Long-term results of a phase 2 study of neoadjuvant chemotherapy with molecularly targeted agents for locally advanced rectal cancer.

BACKGROUND: We previously reported the feasibility and efficacy of neoadjuvant chemotherapy without radiotherapy for locally advanced rectal cancer. Here, we report the results of a long-term follow-up study. METHODS: This was a multi-institutional, prospective phase 2 study of patients with locally advanced rectal cancer. Patients received neoadjuvant chemotherapy with molecularly targeted agents before undergoing total mesorectal excision. Six cycles of modified FOLFOX (mFOLFOX6) with bevacizumab were administered to KRAS-mutant patients, and mFOLFOX6 with cetuximab was administered to KRAS-wild-type patients. Here, we report the secondary end points of overall survival, relapse-free survival, and local recurrence rate. RESULTS: Sixty patients were enrolled in this study. R0 resection was achieved in 98.3% (59/60) patients, and pathological complete response was achieved in 16.7% (10/60) patients. After a median follow-up of 5.4 years, the 5 year overall survival was 81.6%, the 5 year relapse-free survival was 71.7%, and the 5 year local recurrence rate was 12.6%. None of the patients who achieved pathological complete response developed recurrence within 5 years. CONCLUSIONS: The use of molecularly targeted agents in the neoadjuvant setting for locally advanced rectal cancer has an acceptable prognosis.

Changes in the sexual function of male patients with rectal cancer over a 2-year period from diagnosis to 24-month follow-up: A prospective, multicenter, cohort study.

BACKGROUND AND OBJECTIVES: This prospective study aimed to identify long-term changes in sexual function of men with rectal cancer from point of diagnosis to 24 months postoperatively. METHODS: Male patients undergoing laparoscopic rectal cancer surgery were prospectively enrolled. International Index of Erectile Function (IIEF) Questionnaire scores were collected at diagnosis; first follow-up; and 6, 12, and 24 months postoperatively. Missing values were managed via multiple imputations using the propensity score method. Paired t tests were applied to examine changes in IIEF scores over time. RESULTS: This study analyzed 115 patients. For erectile function, there were no significant changes in scores from the point of diagnosis to first treatment (9.4 vs. 9.8 as mean scores; p = .227). Scores deteriorated postoperatively and recovered until 12 months post-surgery, but did not improve significantly from 12 months to 24 months post-surgery (8.7 vs. 8.2 as mean scores; p = .440). This pattern of change was observed in all other domains: orgasmic function, sexual desire, orgasmic satisfaction, and overall satisfaction. CONCLUSIONS: Sexual function was not influenced by a rectal cancer diagnosis. Sexual function deteriorated following surgery and recovered until 12 months post-surgery; however, it did not significantly improve from 12 months to 24 months postoperatively.

A Multicenter Phase 2 Study on the Feasibility and Efficacy of Neoadjuvant Chemotherapy Without Radiotherapy for Locally Advanced Rectal Cancer.

BACKGROUND: This prospective multicenter phase 2 study aimed to evaluate the feasibility and efficacy of neoadjuvant chemotherapy (NAC) without radiotherapy for locally advanced rectal cancer (LARC). METHODS: Patients with LARC (cStage II and III) were included in the study. Those with cT4b tumor were excluded. Six cycles of modified FOLFOX6 (mFOLFOX6) plus either bevacizumab or cetuximab, depending on KRAS status, were administered before surgery. The primary end point of the study was the R0 resection rate. The secondary end points were adverse effect, rate of NAC completion, postoperative complications, and pathologic complete response (pCR) rate. RESULTS: The study enrolled 60 patients from eight institutions. For the study, mFOLFOX6 was administered with cetuximab to 40 patients who had wild-type KRAS and with bevacizumab to 20 patients who had KRAS mutations. The completion rate for NAC was 88.4%. Sphincter-preserving surgery was performed for 43 patients and abdominoperineal resection for 17 patients. The median operation time was 335 min, and the median blood loss was 40 g. The R0 resection rate was 98.3%, and the pCR rate was 16.7%. The overall postoperative complication rate (≥grade 2) was 21.7%. The complications included anastomotic leakage (11.6%), surgical-site infection (6.7%), and urinary dysfunction (3.3%). The patients with wild-type KRAS did not differ significantly from those with KRAS mutations in terms of response rate, postoperative complication rate, and pCR rate. CONCLUSION: The findings show that NAC is a feasible and promising treatment option for LARC (This study is registered with UMIN-CTR, UMIN000005654).

