Long-term follow-up of the randomized trial of mesorectal excision with or without lateral lymph node dissection in rectal cancer (JCOG0212).

BACKGROUND: Japan Clinical Oncology Group (JCOG) 0212 (ClinicalTrials.gov NCT00190541) was a non-inferiority phase III trial of patients with clinical stage II-III rectal cancer without lateral pelvic lymph node enlargement. The trial compared mesorectal excision (ME) with ME and lateral lymph node dissection (LLND), with a primary endpoint of recurrence-free survival (RFS). The planned primary analysis at 5 years failed to confirm the non-inferiority of ME alone compared with ME and LLND. The present study aimed to compare ME alone and ME with LLND using long-term follow-up data from JCOG0212. METHODS: Patients with clinical stage II-III rectal cancer below the peritoneal reflection and no lateral pelvic lymph node enlargement were included in this study. After surgeons confirmed R0 resection by ME, patients were randomized to receive ME alone or ME with LLND. The primary endpoint was RFS. RESULTS: A total of 701 patients from 33 institutions were assigned to ME with LLND (351) or ME alone (350) between June 2003 and August 2010. The 7-year RFS rate was 71.1 per cent for ME with LLND and 70·7 per cent for ME alone (hazard ratio (HR) 1·09, 95 per cent c.i. 0·84 to 1·42; non-inferiority P = 0·064). Subgroup analysis showed improved RFS among patients with clinical stage III disease who underwent ME with LLND compared with ME alone (HR 1·49, 1·02 to 2·17). CONCLUSION: Long-term follow-up data did not support the non-inferiority of ME alone compared with ME and LLND. ME with LLND is recommended for patients with clinical stage III disease, whereas LLND could be omitted in those with clinical stage II tumours.

ANTECEDENTES: El JCOG0212 (ClinicalTrials.gov: NCT00190541) fue un ensayo fase III de no inferioridad en pacientes con cáncer de recto en estadio clínico II/III sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. El ensayo comparó la escisión del mesorrecto (mesorectal excision, ME) con la ME con disección de los ganglios linfáticos laterales (lateral lymph node dissection, LLND), siendo el criterio de valoración principal la supervivencia libre de recidiva (recurrence free survival, RFS). El análisis primario planificado a los 5 años de seguimiento no pudo confirmar la no inferioridad de la ME frente a la ME con LLND. Este estudio tuvo como objetivo comparar la ME como procedimiento único y la ME con LLND utilizando datos de seguimiento a largo plazo del ensayo JCOG0212. MÉTODOS: En este estudio se incluyeron pacientes con cáncer de recto en estadio clínico II/III por debajo de la reflexión peritoneal sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. Después de que los cirujanos confirmaran la resección R0 mediante la ME, los pacientes fueron asignados al azar al brazo de ME sola o al brazo de ME con LLND. El criterio de valoración principal fue la supervivencia libre de recidiva (RFS). RESULTADOS: Un total de 701 pacientes de 33 instituciones fueron asignados al azar para ser tratados mediante una ME con LLND (n = 351) o EM sola (n = 350) entre junio de 2003 y agosto de 2010. Las tasas de RFS a 7 años fueron del 71,1% para ME con LLND y 70,7 % para ME sola (cociente de riesgos instantáneos, hazard ratio, HR: 1,09 (i.c. del 95% 0,84-1,42), no inferioridad P = 0,064)). El análisis de subgrupos mostró una mejor RFS entre los pacientes en estadio clínico III que se sometieron a ME con LLND en comparación con ME sola (HR: 1,49 (i.c. del 95%: 1,02-2,17)). CONCLUSIÓN: Los datos de seguimiento a largo plazo no justificaron la no inferioridad de la ME en comparación con la ME con LLND. Se recomienda la ME con LLND para pacientes en estadio clínico III, mientras que LLND podría omitirse para pacientes en estadio clínico II.

The significance of abdominal para-aortic lymph node metastasis in patients with lower thoracic esophageal cancer.

