Plan quality association between dummy run and individual case review in a prospective multi-institutional clinical trial of postoperative cervical cancer patients treated with intensity-modulated radiotherapy: Japan clinical Oncology Group study (JCOG1402).

BACKGROUND AND PURPOSE: The Japan Clinical Oncology Group (JCOG) 1402 conducted a multicenter clinical trial of postoperative intensity-modulated radiotherapy (IMRT) for high-risk uterine cervical cancer patients. We assess effectiveness of the quality assurance (QA) program in central review through dummy runs (DRs) performed before patient enrollment and post-treatment individual case review (ICR), and clarify the pitfalls in treatment planning. MATERIAL AND METHODS: The ICRs were conducted using the same QA program as the DR for 214 plans. The deviations were compared with those demonstrated in the DRs, and the pitfalls were clarified. Fifteen face-to-face meetings were held with physicians at participating institutions to provide feedback. RESULTS: Two-hundred and eighty-eight deviations and nine violations were detected in the 214 plans. The patterns of the deviations observed in the ICRs were similar to that in the DR. Frequent deviations were observed in clinical target volume (CTV) delineations, 50% in the DRs and 37% in the ICRs, respectively. In the ICRs, approximately 1.4 deviations/violations were observed per plan, which was lower than DR. Nine violations included inaccurate CTV delineation and improper PTV (planning target volume) margin, which had risks in loco-regional failures by inadequate dose coverage. CONCLUSIONS: Our developed QA program commonly used in DR and ICR clarified the pitfalls in treatment plans. Although the frequent deviations in CTV delineations were observed in the ICR, the deviations decreased compared to that in the DR. More specified face-to-face meetings with participating institutions will be necessary to maintain the quality of IMRT in the clinical protocol.

Stage III disease of ovarian, tubal and peritoneal cancers can be accurately diagnosed with pre-operative CT. Japan Clinical Oncology Group Study JCOG0602.

PURPOSE: Computed tomography of the abdomen and pelvis is a useful imaging modality for identifying origin and extent of ovarian cancer before primary debulking surgery. However, the International Federation of Gynecology and Obstetrics staging for ovarian cancer is determined based on surgico-pathological findings. The purpose of this study is to determine whether computed tomography staging can be the surrogate for surgico-pathological International Federation of Gynecology and Obstetrics staging in advanced ovarian cancer undergoing neoadjuvant chemotherapy. METHODS: Computed tomography staging was compared with surgico-pathological International Federation of Gynecology and Obstetrics staging in primary debulking surgery arm patients in a randomized controlled trial comparing primary debulking surgery and neoadjuvant chemotherapy (JCOG0602). The cancer of primary debulking surgery arm was identically diagnosed regarding the origin and extent with the cancer of neoadjuvant chemotherapy arm before accrual, using imaging studies (computed tomography and/or magnetic resonance imaging), cytological examination (ascites, pleural effusion or tumor contents fluid) and tumor marker (CA125 > 200 U/mL and CEA < 20 ng/mL). Institutional computed tomography staging was also compared with computed tomography staging by central review. RESULTS: Among 149 primary debulking surgery arm patients, 147 patients who underwent primary debulking surgery immediately were analyzed. Positive predictive values and sensitivity of computed tomography staging for surgical stage III disease (extra-pelvic peritoneal disease and/or retroperitoneal lymph node metastasis) were 99%. Meanwhile, positive predictive values for the presence of small (≤2 cm) extra-pelvic peritoneal disease were low; <20% in omentum. Accuracy of institutional computed tomography staging was comparable with computed tomography staging by central review. CONCLUSIONS: Preoperative computed tomography staging in each institution can be the surrogate for surgico-pathological diagnosis in stage III disease of ovarian cancer patients undergoing neoadjuvant chemotherapy without diagnostic surgery, but reliability of diagnosis of stage IIIB disease is inadequate.Clinical trial registration: UMIN000000523(UMIN-CTR).