Oral and Parenteral Versus Parenteral Antibiotic Prophylaxis in Elective Laparoscopic Colorectal Surgery (JMTO PREV 07-01): A Phase 3, Multicenter, Open-label, Randomized Trial.

OBJECTIVE: To confirm the efficacy of oral and parenteral antibiotic prophylaxis (ABX) in the elective laparoscopic colorectal surgery. BACKGROUND: There is no evidence for the establishment of an optimal ABX regimen for laparoscopic colorectal surgery, which has become an important choice for the colorectal cancer patients. METHODS: The colorectal cancer patients scheduled to undergo laparoscopic surgery were eligible for this multicenter, open-label, randomized trial. They were randomized to receive either oral and parenteral prophylaxis (1 g cefmetazole before and every 3 h during the surgery plus 1 g oral kanamycin and 750 mg metronidazole twice on the day before the surgery; Oral-IV group) or parenteral prophylaxis alone (the same IV regimen; IV group). The primary endpoint was the incidence of surgical site infections (SSIs). Secondary endpoints were the incidence rates of Clostridium difficile colitis, other infections, and postoperative noninfectious complications, as well as the frequency of isolating specific organisms. RESULTS: Between November 2007 and December 2012, 579 patients (289 in the Oral-IV group and 290 in IV group) were evaluated for this study. The incidence of SSIs was 7.26% (21/289) in the Oral-IV group and 12.8% (37/290) in the IV group with an odds ratio of 0.536 (95% CI, 0.305-0.940; P = 0.028). The 2 groups had similar incidence rates of C difficile colitis (1/289 vs 3/290), other infections (6/289 vs 5/290), and postoperative noninfectious complications (11/289 vs 12/290). CONCLUSIONS: Our oral-parenteral ABX regimen significantly reduced the risk of SSIs following elective laparoscopic colorectal surgery.

Differences in surgical site infection between laparoscopic colon and rectal surgeries: sub-analysis of a multicenter randomized controlled trial (Japan-Multinational Trial Organization PREV 07-01).

BACKGROUND: The incidence of surgical site infection (SSI) is reportedly lower in laparoscopic colorectal surgery than in open surgery, but data on the difference in SSI incidence between colon and rectal laparoscopic surgeries are limited. METHODS: The incidence and risk factors for SSI, and the effect of oral antibiotics in colon and rectal laparoscopic surgeries, were investigated as a sub-analysis of the JMTO-PREV-07-01 (a multicenter, randomized, controlled trial of oral/parenteral vs. parenteral antibiotic prophylaxis in elective laparoscopic colorectal surgery). RESULTS: A total of 582 elective laparoscopic colorectal resections, comprising 376 colon surgeries and 206 rectal surgeries, were registered. The incidence of SSI in rectal surgery was significantly higher than in colon surgery (14 vs. 8.2 %, P = 0.041). Although the incidence of incisional SSI was almost identical (7 %) between the surgeries, the incidence of organ/space SSI in rectal surgery was significantly higher than in colon surgery (6.3 vs. 1.1 %, P = 0.0006). The lack of oral antibiotics was significantly associated with the development of SSI in colon surgery. Male sex, stage IV cancer, and abdominoperineal resection were significantly associated with SSI in rectal surgery. The combination of oral and parenteral antibiotics significantly reduced the overall incidence of SSI in colon surgery (relative risk 0.41, 95 % confidence interval 0.19-0.86). CONCLUSION: The incidence of SSI in laparoscopic rectal surgery was higher than in colon surgery because of the higher incidence of organ/space SSI in rectal surgery. The risk factors for SSIs and the effect of oral antibiotics differed between these two procedures.

Suppressed complement activation in human decay accelerating factor transgenic porcine liver cross-circulated with nonhuman primates.