Para-aortic lymph node (PALN) recurrence is often seen in patients with lower thoracic esophageal cancer treated by esophagectomy with extended lymph node dissection. However, the clinicopathological characteristics of patients with PALN metastasis and the significance of PALN dissection are unknown. A total of 283 patients with lower thoracic esophageal cancer underwent esophagectomy with lymphadenectomy at our hospital between April 1984 and March 2007. Among these 283 patients, 60 patients were enrolled in this retrospective study according to following criteria: (i) clinical T2 to T4 tumor, (ii) no clinical PALN metastasis, and (iii) received PALN dissection. PALN dissection was indicated by a tumor depth of at least T2 and no severe complications. The clinicopathological data, recurrence pattern, and overall survival were compared between patients with PALN and without PALN metastasis. The mean length of surgery was 587 min and the mean blood loss was 1383 mL. The morbidity was 33.3% and mortality was 5% in this series. Sixteen patients (26.7%) had PALN metastasis; these showed significantly more lymph node metastases (15.8 ± 13.2 vs. 3.0 ± 3.2, P < 0.0001) and significantly worse survival rates (53.3% vs. 79.9% at 1 year, 6.7% vs. 62.0% at 3 years, P < 0.0001) than patients without PALN metastasis. The incidence of lymph node recurrence (P < 0.0001) and hematogenous recurrence (P= 0.0487) was also higher in patients with PALN metastasis than in patients without PALN metastasis. Among the 16 patients with PALN metastasis, a univariate analysis revealed total number of metastatic nodes < 8 (P= 0.0325) to be a significant prognostic factor. A multivariate logistic regression analysis of the regional lymph nodes identified the invasion of the lower mediastinal nodes (hazard ratio = 6.120) and retroperitoneal nodes (hazard ratio = 15.167) to be significantly correlated with PALN metastasis. PALN metastasis is suggested to be related to the systemic spread of lymphatic metastasis even in lower thoracic esophageal cancer. PALN dissection for pathological PALN(+) patients should not be performed. It remains to be determined in future prospective studies whether patients without pathological PALN metastasis, but showing PALN micrometastasis, could achieve improved survival with PALN dissection.

Prevention of gastroduodenal content reflux and delayed gastric emptying after esophagectomy: gastric tube reconstruction with duodenal diversion plus Roux-en-Y anastomosis.

Reflux of gastroduodenal contents and delayed gastric emptying are the most common and serious problems after esophagectomy with gastric reconstruction. However, attempts to reduce the above symptoms, surgically as well as non-surgically, had no or limited effect. To address this issue, we performed retrosternal gastric reconstruction with duodenal diversion plus Roux-en-Y anastomosis (RY) in eight patients with thoracic esophageal cancer and compared the outcomes with control patients who underwent standard reconstruction. The procedure is simple, safe, and not associated with any postoperative complications. The pancreatic amylase concentrations in the gastric juice samples on postoperative day 2 were slightly lower in the non-RY group than in the RY group (1884 ± 2152 vs. 25,790 ± 23,542IU/mL, respectively, P= 0.07). Postoperative endoscopic examination showed neither reflux esophagitis nor residual gastric content in the RY group. Quality of life assessed by the Dysfunction After Upper Gastrointestinal Surgery-32 questionnaire postoperatively was significantly better in the RY group than in the non-RY group for 'decreased physical activity,''symptoms of reflux,''nausea and vomiting,' and 'pain.' The results of this pilot study suggest that gastric reconstruction with duodenal diversion plus RY seems effective in improving both the reflux and delayed gastric emptying. The benefits of this procedure need to be further assessed in a large-scale, randomized controlled trial.

The indications for a diverting stoma in low anterior resection for rectal cancer: a prospective multicentre study of 222 patients from Japanese cancer centers.

AIM: The aim of the study was to determine the present state of diverting stoma construction in Japanese cancer centres and to investigate the relationship between symptomatic leakage and diverting stoma after low anterior resection for rectal cancer. METHOD: Two hundred and twenty-two consecutive patients undergoing low anterior resection for rectal cancer located within 10 cm from the anal verge were investigated in a prospective, multicenter study. RESULTS: The overall leakage rate was 9.0% (20/222). Of 31 cases with an anastomosis within 2.0 cm from the anal verge, 22 (71%) had a diverting stoma. Of cases anastomosed within 5.0 cm, the absence of a diverting stoma and tumour size were significantly related to an increased rate of leakage [leakage in 13 (12.7%) of 102 cases without a diverting stoma; in three (3.8%) of 80 cases with a diverting stoma]. Among anastomoses within 2.0 cm from the anal verge, leakage occurred in four (44.4%) of nine cases without and in none (0%) of 22 cases with a diverting stoma. CONCLUSION: We recommend a diverting stoma for an anastomosis within 5.0 cm of the anal verge and strongly recommend it for a very low anastomosis within 2.0 cm.