A multi-institutional survey of the quality of life after treatment for uterine cervical cancer: a comparison between radical radiotherapy and surgery in Japan.

This study aimed to research the post-treatment quality of life (QOL) between radiotherapy (RT)- and operation (OP)-treated early cervical cancer survivors, using separate questionnaires for physicians and patients. We administered an observational questionnaire to patients aged 20-70 years old with Stages IB1-IIB cervical cancer who had undergone RT or OP and without recurrence as outpatients for ≥6 months after treatment. We divided 100 registered patients equally into two treatment groups (n = 50 each). The average age was 53 and 44 years in the RT and OP groups, respectively. The RT group included 34 and 66% Stage I and II patients, respectively, whereas the OP group included 66 and 34% Stage I and II patients, respectively. The OP group included 58% of patients with postoperative RT. Combination chemotherapy was performed in 84 and 48% of patients in the RT and OP groups, respectively. On the physicians' questionnaire, we observed significant differences in bone marrow suppression (RT) and leg edema (OP). On the patients' questionnaire, significantly more patients had dysuria and leg edema in the OP group than in the RT group, and severe (Score 4-5) leg edema was significantly higher in the post-operative RT group than in the OP only group. The frequency of sexual intercourse decreased after treatment in both groups. On the patients' questionnaire, there were no significant differences between the two groups regarding sexual activity. These findings are useful to patients and physicians for shared decision-making in treatment choices. The guidance of everyday life and health information including sexual life after treatment is important.

Single-arm confirmatory trial of postoperative concurrent chemoradiotherapy using intensity modulated radiation therapy for patients with high-risk uterine cervical cancer: Japan Clinical Oncology Group study (JCOG1402).

A single-arm multi-center confirmatory trial was started in Japan to confirm the efficacy and safety of post-radical hysterectomy concurrent chemoradiotherapy using intensity-modulated radiation therapy (IMRT-CCRT) for patients with high-risk uterine cervical cancer, for which the current standard treatment is CCRT using three-dimensional conformal radiation therapy (3DCRT-CCRT). This study began in April 2017 and a total of 220 patients will be accrued from 44 institutions within 3.5 years. The primary endpoint is 3-year relapse-free survival. The secondary endpoints are overall survival, loco-regional relapse-free survival, proportion of late lower gastrointestinal adverse events greater than or equal to grade 3, proportion of lower edema limbs, adverse events, and serious adverse events. This trial was registered at the Japan Registry of Clinical Trials as jRCTs031180194 (https://jrct.niph.go.jp/).

Dummy-run for standardizing plan quality of intensity-modulated radiotherapy for postoperative uterine cervical cancer: Japan Clinical Oncology Group study (JCOG1402).

BACKGROUND: The purpose of this study was to assess compliance with treatment planning in a dummy-run for a multicenter clinical trial involving patients with high-risk postoperative uterine cervical cancer using intensity-modulated radiation therapy (IMRT) (JCOG1402 trial). METHODS: For the dummy-run, we prepared a computed tomography dataset comprising two anonymized cases of post-hysterectomy cervical cancer. These were sent to the 47 participating institutions to assess institutional plan quality such as delineations and dose distributions. RESULTS: Central review showed 3 and 4 deviations per treatment plan on average. The deviations related to the nodal and vaginal cuff clinical target volume (CTV) delineation, which accounted for approximately 50% of the total deviations. The CTV vaginal cuff showed considerable differences in delineation compared with the nodal CTV. For the Dice similarity coefficient, case 1 showed a mean ± 1σ of 0.81 ± 0.03 and 0.60 ± 0.09 for the nodal and the CTV vaginal cuff, respectively, while these were 0.81 ± 0.04 and 0.54 ± 0.14, respectively, for case two. Of the 47 institutions, 10 were required to resubmit their treatment plan because the delineations, planning target volume margin, and required dose distributions were not in accordance with the JCOG1402 protocol. CONCLUSIONS: The dummy-run test in postoperative uterine cervical cancer demonstrated substantial deviations in the delineations, particularly for the CTV vaginal cuff. The analysis data could provide helpful information on delineation and planning, allowing standardization of IMRT planning for postoperative uterine cervical cancer. TRIAL REGISTRATION: Japanese Clinical Trial Registry #: UMIN000027017 at https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000030672;language=J.