BACKGROUND: We developed an extracorporeal liver perfusion (ECLP) system as a liver-assist device. In this study, we evaluated the safety of the ECLP using human decay accelerating factor (hDAF) transgenic porcine livers in healthy baboons. METHODS: Livers were isolated from five hDAF transgenic pigs and five nontransgenic pigs for the ECLP. Ten cross-circulations between the ECLP and healthy baboons were performed without immunosuppressive agents. Cross-circulation was discontinued in any of the following circumstances: elevated hepatic arterial (>200 mm Hg) or portal (>60 mm Hg) perfusion pressure, massive exudate from the graft liver, mild macroscopic hemolysis, thrombocytopenia, or 24-hr well-conditioned cross-circulation. RESULTS: The cross-circulations with nontransgenic porcine livers were discontinued at 4.4+/-1.2 hr (mean+/-standard deviation) because of high perfusion pressure (n=2) or hemolysis (n=3). Three cross-circulations with hDAF transgenic porcine livers were performed for 24 hr; the other two cross-circulations were discontinued at 13 and 17 hr because of massive exudate and thrombocytopenia, respectively. The duration was 20.4+/-5.1 hr. Deposition of membrane attack complex in the hDAF transgenic porcine liver was less than that in the nontransgenic liver, although immunoglobulin-M deposition was comparable. The porcine livers showed no apparent interlobular bleeding or lobular necrosis. All porcine livers maintained bile production during the cross-circulation. No baboons showed any serious complications after the cross-circulation. CONCLUSION: The hDAF transgenic porcine liver reduced complement activation in xenoperfusion with healthy nonhuman primate blood and led to extended duration of cross-circulation.

Relocation of truncated bid plays an important role in suppression of tumor necrosis factor alpha induced apoptosis in hepatocytes isolated from transgenic mouse.

BACKGROUND/AIMS: Tumor necrosis factor alpha (TNFalpha) induces apoptosis in murine hepatocytes pretreated with Actinomycin D (ActD) in vitro. This study sought to clarify the relationship between hepatic energy status and TNFalpha-induced hepatocyte apoptosis, using mice transgenic for creatine kinase (CK) expression. MATERIALS AND METHODS: Hepatocytes from CK transgenic mice were cultured with or without creatine (Cr). The concentrations of ATP and phosphocreatine (PCr) in hepatocytes were measured by high-pressure liquid chromatography. Sixteen hours after treatment with ActD and TNFalpha, we evaluated cell viability of these hepatocytes. We examined truncated Bid and cytochrome c by immunoblot analysis. RESULTS: Six hours after cell isolation, the concentration of PCr in CK transgenic hepatocytes cultured with Cr increased to 8.23 +/- 0.01 microg/mg protein, while that of hepatocytes cultured without Cr was lower than 0.1 microg/mg protein. In hepatocytes cultured without Cr, ActD and TNFalpha treatment induced massive cell death, while hepatocytes cultured with Cr maintained greater than 80% viability. In CK transgenic hepatocytes cultured with Cr, truncated Bid relocation to mitochondria was highly suppressed, compared to CK transgenic hepatocytes cultured without Cr. CONCLUSION: PCr accumulation may prevent TNFalpha-induced apoptosis in murine hepatocytes by suppression of truncated Bid targeting to mitochondria.

Asialo GM1 positive CD8+ T cells induce skin allograft rejection in the absence of the secondary lymphoid organs.

BACKGROUND: Secondary lymphoid organs are considered to be the only organs in which APCs and naïve T cells interact to initiate adaptive immune responses. Aly/aly mice are autosomal recessive mutants of C57BL/6 mice, and lack lymph nodes and Peyer's patches. In this study, we investigated immune responses to skin allografts in splenectomized aly/aly mice, which lack secondary lymphoid organs completely, and examined the effect of anti-asialo GM1 (AsGM1) antibodies on these responses. METHODS: Skin allografts were transplanted to 1) heterozygous aly/+ mice, which had normal secondary lymphoid organs, 2) splenectomized aly/+ mice, 3) aly/aly mice, and 4) splenectomized aly/aly mice, with and without anti-AsGM1 antibody treatment. Graft survival time and alloreactive antibody production were investigated. RESULTS: Heterozygous aly/+ mice and splenectomized aly/+ mice rejected skin allografts acutely. Aly/aly mice also rejected skin allografts, but at a later time than aly/+ mice. Sixty percent of splenectomized aly/aly mice rejected skin allografts within 120 days. Serial administration of anti-AsGM1 antibodies prevented skin allograft rejection in splenectomized aly/aly mice during the same 120-day period of observation. After ceasation of anti-AsGM1 antibody treatment, skin allografts were rejected; we observed a simultaneous increase in AsGM1 expression on CD8+ T cells. Alloreactive antibodies were detected in both splenectomized aly/aly mice that rejected skin allografts and in splenectomized aly/aly mice that accepted skin allografts after treatment with anti-AsGM1 antibodies. CONCLUSIONS: Cytotoxic and humoral immune responses to skin allografts could be initiated despite the absence of secondary lymphoid organs. AsGM1+ cells were important effector cells in secondary lymphoid organ-independent skin allograft rejection.

