Immunogenomic profiles associated with response to neoadjuvant chemoradiotherapy in patients with rectal cancer.

BACKGROUND: Accumulating evidence suggests that radiotherapy success has an immune-associated component. The immunogenomic profiles associated with responses to chemoradiotherapy (CRT) were assessed in patients with locally advanced rectal cancer in this study. METHODS: CD8+ tumour-infiltrating lymphocyte (TIL) and stromal lymphocyte densities were assessed by immunohistochemistry using pretreatment biopsies from patients with advanced rectal cancer who had preoperative CRT. Whole-exome sequencing and gene expression microarray analysis were conducted to investigate the genomic properties associated with the response to CRT and CD8+ TIL density. Response to CRT was determined based on Dworak tumour regression grade (TRG); tumours with complete (TRG 4) or near-complete (TRG 3) regression were grouped as good responders, and those with TRG 1 as non-responders. RESULTS: Immunohistochemical examinations (275 patients) showed that pre-CRT CD8+ TIL density was associated with better response to CRT and improved recurrence-free survival, whereas pre-CRT stromal CD8+ cell density was not associated with either response to CRT or recurrence-free survival. Whole-exome sequencing (74 patients) showed that the numbers of single-nucleotide variations (SNVs) and neoantigens predicted from SNVs were higher in good responders than in non-responders, and these correlated positively with CD8+ TIL density (rS = 0·315 and rS = 0·334 respectively). Gene expression microarray (90 patients) showed that CD8A expression correlated positively with the expression of programmed cell death 1 (PDCD1) (rS = 0·264) and lymphocyte-activation gene 3 (LAG3) (rS = 0·507). CONCLUSION: Pre-CRT neoantigen-specific CD8+ T cell priming may be a key event in CRT responses where immune checkpoint molecules could be useful targets to enhance tumour regression.

ANTECEDENTES: Las evidencias existentes sugieren que el éxito de la radioterapia tiene un componente asociado con el sistema inmunitario. En este estudio se evaluaron los perfiles inmunogenómicos asociados con la respuesta a la quimiorradioterapia (chemoradiotherapy, CRT) en pacientes con cáncer de recto localmente avanzado. MÉTODOS: Las densidades de los linfocitos infiltrantes de tumor CD8+ (tumour-infiltrating lymphocyte, TIL) y de los linfocitos del estroma se evaluaron por inmunohistoquímicas en las biopsias antes del tratamiento de pacientes con cáncer de recto localmente avanzado que recibieron CRT preoperatoria. Se realizó secuenciación de todo el exoma, así como microarrays de expresión génica, para investigar las propiedades genómicas asociadas con la respuesta a la CRT y a la densidad de los TIL CD8+. La respuesta a la CRT se determinó según el grado de regresión del tumor de Dworak (tumour regression grade, TRG), agrupándose como buenos respondedores los casos de regresión tumoral completa (TRG4) o casi completa (TRG3) y como no respondedores, los casos de grado TRG1. RESULTADOS: Los exámenes inmunohistoquímicos (n = 275) mostraron que la densidad pre-CRT de TIL CD8+ se asoció con una mejor respuesta a la CRT y una mejor supervivencia libre de recidiva, aunque la densidad de células CD8+ del estroma previa a la CRT no se asoció con la respuesta a la CRT ni con la supervivencia libre de recidiva. La secuenciación de todo el exoma (n = 74) mostró que el número de variaciones de nucleótidos únicos (single nucleotide variations, SNVs) y los neoantígenos predichos a partir de los SNVs fueron mayores en los que respondieron bien que en los que no respondieron, y éstos se correlacionaron positivamente con la densidad de los TIL CD8+ (Spearman r = 0,315 y r = 0,334 respectivamente). Los microarrays de expresión génica (n = 90) mostraron que la expresión CD8A se correlacionó positivamente con la expresión del ligando de muerte programada-1 (r = 0,264) y con el antígeno linfocitario del gen 3 (r = 0,507). CONCLUSIÓN: La activación de células T CD8+ específicas para neoantígenos previa a la CRT puede ser un evento clave en la respuesta a la misma donde las moléculas del punto de control inmunitario podrían ser dianas útiles para intensificar la regresión del tumor.