Computer vision for total laparoscopic hysterectomy.

INTRODUCTION: Laparoscopic surgery is widely performed in various surgical fields, but this technique requires time for surgeons to master. However, at the same time, there are many advantages in visualizing the operative field through a camera. In other words, we can visualize what we cannot see with our own eyes by using augmented reality and computer vision. Therefore, we investigated the possibilities and usefulness of computer vision in total laparoscopic hysterectomy. METHODS: This study was approved by the Mitsui Memorial Hospital ethics committee. Patients who underwent total laparoscopic hysterectomy at Mitsui Memorial Hospital from January 2015 to December 2015 were enrolled. We evaluated 19 cases in which total laparoscopic hysterectomy was performed by the same operator and assistant. We used the Open Source Computer Vision Library for computer vision analysis. The development platform used in this study was a computer operating on Mac OS X 10.11.3. RESULTS: We created panoramic images by matching features with the AKAZE algorithm. Noise reduction methods improved haziness caused by using energy devices. By abstracting the color of the suture string, we succeeded in abstracting the suture string from movies. We could not achieve satisfactory results in detecting ureters, and we expect that creative ideas for ureter detection may arise from collaborations between surgeons and medical engineers. CONCLUSIONS: Although this was a preliminary study, the results suggest the utility of computer vision in assisting laparoscopic surgery.

A multi-institutional observational study on the effects of three-dimensional radiotherapy and weekly 40-mg/m2 cisplatin on postoperative uterine cervical cancer patients with high-risk prognostic factors.

BACKGROUND: The aim of this study was to evaluate the effects of treatment with both three-dimensional radiotherapy (3DRT) and weekly 40-mg/m2 cisplatin on postoperative uterine cervical cancer patients with high-risk prognostic factors. METHODS: We conducted a retrospective multi-institutional chart review of postoperative uterine cervical cancer patients with high-risk prognostic factors who had been treated with both 3DRT and weekly 40-mg/m2 cisplatin from 2007 to 2012. Each participating hospital provided detailed information regarding patient characteristics, treatment outcomes, and treatment complications. RESULTS: The eligible 96 patients were analyzed. The median follow-up period was 61 months. The 3-year relapse-free survival, overall survival (OS), and locoregional relapse-free survival (LRFS) rates were 76%, 90%, and 88%, respectively. In multivariate analysis, the histological finding of either adenocarcinoma or adenosquamous carcinoma was a significant risk factor for both OS and LRFS. The percentage of patients with grade ≥ 3 acute hematologic toxicity, acute lower gastrointestinal toxicity (GIT), and late lower GIT were 45%, 19%, and 17%, respectively. CONCLUSIONS: The outcomes of concurrent chemoradiotherapy (CCRT) using weekly 40-mg/m2 cisplatin are similar to those in the previous studies that used several chemotherapy regimens. However, postoperative CCRT using 3DRT had a high level of late GIT.

Retained Barium in the Appendix Is Difficult to Distinguish from Surgical Remnants following Laparoscopic Surgery.

Surgical materials, such as gauze, can be accidentally left inside of patients following surgery. This iatrogenic complication should be avoided and is often prevented by routine X-ray analysis after surgical abdominal procedures. We report a case of retained barium in the appendix that was difficult to distinguish from surgical remnants. A 41-year-old Japanese female was diagnosed with uterine leiomyoma and underwent laparoscopic myomectomy. The postoperative X-ray test showed a cord-like material in the lower right abdomen that was not captured in the preoperative X-ray test two months prior to the operation. Because of this difference, the area was reexamined laparoscopically. After examination, we concluded that the cord-like material in X-ray tests was in fact retained barium in the appendix. Barium can be retained in the appendix for long periods of time, and retained barium in the appendix can be captured radiographically and can mimic the appearance of surgical remnants, appearing as a cord-like material. The knowledge above combined with detailed interviews before surgery could prevent such confusion during interpretation of X-ray tests after surgery.