Absence of PERV infection in baboons after transgenic porcine liver perfusion.

BACKGROUND: Xenotransplantation offers great promise to supplement the shortage of human organs available for transplant, but cross-species infection is a substantial concern. Porcine endogenous retrovirus (PERV), in particular, is thought to pose a risk as a potential pathogen to humans. We evaluated whether PERV is capable of infecting nonhuman primates in vivo after extracorporeal porcine liver perfusion (ECLP). METHODS: Livers were harvested from six human decay-accelerating factor (h-DAF) transgenic piglets and perfused with fresh baboon blood via the portal vein and the hepatic artery. Six healthy baboons underwent direct cross-circulation with the ECLP for 13 to 24 h without immunosuppression. Peripheral blood and bone marrow of baboons were sampled periodically until the baboons were euthanized for the examination of various organ tissue samples. Genomic DNA was extracted from those samples and tested for PERV and pig-specific centromeric DNA sequences by quantitative PCR. Validation showed that the assay could detect one copy of PERV in a background of 150,000 baboon cells, and it was quantitative over a range from 10 to 10(6) copies of PERV. RESULTS: PERV sequences were detected in a high number (4.4 x 10(3)-1.6 x 10(4)/1 microg) in peripheral leukocyte DNA during the initial phases of ECLP, but they disappeared within 1 week. Bone marrow DNA contained PERV sequences longer than peripheral blood, but PERV signals became negative within 1 month. No PERV DNA relapse was seen over the course of this study. Pig-specific centromeric sequences were also detected in the same manner. At 6 months or 1 year after ECLP, no PERV or pig-specific centromeric sequences were detected in the genomic DNA obtained from the following organs: skin, lymph nodes, spleen, liver, pancreas, kidney, heart, and lung. CONCLUSIONS: ECLP did not result in PERV infection or pig-cell microchimerism in baboons.

Long-term culture of primary human hepatocytes with preservation of proliferative capacity and differentiated functions.

BACKGROUND: The aim of this study was to develop a suitable method for the prolonged culture and maintenance of human hepatocytes with preservation of both proliferative capacity and differentiated functions. MATERIALS AND METHODS: Primary human hepatocytes were isolated from small pieces of liver tissue obtained from 15 patients who underwent hepatic resection. Hepatocytes were cultured in keratinocyte-stimulating factor medium supplemented with 10% human serum, 10 mM nicotinamide, 10 ng/ml epidermal growth factor, 0.5 microg/ml insulin, 10(-7) M dexamethasone, and antibiotics. Hepatic differentiation and function were analyzed by immunocytochemistry, Western blot, ELISA, lidocaine metabolism, and urea synthesis. Ultrastructural analysis of cultured hepatocytes was performed by electron microscopy. RESULTS: Many primary hepatocytes were maintained for more than 56 days. Hepatocytes proliferated during the initial 14 days, and bromodeoxyuridine labeling indices were 15.2, 12.2, and 6.2% at days 5, 10, and 15, respectively. Electron micrographs of the hepatocytes at day 28 demonstrated numerous mitochondria, rough endoplasmic reticulum, large peroxisomes, and glycogen granules. Albumin secretion increased for the first 14 days and then gradually decreased thereafter but was maintained at levels greater than 2 microg/ml/h until day 56. alpha(1)-Antitrypsin, alpha(1)-antichymotrypsin, and ceruloplasmin production was also observed at day 56, while lidocaine metabolism and urea synthesis were maintained for a long time. CONCLUSION: This hepatocyte culture method facilitates the prolonged culture of primary human hepatocytes with preservation of hepatocyte differentiation, function, and proliferative capacity.