Can ultrasonographic evaluation of subcutaneous fat predict pressure ulceration?

High-resolution ultrasound may be able to detect pressure ulcers before clinical signs emerge. This retrospective study found that one such early indicator is inflammatory oedema in the subcutaneous fat, which resolves as healing occurs.

Correlation between pretherapeutic d-dimer levels and response to neoadjuvant chemotherapy in patients with advanced esophageal cancer.

Neoadjuvant chemotherapy may improve survival of responders in esophageal cancer patients but is useless and harmful in non-responders. Thus, it is important to predict the effect of the chemotherapy, and that any predictor must be applicable clinically. The aim of this study is to examine the correlation between pretherapeutic hypercoagulopathy as determined by plasma d-dimer levels and response to chemotherapy. In 71 patients with esophageal cancer who underwent neoadjuvant chemotherapy (cisplatin, adriamycin and 5-fluorouracil) followed by surgery, plasma d-dimer levels were measured before chemotherapy and the clinical and pathological responses to chemotherapy were assessed at 4 weeks after therapy (after surgery). Pretherapeutic plasma d-dimer level was significantly lower in clinical responders (complete response/partial response [CR/PR]; 0.62 +/- 1.10 microg/mL, mean +/- SD) than in non-responders (no change/progressive disease [NC/PD]; 1.15 +/- 1.08 microg/mL, P = 0.0491), and in pathological responders (Grade 1b-3; 0.62 +/- 1.11 microg/mL) and non-responders (Grade 0-1a; 1.15 +/- 1.05 microg/mL, P = 0.0107). The optimal cut-off level of the plasma d-dimer levels for predicting clinical and pathological responses was 0.6 microg/mL. Then, sensitivity and specificity for the prediction of CR/PR were 68% and 73%, and those for Grade 1b-3 were 91% and 69%, respectively. Our results suggested that pretherapeutic plasma d-dimer level correlated significantly with clinical and pathological responses to chemotherapy. Pretherapeutic plasma d-dimer level can be used as a predictor for chemosensitivity.

Analgesic and chondroprotective effects of risedronate in osteoarthritis assessed by electroalgometry and measurement of collagen type II fragments in urine.

We evaluated the effect of the bisphosphonate, risedronate, on pain and cartilage metabolism in 33 patients with osteoarthritis of the knee, randomized into two groups. Group RC was treated with risedronate (2.5 mg/day) and calcium (900 mg/day); group C received calcium (900 mg/day) alone. Pain on exercise was estimated using a subjective visual rating scale (VRS) and an electroalgometric method of measuring decrease in skin impedance, previously shown to be indicative of pain. We measured urinary excretion of cartilage-specific collagen type II fragments as a marker of cartilage degradation. Multiple regression analysis revealed that pain alleviation as measured by skin impedance, but not VRS, was associated with a decrease in collagen fragment excretion. This suggests that, for pain evaluation, reduction in skin impedance may have a greater physiological basis compared with VRS-based evaluation. We consider that the chondroprotective and analgesic effects of risedronate may be related.

Evaluation of the technical difficulty performing laparoscopic resection of a rectosigmoid carcinoma: visceral fat reflects technical difficulty more accurately than body mass index.

BACKGROUND: In general, visceral fat and adhesion greatly influence the technical difficulty in performing abdominal surgery. Body mass index (BMI) has been widely used to express the degree of obesity, but it does not always properly reflect the degree of visceral fat. This retrospective study investigated the impact of visceral fat on the operation time to examine whether a quantified visceral fat area (VFA) could be used as a sensitive predictor of technical difficulty in performing a laparoscopic resection of rectosigmoid carcinoma. METHODS: Between February 1999 and April 2004, 58 consecutive patients underwent a laparoscopically assisted sigmoidectomy or anterior resection. After a review of the medical charts, the relationship between the operation time and the following variables was analyzed: sex, depth of invasion, approach (medial-to-lateral, lateral-to-medial), subjectively graded degree of visceral fat and adhesion, history of previous abdominal surgery, and BMI. The correlations between VFA, VFA/body surface area (BSA) measured by the "FatScan," software package for quantifying the VFA from the preoperative CT images, and operation time were investigated. Next, the impact of the VFA amount on the early surgical outcome was examined. RESULTS: According to the intraoperative findings, two patients with a severe adhesion required a significantly longer operation time. A history of previous abdominal surgery was not a significant factor in the operation time. Instead, the VFA/BSA had a stronger correlation with the operation time than the BMI. A significantly longer operation time (209 +/- 42 vs 179 +/- 37 min; p = 0.031) was observed for the patients in the high VFA/BSA group (> or =85 cm(2)/m(2)) group than in the normal VFA/BSA group (<85 cm(2)/m(2)). CONCLUSION: For predicting the technical difficulty of performing a laparoscopic resection of rectosigmoid carcinoma, VFA/BSA may be a more useful index than BMI.