Exploration of assistive technology for uniform laparoscopic surgery.

INTRODUCTION: Laparoscopic surgery is less invasive than open surgery and is now common in various medical fields. However, laparoscopic surgery is more difficult than open surgery and often requires additional time for the operator to achieve mastery. Therefore, we investigated the use of assistive technology for uniform laparoscopic surgery. METHODS: We used the OpenCV2 library for augmented reality with an ArUco marker to detect and estimate forceps positioning. We used Sense HAT as the gyro sensor. The development platforms used were Mac OS X 10.11.3 and Raspberry Pi 3, model B. RESULTS: By attaching the ArUco marker to the needle holder, we could draw a line vertically to the marker. When the needle was held, a cube could be imagined, and both the needle and lines could be used to determine the appropriate position. By attaching the gyro sensor to the camera, we could detect its angle of rotation. We obtained stabilized images by rotating the image by the detected degrees; this was possible for any camera position. CONCLUSIONS: Assistive technology allowed us to obtain consecutive converted images in real time and may be readily applicable to clinical practice.

Gynecologists May Underestimate the Amount of Blood Loss during Total Laparoscopic Hysterectomy.

BACKGROUND: We considered the possibility of underestimation of the amount of bleeding during laparoscopic surgery, and we investigated comparing the amount of bleeding between laparoscopic surgery and open surgery by considering the concentration of hemoglobin before and after surgery as indicators. METHODS: The following procedures were included: A, surgery for ovarian tumor; B, myomectomy; and C, hysterectomy either by laparoscopic surgery or open surgery. Patients who underwent the above procedures in between January 1, 2010, and December 31, 2017, were enrolled. We identified 1749 cases (A: 90, B: 105, and C: 325 of open surgery and A: 667, B: 437, and C: 125 of laparoscopic surgery). We considered the sum as an estimation of blood loss during surgery and the change in the value of hemoglobin in laboratory testing one day before and after surgery. RESULTS: During laparoscopic surgery, the measurements of blood loss included the following: A: 59.8 ml; B: 168.6 ml; and C: 206.8 ml. During open surgery, measurements of blood loss included the following: A: 130.7 ml; B: 236.7 ml; and C; 280.9 ml. The reduction of hemoglobin after surgery compared with that before surgery was less in laparoscopic surgery than that in open surgery in A and B; however, this reduction was not significantly different in C. CONCLUSION: Our results suggest that the estimation of the bleeding in A and B was appropriate; however, the estimation might be underestimated in C during laparoscopic surgery.

Prognostic factors and optimal therapy for stages I-II neuroendocrine carcinomas of the uterine cervix: A multi-center retrospective study.

PURPOSE: We aimed to determine appropriate treatment guidelines for patients with stages I-II high-grade neuroendocrine carcinomas (HGNEC) of the uterine cervix in a multicenter retrospective study. PATIENTS AND METHODS: We reviewed the clinicopathological features and prognoses of 93 patients with HGNEC of International Federation of Gynecology and Obstetrics (FIGO) stages I and II. All patients were diagnosed with HGNEC by central pathological review. RESULTS: The median overall survival (OS) and disease-free survival (DFS) were 111.3months and 47.4months, respectively. Eighty-eight patients underwent radical surgery, and five had definitive radiotherapy. The hazard ratio (HR) for death after definitive radiotherapy to death after radical surgery was 4.74 (95% confidence interval [CI], 1.01-15.90). Of the surgery group, 18 received neoadjuvant chemotherapy. Pathological prognostic factors and optimal adjuvant therapies were evaluated for the 70 patients. Forty-one patients received adjuvant chemotherapy with etoposide-platinum (EP) or irinotecan-platinum (CPT-P). Multivariate analyses identified the invasion of lymphovascular spaces as a significant prognostic factor for both OS and DFS. Pelvic lymph node metastasis was also a prognostic factor for DFS. Adjuvant chemotherapy with an EP or CPT-P regimen appeared to improve DFS (HR=0.27, 95% CI, 0.10-0.69). A trend toward improved OS was also observed, but was not statistically significant (HR=0.39, 95% CI, 0.15-1.01). CONCLUSION: Radical surgery followed by adjuvant chemotherapy with an EP or CPT-P regimen was optimal treatment for stages I and II HGNEC of the uterine cervix.