FOLFIRI plus bevacizumab as second-line therapy in patients with metastatic colorectal cancer after first-line bevacizumab plus oxaliplatin-based therapy: the randomized phase III EAGLE study.

BACKGROUND: A targeted agent combined with chemotherapy is the standard treatment in patients with metastatic colorectal cancer (mCRC). The present phase III study was conducted to compare two doses of bevacizumab combined with irinotecan, 5-fluorouracil/leucovorin (FOLFIRI) in the second-line setting after first-line therapy with bevacizumab plus oxaliplatin-based therapy. PATIENTS AND METHODS: Patients were randomly assigned to receive FOLFIRI plus bevacizumab 5 or 10 mg/kg in 2-week cycles until disease progression. The primary end point was progression-free survival (PFS), and secondary end points included overall survival (OS), time to treatment failure (TTF), and safety. RESULTS: Three hundred and eighty-seven patients were randomized between September 2009 and January 2012 from 100 institutions in Japan. Baseline patient characteristics were well balanced between the two groups. Efficacy was evaluated in 369 patients (5 mg/kg, n = 181 and 10 mg/kg, n = 188). Safety was evaluated in 365 patients (5 mg/kg, n = 180 and 10 mg/kg, n = 185). The median PFS was 6.1 versus 6.4 months (hazard ratio, 0.95; 95% confidence interval [CI] 0.75-1.21; P = 0.676), and median TTF was 5.2 versus 5.2 months (hazard ratio, 1.01; 95% CI 0.81-1.25; P = 0.967), respectively, for the bevacizumab 5 and 10 mg/kg groups. Follow-up of OS is currently ongoing. Adverse events, including hypertension and hemorrhage, occurred at similar rates in both groups. CONCLUSION: Bevacizumab 10 mg/kg plus FOLFIRI as the second-line treatment did not prolong PFS compared with bevacizumab 5 mg/kg plus FOLFIRI in patients with mCRC. If bevacizumab is continued after first-line therapy in mCRC, a dose of 5 mg/kg is appropriate for use as second-line treatment. CLINICAL TRIAL IDENTIFIER: UMIN000002557.

Prognostic significance of epithelial-mesenchymal transition-related markers in extrahepatic cholangiocarcinoma: comprehensive immunohistochemical study using a tissue microarray.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is characterised by the loss of cell-to-cell adhesion and gaining of mesenchymal phenotypes. Epithelial-mesenchymal transition is proposed to occur in various developmental processes and cancer progression. 'Cadherin switch', a process in which cells shift to express different isoforms of the cadherin transmembrane protein and usually refers to a switch from the expression of E-cadherin to N-cadherin, is one aspect of EMT and can have a profound effect on tumour invasion/metastasis. The aim of this study was to investigate the clinicopathological significance of EMT-related proteins and cadherin switch in extrahepatic cholangiocarcinoma (EHCC). METHODS: We investigated the association between altered expression of 12 EMT-related proteins and clinical outcomes in patients with EHCC (n=117) using immunohistochemistry on tissue microarrays. RESULTS: Univariate and multivariate analyses revealed that, in addition to N classification (P=0.0420), the expression of E-cadherin (P=0.0208), N-cadherin (P=0.0038) and S100A4 (P=0.0157) was each an independent and a significant prognostic factor. We also demonstrated that cadherin switch was independently associated with poor prognosis (P=0.0143) in patients with EHCC. CONCLUSIONS: These results may provide novel information for selection of patients with EHCC who require adjuvant therapy and strict surveillance.

Long-term outcome in Japanese patients with lupus nephritis.