FDG-PET predicts treatment efficacy and surgical outcome of pre-operative chemoradiation therapy for resectable and borderline resectable pancreatic cancer.

BACKGROUND: The efficacy of neoadjuvant chemoradiotherapy (NACRT) for resectable and borderline resectable pancreatic cancer is important for predicting outcomes after radical surgery, but few clinical indicators predict outcome before resection. This study examined the utility of FDG-PET in predicting the efficacy of NACRT and outcome after radical surgery. METHODS: Eighty-three pancreatic cancer patients who underwent FDG-PET before and after NACRT and had positive standard uptake values (SUVs) before NACRT were enrolled in this study. Peri-operative clinical factors, including FDG-PET findings, were examined to predict the efficacy of NACRT and outcome after surgery. RESULTS: Evans grade I, IIA, IIB, III, and IV was determined in 11, 31, 27, 11, and 3 patients, respectively. The maximum SUVs after NACRT (post SUV-max) and tumor size were significantly decreased compared to pretreatment values (p < 0.001 and p = 0.007, respectively). The post SUV-max and regression index were significantly related to grade III/IV (p = 0.04 and p < 0.001, respectively), but only the regression index predicted NACRT efficacy (p = 0.002). The AUC of the regression index for the detection of grade III/IV was 0.822, and 13 of 14 grade III/IV patients were picked up using 50% as the threshold (p < 0.001). Patients with a regression index >50% had a significantly better prognosis after radical resection than patients with <50% (p = 0.032). Regression index as well as pathological lymph node status and resectability status were independent prognostic factors in multivariate analysis (exp 2.086, p = 0.043). CONCLUSION: The regression index is potentially a good indicator of the efficacy of NACRT and outcome after radical resection for pancreatic cancer.

A frequent alteration of p53 gene in carcinoma in adenoma of colon.

In general, colorectal carcinoma is thought to originate mainly from adenoma, and this pathway is called the adenoma-carcinoma sequence. Carcinoma in adenoma is an appropriate model for analysis of this mechanism, because adenoma and carcinoma tissues coexist in the same polyp and the carcinoma is thought to have originated from the surrounding adenoma. Expression of the p53 protein was analyzed in 36 cases of carcinoma in adenoma in the colon by immunohistochemistry using an anti-human p53 monoclonal antibody (PAb1801). Alterations of the p53 gene were analyzed by the polymerase chain reaction for microanalysis of normal mucosa, adenoma, and carcinoma from histological slides. Mutations were assessed by the polymerase chain reaction-single strand conformation polymorphism analysis and identified by DNA sequencing in some cases. Loss of heterozygosity was studied by polymerase chain reaction-restriction fragment length polymorphism analysis. Positive staining for p53 was detected in three (8%) of 37 adenomas and 20 (53%) of 38 focal carcinomas. One (7%) of 15 adenomas with mild dysplasia, three (14%) of 22 adenomas with moderate dysplasia, and 16 (42%) of 38 focal carcinomas had a mutation in exon 5 through exon 8 of the p53 gene. As for allelic loss in the p53 gene locus, only one adenoma with moderate dysplasia had loss of heterozygosity, whereas six (40%) of 15 focal carcinomas had loss of heterozygosity. Of those tumors (3 of 37 adenomas and 20 of 38 focal carcinomas) that reacted with PAb1801, 78% (18 of 23) showed genetic alterations. Among 52 tumors which showed negative staining, five tumors had a p53 mutation and four of them were nonsense mutations. Putting all of these results together, 71% (24 of 34) of the cases underwent p53 gene and protein alterations during the conversion from adenoma to focal carcinoma. These data clearly indicate that genetic alterations of p53 are involved mainly in the malignant transformation from adenoma to focal carcinoma in colon carcinogenesis. In addition, some cases show heterogeneity of the p53 gene in carcinoma in adenoma of the colon. There may be other pathways than p53 responsible for malignant change in the colon.