Radical hysterectomy with or without para-aortic lymphadenectomy for patients with stage IB2, IIA2, and IIB cervical cancer: outcomes for a series of 308 patients.

BACKGROUND: Although many studies have already shown that lymph node metastasis is one of the major prognostic factors for cervical cancer, the therapeutic significance of para-aortic lymphadenectomy for the surgical treatment of cervical cancer remains controversial. METHODS: A total of 308 patients diagnosed with stage IB2, IIA2, or IIB cervical cancer and treated with radical hysterectomy were retrospectively investigated to assess the incidence of para-aortic lymph node metastasis and the clinicopathological factors linked to cervical cancer prognosis. RESULTS: Para-aortic lymph node metastases were pathologically confirmed in 13 of the 136 patients (9.6 %) who underwent para-aortic lymphadenectomy. The incidence of para-aortic lymph node metastasis was significantly higher in the patients who had common iliac lymph node metastases (odds ratio 31.5, p < 0.001) according to logistic regression analysis. Common iliac lymph node metastasis was related to risk of recurrence (hazard ratio 2.43, p = 0.003) and death (hazard ratio 2.62, p = 0.007) in Cox regression analysis. Kaplan-Meier analysis and Cox regression analysis showed that para-aortic lymphadenectomy did not have a positive impact on survival in 308 patients or 140 pN1 patients, but para-aortic lymphadenectomy was related to better overall survival with a marginal trend toward significance (p = 0.053) in 30 patients with common iliac lymph node metastasis. CONCLUSIONS: Indication for para-aortic lymphadenectomy in the surgical treatment of stage IB2, IIA2, or IIB cervical cancer needs to be individualized. Patients with common iliac lymph node metastasis are possible candidates, and a prospective study is needed to clarify this issue.

What is an appropriate second-line regimen for recurrent endometrial cancer? Ancillary analysis of the SGSG012/GOTIC004/Intergroup study.

PURPOSE: We previously reported that the concept of "platinum sensitivity" could be applied to recurrent endometrial cancer. We conducted an ancillary analysis to determine an appropriate second-line regimen for patients who received a platinum agent as first-line chemotherapy. METHODS: We extracted and reanalyzed data of patients treated with doxorubicin and cisplatin (AP), paclitaxel and carboplatin (TC), or docetaxel and carboplatin (DC) as first- and second-line chemotherapies from the SGSG012/GOTIC004/Intergroup study. RESULTS: We identified 216 patients: 38 received AP as first-line chemotherapy, of which 36 received TC or DC (Tax-C) as second-line chemotherapy; and 178 received Tax-C as first-line chemotherapy, of which 51 received AP and 127 received Tax-C as second-line chemotherapy. Median progression-free survival (PFS) and overall survival (OS) after second-line chemotherapy decreased in the order of Tax-C followed by Tax-C (10 and 48 months, respectively), AP followed by Tax-C (9 and 23 months, respectively), and Tax-C followed by AP (3 and 12 months, respectively). Median PFS and OS after second-line chemotherapy for platinum-resistant patients receiving Tax-C as first-line chemotherapy were longer in Tax-C than in AP (7 and 23 vs. 3 and 10 months, respectively) as second-line chemotherapy [hazard ratio (HR) 3.255, 95 % confidence interval (CI) 1.908-5.555, p < 0.0001; HR 3.179, 95 % CI 1.835-5.507, p < 0.0001, respectively]. Median PFS and OS after second-line chemotherapy for platinum-sensitive patients receiving Tax-C as first-line chemotherapy were almost equivalent to those receiving Tax-C or AP as second-line chemotherapy. CONCLUSIONS: For platinum-resistant recurrent endometrial cancer patients, Tax-C may be preferred over AP as second-line chemotherapy.