The objective of this study was to clarify the long-term outcome in patients with lupus nephritis (LN) according to the International Society of Nephrology and Renal Pathology Society classification. This retrospective analysis comprised 186 Japanese patients given a diagnosis of LN by renal specimen with a mean observation period of 12 years. Primary end point was defined as death or end-stage renal disease, and standardized mortality ratios were calculated. Five patients presented with histopathological class I, 62 with II, 21 with III or III+V, 73 with IV or IV+V and 25 with V. Fourteen deaths occurred, corresponding to an overall standardized mortality ratio of 3.59 (95% confidence interval 2.02-5.81, p < 0.0001). Kaplan-Meier analysis revealed a 10-year overall survival of 95.7%. Nephrotic proteinuria (≥3.5 g/day) at baseline was identified as an independent poor prognostic factor for overall survival in Cox regression analysis. Kaplan-Meier analysis revealed a 10-year renal survival as 94.3%. Male gender and nephrotic proteinuria at baseline were identified as independent poor prognostic factors for renal survival in Cox regression analysis. In conclusion, LN was associated with a 3.59-fold increase in mortality compared with the general population. Male gender and nephrotic proteinuria were predictive for poor renal outcome.

Phase II study of trifluridine/tipiracil plus bevacizumab by RAS mutation status in patients with metastatic colorectal cancer refractory to standard therapies: JFMC51-1702-C7.

BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status. PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort. RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals. CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.

Multicenter phase II study of FOLFOX for metastatic colorectal cancer (mCRC) in Japan; SWIFT-1 and 2 study.

BACKGROUND/AIMS: This study assessed the efficacy and toxicity of the FOLFOX4 (SWIFT1) and mFOLFOX6 (SWIFT2) regimens in Japanese patients with metastatic colorectal cancer (mCRC). METHODOLOGY: Patients with mCRC were required to have ECOG performance status of 0 to 1, and to have adequate organ function. Two multicenter Phase II studies (SWIFT1/SWIFT2) were conducted in chemotherapy naive patients with mCRC. RESULTS: 112 patients were enrolled in these studies (SWIFT1: 54 patients / SWIFT2: 58 patients). The disease sites for each study were the colon in 27 patients and 28 patients, and the rectum in 27 patients and 30 patients, respectively. All patients received a median of 8 courses. After a median follow-up period of 35 months, 54 patients and 58 patients were evaluable in the respective studies, and the overall response rate was 50.0% (CR:31 PR:53). The response rate according to the sites of metastasis were as follows: liver, 54.1% (46/85); lung, 17.4% (4/23); and lymph node, 23.3% (7/30). Grade 3/4 neutropenia occurred in 14 patients (12.5%), while Grade 3/4 non-hematological toxicities were observed in 16 patients (31.0%) and Grade 3 neurotoxicity was observed in 6patients (5.4%) and 5 patients (4.5%), respectively. CONCLUSIONS: FOLFOX4 (SWIFT1) and mFOLFOX6 (SWIFT2) regimens complying with the international standard dosage and schedule can also be administered safely and effectively in Japan.

Prognostic impact of residual lateral lymph node metastasis after neoadjuvant (chemo)radiotherapy in patients with advanced low rectal cancer.

Background: There is a lack of large studies focusing on the prognostic significance of lateral lymph node (LLN) metastasis following LLN dissection (LLND) in rectal cancer. The aim of this study was to evaluate the prognostic impact of LLN metastases on survival of patients with advanced low rectal cancer. Methods: Consecutive patients with locally advanced, but not metastatic, extraperitoneal rectal cancer treated with neoadjuvant (chemo)radiotherapy plus total mesorectal excision between 2004 and 2015 were included in the study. LLND was performed when pretreatment imaging documented enlarged LLNs (7 mm or greater in size). Localization of nodal metastases and long-term outcomes were analysed. Kaplan-Meier analysis was used to compare the survival of patients with ypN0 disease with that of patients with mesorectal ypN+/LLN- status and patients with positive LLNs. The Cox proportional hazards model was used to evaluate predictors of disease-free survival (DFS) and local recurrence. Results: A total of 613 patients were included in the study; LLND was performed in 212 patients (34·6 per cent) and 57 (9·3 per cent) had LLN metastasis. Patients with LLN metastasis had improved DFS and local recurrence cumulative incidence rates compared with patients with mesorectal ypN2+/LLN- disease (DFS: P = 0·014; local recurrence: P = 0·006). Although the DFS rate of patients with LLN metastasis was worse than that of patients with ypN0 disease (P < 0·001), the cumulative incidence of local recurrence was similar (P = 0·491). In multivariable analysis, residual LLN metastasis was not an independent predictor of worse DFS or local recurrence. Conclusion: LLN metastasis is not an independent predictor of local recurrence or survival. Survival of patients presenting with LLN metastasis after (chemo)radiotherapy was intermediate between that of patients with ypN0 status and those with mesorectal ypN2 positivity.