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy.

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.

Overexpression of CDC25B phosphatase as a novel marker of poor prognosis of human colorectal carcinoma.

There is evidence to suggest that CDC25B phosphatase is an oncogenic protein. To elucidate the role of CDC25B in colorectal carcinoma, we examined the expression of CDC25B at the mRNA and protein levels. Reverse transcription-PCR assay indicated that CDC25B was overexpressed in tumor tissues relative to normal mucosa in 6 of 10 cases. Using immunohistochemistry, we identified high expression of CDC25B in 77 of 181 colorectal cases (43%). Univariate analysis showed that high expression was a significant predictor for poor prognosis compared with low expression (5-year survival rate; 59% versus 82%, respectively; P < 0.0001). Multivariate analysis indicated that CDC25B was an independent prognostic marker (risk ratio for death, 3.7; P < 0.0001) even after controlling for various factors such as lymph node metastasis, tumor size, degree of differentiation, and depth of invasion. Furthermore, the level of CDC25B expression clearly predicted the outcome of patients with Dukes' B and Dukes' C tumors. On the other hand, CDC25A mRNA was overexpressed in 9 of 10 colorectal cancer cases, and immunohistochemistry for CDC25A showed high expression in 52 of 111 cases (47%), but no significant correlation with prognosis. Our findings suggest that CDC25B is a novel independent prognostic marker of colorectal carcinoma and that it may be clinically useful for selecting patients who could benefit from adjuvant therapy.

Novel prognostic prediction models for patients with stage IV colorectal cancer after concurrent curative resection.

BACKGROUND: We developed a prediction tool for recurrence and survival in patients with stage IV colorectal cancer (CRC) following surgically curative resection. PATIENTS AND METHODS: From January 1983 to December 2012, 113 patients with CRC and synchronous liver and/or lung metastatic CRC were investigated at the Osaka Medical Center for Cancer and Cardiovascular Diseases. All patients underwent curative resection of primary and metastatic lesions. In the group of patients who underwent surgery from 1983 to 2008, a Cox regression model was used to develop prediction models for 1-year, 3-year and 5-year cancer-specific survival (CSS) and relapse-free survival (RFS). In the other group of patients who underwent surgery from 2009 to 2012, the developed prediction model was validated. RESULTS: Univariate analysis of clinicopathological factors showed that the following factors were significantly correlated with CSS and RFS: preoperative serum carcinoembryonic antigen level, tumour location, pathologically defined tumour invasion and lymph node metastasis, and synchronous metastatic lesions. Using these variables, novel prediction models predicting CSS and RFS were constructed using the Cox regression model with concordance indexes of 0.802 for CSS and 0.631 for RFS. The prediction models were validated by external data sets in an independent patient group. CONCLUSIONS: We developed novel and reliable personalised prognostic models, integrating tumour, node, metastasis (TNM) factors as well as the preoperative serum carcinoembryonic antigen level, tumour location and metastatic lesions, to predict patients' prognosis following surgically curative resection. This individualised prediction model may help clinicians in the treatment of postoperative stage IV CRC following surgically curative resection.

Male sexual dysfunction after rectal cancer surgery: Results of a randomized trial comparing mesorectal excision with and without lateral lymph node dissection for patients with lower rectal cancer: Japan Clinical Oncology Group Study JCOG0212.