Combination therapy using imatinib and vatalanib improves the therapeutic efficiency of paclitaxel towards a mouse melanoma tumor.

Melanomas respond poorly to chemotherapy. In this study, we investigated the sensitization of B16 mouse melanoma tumors to paclitaxel by a combination of two tyrosine kinase inhibitors: vatalanib, targeting vascular endothelial growth factor receptors, and imatinib, an inhibitor targeting for example, Abl/BCR-ABL, the platelet-derived growth factor receptor, and stem cell factor receptor c-Kit. C57Bl6/J mice carrying B16/PDGF-BB mouse melanoma tumors were treated daily with vatalanib (25 mg/kg), imatinib (100 mg/kg), or a combination of these drugs. Paclitaxel was given subcutaneously twice during the study. The effects of the drugs on tumor cell proliferation in vitro were determined by counting cells. B16/PDGF-BB mouse melanoma tumors were not sensitive to paclitaxel at doses of either 5 or 20 mg/kg. However, the tumor growth was significantly reduced by 58%, in response to paclitaxel (5 mg/kg) when administered with daily doses of both vatalanib and imatinib. Paclitaxel only inhibited the in-vitro growth of B16/PDGF-BB tumor cells when given in combination with imatinib. Imatinib presumably targets c-Kit, as the cells do not express platelet-derived growth factor receptor and as another c-Abl inhibitor was without effect. This was supported by data from three c-Kit-expressing human melanoma cell lines showing varying sensitization to paclitaxel by the kinase inhibitors. In addition, small interfering RNA knockdown of c-Kit sensitized the cells to paclitaxel. These data show that combination of two tyrosine kinase inhibitors, imatinib and vatalanib, increases the effects of paclitaxel on B16/PDGF-BB tumors, thus suggesting a novel strategy for the treatment of melanomas expressing c-Kit.

Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers.

Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q10) and ubiquinone-10 (oxidized coenzyme Q10) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q10 (CoQ10) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ10 ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.

Combined anti-angiogenic therapy targeting PDGF and VEGF receptors lowers the interstitial fluid pressure in a murine experimental carcinoma.

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling.

The aim of our study was to further explore the use of anti-angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet-derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF-BB (B16/PDGF-BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size-matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF-BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF-BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of alpha-smooth muscle actin and PDGFRbeta-positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF-BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.

Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector.

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.

Soluble FLT-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer.

Vascular endothelial growth factor (VEGF), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. To investigate the effects of stable expression of the soluble form of Flt-1 VEGF receptor (sFlt-1), a known endogenous inhibitor of VEGF, on the malignant ascites tumors, we cotransduced RMG-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sFlt-1 cDNA and Neo gene or Neo gene alone and isolated both the sFlt-1-expressing clone and the Neo-expressing clone. In vitro growth characteristics were essentially the same. As expected, conditioned medium collected from the sFlt-1-expressing cells significantly inhibited the human umbilical vein endothelial cell proliferation in the presence of recombinant VEGF. Expression of sFlt-1 significantly suppressed RMG-1 cell-induced angiogenesis in vivo in the mouse dorsal air sac assay model. We then inoculated sFlt-1- or Neo alone-expressing cells i.p. into female BALB/c nude mice. The average volume of ascites fluid, number of leaked RBCs, and number of cancer cells were significantly lower in mice injected with sFlt-1-expressing cells than in the controls. Survival time was significantly prolonged in mice injected with sFlt-1-expressing cells. These results suggest that inhibition of VEGF activity by sFlt-1 expression may provide a means to control carcinomatous ascites and angiogenesis of malignant ascites tumors.