grade 3 or 4 toxicity was neutropenia (25.0%). No treatment‐related deaths were observed, and the incidence of operative morbidity was tolerable. The overall response rate after chemotherapy was 83.3%. This DGS regimen was well tolerated and highly active. This trial is registered with the University Hospital Medical Information Network (UMIN ID: 000014626).

Novel therapy for locally advanced triple-negative breast cancer

To evaluate a novel therapy for triple-negative breast cancer (TNBC), the biological responses to vitamin K3 (VK3) should be considered with the understanding of the features of breast cancer. In human breast cancer cell lines, the effects of VK3 on cell growth inhibition and the cellular signaling pathway were determined by MTT assay and western blotting. In the in vivo study, a subcutaneous tumor model of breast cancer was created, VK3 was injected into the subcutaneous tumors, and tumor size was measured. The IC50 of VK3 for breast cancer cells was calculated to be 11.3–25.1 μM. VK3 induced phosphorylation of whole tyrosine and epidermal growth factor receptor. VK3 mediated phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) for 30 min. ERK but not JNK phosphorylation was maintained for at least 6 h. In contrast, another antioxidant agent, catalase, showed no effect on either ERK phosphorylation or growth inhibition. On built-up tumors under the skin of mice, local treatment with VK3 was effective in a time- and dose-dependent manner, and the experiments for total tumor volume also showed a dose-dependent effect of VK3. The expression of phosphorylated ERK was clearly detected at 10.9 times the control in tumor tissue, whereas ethanol itself showed no effect. In conclusion, ERK plays a critical role in VK3-induced growth inhibition, and it will be the focus of next steps in the development of molecular therapy for TNBC.

5FU resistance caused by reduced fluoro‑deoxyuridine monophosphate and its reversal using deoxyuridine

The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine monophosphate (FdUMP). MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU and fluoro-deoxyuridine (FdU) in combination with deoxyuridine (dU) and thymidine (dT). Subsequently, the levels of ternary complex were determined by western blotting and the cell viability was calculated using an MTT assay. MKN45/F2R cells exhibited 5FU resistance (56.2-fold relative to MKN45 cells), and demonstrated decreased orotate phosphoribosyltransferase (OPRT) and increased TS levels, requiring a higher concentration of 5FU to induce ternary complex formation than MKN45 cells. Following transfection of small interfering RNA against OPRT, MKN45 exhibited increased resistance to 5FU and decreased ternary complex formation subsequent to treatment with 5FU, indicating that decreased OPRT led to increased 5FU resistance. However, MKN45/F2R also exhibited resistance to FdU, which can be converted to FdUMP without OPRT, and there was decreased ternary complex formation after treatment with FdU, indicating that the 5FU-resistant cells had the ability to decrease intracellular FdUMP. The addition of dU and thymidine dT to 5FU promoted the formation of ternary complexes and reversed 5FU resistance in MKN45/F2R cells, although dT inhibited the efficacy of raltitrexed (another TS inhibitor). These results suggested that 5FU-resistant cells had the ability to reduce intracellular FdUMP irrespective of decreased OPRT, which led to resistance to 5FU. This resistance was then inhibited by treatment with dT or dU.

Safety and feasibility of laparoscopic intersphincteric resection for a lower rectal tumor

The aim of the present study was to evaluate the short-term surgical outcomes of laparoscopic intersphincteric resection (ISR) for a lower rectal tumor in comparison with a case-control series of patients undergoing open ISR. Quality of life factors and anal function were also evaluated. Between July 2008 and April 2013, 103 patients with lower rectal cancer underwent laparoscopic surgery at the Surgical Oncology Department of Gifu University School of Medicine. A total of 25 patients with lower rectal cancer underwent ISR, and 19/25 patients who underwent laparoscopic ISR were compared with the control group of 6 patients who underwent open ISR. The technical feasibility and safety of ISR, and the short- and long-term outcomes following laparoscopic ISR were evaluated. Additional data associated with fecal incontinence conditions of the postoperative patients were evaluated using the Modified Fecal Incontinence Quality of Life scale. There was no recorded perioperative mortality, three complications were observed to occur in three patients and the morbidity rate was 15.8%. The postoperative complications detected included bleeding in one patient and ileus in two patients of the laparoscopic ISR group. The rate of severe complications of grade ≥3a was 15.8% and that of grade ≥3b was 5.3%. In the matched case-control study, blood loss was significantly lower in the laparoscopic ISR group. The median postoperative hospital stay was 14.1 days in the laparoscopic ISR group, which was significantly shorter compared with in the open ISR group (18.7 days). Cancer recurrence was detected in one (5%) patient in a single inguinal lymph node. No significant differences between the ISR and ultra-low anterior resection (ULAR) groups were observed in the maximum resting and maximum squeeze pressures; the outcomes for anal function and fecal incontinence were the same for ISR and ULAR. Thus, laparoscopic ISR for lower rectal cancer may provide a benefit in the early postoperative period without increasing morbidity or mortality.