BACKGROUND: We conducted a randomized controlled trial (JCOG0212) to determine whether the outcome of mesorectal excision (ME) alone for rectal cancer is not inferior to that of ME with lateral lymph node dissection (LLND). The present study focused on male sexual dysfunction after surgery. METHODOLOGY: Eligibility criteria included clinical stage II/III rectal cancer, the lower margin of the lesion below the peritoneal reflection, the absence of lateral pelvic lymph node enlargement, and no preoperative radiotherapy. After confirmation of R0 resection by ME, patients were intraoperatively randomized. Questionnaires using the International Index of Erectile Function (IIEF-5) about the sexual function of men were collected before and 1 year after surgery. Sexual dysfunction incidence was defined as the ratio of patients showing sexual dysfunction after surgery relative to the number who had no erectile dysfunction before surgery. RESULTS: Among 701 patients enrolled between June 2003 and August 2010, 472 males were included. Among them, 343 (73%) completed preoperative and postoperative questionnaires. According to the study protocol, the incidences of sexual dysfunction in patients who underwent ME alone and ME with LLND were 68% (17/25; 95%CI, 47-85%) and 79% (23/29; 95%CI, 60-92%), respectively (p = 0.37). Incidences of sexual dysfunction in patients with no or only mild erectile dysfunction before surgery who underwent ME alone and ME with LLND were 59% (48/81) and 71% (67/95), respectively (p = 0.15). Multivariate analysis identified age as the only risk factor for sexual dysfunction after surgery (p = 0.02). CONCLUSIONS: LLND may not increase sexual dysfunction incidence after rectal cancer surgery. This incidence is associated with increased age. This trial is registered with ClinicalTrials.gov, number NCT00190541 and University Hospital Medical Information Network Clinical Trials Registry, number C000000034.

Mechanism of amelioration of insulin resistance by beta3-adrenoceptor agonist AJ-9677 in the KK-Ay/Ta diabetic obese mouse model.

The mechanism by which the specific beta3-adrenoceptor agonist AJ-9677 relieves insulin resistance in vivo was investigated by studying its effects in the white and brown adipose tissues of the KK-Ay/Ta diabetic obese mouse model. AJ-9677 reduced the total weight of white adipose tissues by reducing the size of the adipocytes, an effect associated with the normalization of tumor necrosis factor-alpha (TNF-alpha) and leptin expression levels. The levels of uncoupling protein (UCP)-1 mRNA in brown adipose tissue were increased threefold. AJ-9677 caused a marked increase (20- to 80-fold) in the expression of UCP-1 in white adipose tissues. The levels of UCP-2 mRNA were increased in both the white and brown adipose tissues of diabetic obese mice, and AJ-9677 further upregulated UCP-2 mRNA levels in brown adipose tissue, but reduced its levels in white adipose tissue. UCP-3 mRNA levels were not essentially changed by AJ-9677. However, AJ-9677 significantly (two- to four-fold) upregulated the GLUT4 mRNA and protein levels in white and brown adipose tissues and the gastrocnemius. The generation of small adipocytes, presumably mediated by increased expression of UCP-1 in addition to increased lipolysis in response to AJ-9677, was associated with decreased TNF-alpha and free fatty acid production and may be the mechanism of amelioration of insulin resistance in KK-Ay/Ta diabetic obese mice.

Extensive micrometastases to lymph nodes as a marker for rapid recurrence of colorectal cancer: a study of lymphatic mapping.

To provide a detailed assessment of micrometastases of colorectal cancer by anatomical mapping of regional lymph nodes (LNs), we analyzed 237 LNs from 11 patients with colorectal cancer by reverse transcription-PCR (RT-PCR) using carcinoembryonic antigen and cytokeratin 20 as genetic markers. All dissected LNs were mapped anatomically and subjected to detection assays for micrometastases. Immunohistochemical analysis was also performed using anti-pancytokeratin antibody AE1/AE3 to confirm the existence of occult cancer cells. By histological analysis, 20 of 237 LNs contained metastatic cells, and they were all positive by both immunohistochemistry and RT-PCR. Of the 217 histologically negative LNs, 14 (6.5%) harbored micrometastases by immunohistochemistry, and 57 (26.2%) were positive for at least one of the two genetic markers. Lymphatic mappings of all patients showed that micrometastases were distributed not only at the pericolic LNs but often at distant LNS: Clinical follow-up study showed that two patients developed recurrence within 1 year after surgery, and both of them had RT-PCR-positive micrometastases in not less than 70% of LNs examined. Moreover, both patients had frequent micrometastases at distant LNs, i.e., those around the root or along the inferior mesenteric artery, when compared with patients with no recurrence. Our findings suggest that genetic diagnosis using the RT-PCR method may be clinically useful along with conventional pathological diagnosis, especially when micrometastases spread to distant LNS:

Microsatellite instability and mutated type II transforming growth factor-beta receptor gene in gliomas.

Microsatellite instability has been reported in familial cancer syndrome and in various kinds of human sporadic tumors. We investigated the replication error (RER) and mutation rate of the transforming growth factor-beta type II receptor (TGF-beta RII) gene to determine the frequency of the RER+ phenotype and elucidate the relation between the mutation of the TGF-beta RII gene and RER in the tumorigenesis of glioma. We screened genomic DNA from 40 gliomas, comprised from 24 glioblastomas (GB), 11 anaplastic astrocytomas (AA) and five astrocytomas (AS) and compared the results with DNA from corresponding leukocytes. Seven of the 40 (18%) gliomas had the RER+ phenotype: five (21%) of 24 GB and two (18%) of 11 AA. In six gliomas we detected mutation of the TGF-beta RII gene. Five (71%) of seven RER+ and one (3%) of 33 RER-tumors had one A deletion in the (A)10 repeat of the TGF-beta RII gene. No mutations were detected in the (GT)3 repeat area of the TGF-beta RII gene. As the normal cells of these glioma patients had no mutations, we concluded that the mutations were somatic. We posit that the observed mutations inactivate the receptor through a frameshift mutation resulting in protein truncation. Our data suggest that the TGF-beta RII (A)10 repeat may be one area of genomic instability in the early stages of malignant glioma tumorigenesis.

Immunohistochemical study of retinoblastoma gene-expression in colorectal carcinomas.

The expression of the retinoblastoma gene (Rb-1) in colorectal carcinomas was studied by Western blotting and immunohistochemistry. Western blot analysis using monoclonal antibodies raised against synthetic peptides of the Rb-1 gene product (pRB) revealed that colorectal carcinomas overexpressed pRB with a molecular weight of around 110 kD when compared to normal mucosa. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections from 48 colorectal carcinomas demonstrated that pRB expression was exclusively localized to the nucleus. More than 50% of the carcinoma cells expressed nuclear pRB in 14 tumors (29.2%), while 10-50% of the carcinoma cells did so in 21 tumors (43.8%), and less than 10% of cells did so in 13 tumors (27.1%). Although there was no clear correlation between pRB expression and clinico-pathologic parameters such as tumor stage, tumor size, depth of invasion, and lymph node metastasis, a higher incidence of pRB expression was observed in well to moderately differentiated adenocarcinoma than in the signet ring cell carcinoma. Thus the present study demonstrated for the first time that the oncosuppressor gene, Rb-1, is overexpressed at the protein level in most colorectal carcinomas.

Quantification of micrometastases in lymph nodes of colorectal cancer using real-time fluorescence polymerase chain reaction.

The expression of carcinoembryonic antigen (CEA) mRNA was assessed in 102 lymph nodes (LNs) obtained from seven colorectal cancer patients by both the conventional non-quantitative RT-PCR and quantitative RT-PCR. The number of CEA-expressing cells was calculated compared with CEA-expressing MKN-45 cell line as a standard control. Using the quantitative RT-PCR, the relative number of CEA-expressing cells ranged between 1.3x103 and 5.7x106 in 16 histologically positive LNs and between 2.3x101 and 8.1x105 in 10 histologically negative and RT-PCR positive LNs. In both histologically and RT-PCR negative LNs, the relative cell number was <4.0x102. Our results demonstrated that quantifying the amount of metastasis might enhance the reliability of RT-PCR detection assay as a diagnostic tool for the detection of cancer micrometastases.

Comparative genomic hybridization defines frequent loss on 16p in human anaplastic thyroid carcinoma.

Anaplastic thyroid cancer (ATC) is one of the malignant tumors with the poor prognosis that is thought to arise from well-differentiated thyroid cancer (DTC). To investigate the molecular mechanism of ATC, we studied genomic alterations of eight ATC cell lines and three DTC cell lines by means of the comparative genomic hybridization (CGH) method. Loss of 16p was observed in five of eight ATC cell lines (62. 5%), but none of the three DTC cell lines showed loss of this chromosome arm. It is notable that loss of 18q [7/8 of ATC (87.5%), 2/3 of DTC (67%)] and gain of 20q [5/8 of ATC (62.5%), 3/3 of DTC (100%)] were frequently seen in both histologic types. Our results suggest that there is a gene in 16p that is closely associated with transformation from well-differentiated thyroid cancer to anaplastic cancer.