Antecedentes: No existen en la literatura grandes estudios dirigidos a investigar la importancia pronóstica de las metástasis en los ganglios linfáticos laterales (lateral lymph nodes, LLN) después de la disección de los mismos (LLN dissection, LLND) en pacientes con cáncer de recto. El objetivo de este estudio fue evaluar el impacto pronóstico de las metástasis en los LLN sobre la supervivencia de los pacientes con cáncer de recto. Métodos: Se analizaron 613 pacientes consecutivos con cáncer de recto localmente avanzado extraperitoneal y no metastásico tratados con (quimio)radioterapia neoadyuvante seguida de resección total del mesorrecto (total mesorectal excision, TME) entre 2004 y 2015. Se realizó una LLND cuando el estudio mediante pruebas de imagen previo el tratamiento mostró LLN aumentados de tamaño ≥ 7 mm. Se analizó la localización de las metástasis ganglionares y los resultados a largo plazo. El análisis de supervivencia se realizó mediante el método de Kaplan­Meier para comparar las supervivencias de los pacientes ypN0 frente a los pacientes ypN con positividad mesorrectal/LLN negativos y frente a los pacientes LLN positivos. Se utilizó el modelo de riesgo proporcional de Cox para evaluar los factores predictivos de supervivencia libre de enfermedad y de recidiva local. Resultados: Se realizó una LLND en 212 (34,6%) pacientes, y 57 (9,3%) pacientes presentaban metástasis en los LLN. Los pacientes con metástasis en los LLN presentaron mejores curvas de incidencia acumulada de recidiva local y de supervivencia libre de enfermedad en comparación con los pacientes con ganglios mesorrectales ypN2 positivos/LLN negativos (respectivamente, P = 0,0135 y P = 0,0060). Aunque la curva de la supervivencia libre de enfermedad de los pacientes con metástasis en los LLN fue peor que la de los pacientes ypN0 (P < 0,0001), la incidencia acumulada de recidiva local fue similar (P = 0,4905). En el análisis multivariable, la metástasis residual en los LLN no fue un factor predictivo independiente de peor supervivencia libre de enfermedad ni de recidiva local. Conclusión: Las metástasis en los LLN no es un factor predictivo independiente de recidiva local o supervivencia. Los pacientes que presentaron metástasis en los LLN después de (quimio)radioterapia mostraron características de supervivencia intermedias entre ypN0 y pacientes con ganglios mesorrectales ypN2 positivos.

SAPPHIRE: a randomised phase II study of planned discontinuation or continuous treatment of oxaliplatin after six cycles of modified FOLFOX6 plus panitumumab in patients with colorectal cancer.

BACKGROUND: Fluorouracil (5-FU), leucovorin (LV) and oxaliplatin (FOLFOX) plus panitumumab therapy is a commonly used first-line chemotherapy for metastatic colorectal cancer (mCRC). However, the long-term administration of oxaliplatin is associated with peripheral neuropathy (PN). We investigated whether the planned discontinuation of oxaliplatin after FOLFOX plus panitumumab therapy can maintain efficacy and reduce PN incidence. PATIENTS AND METHODS: Chemotherapy-naive patients with RAS wild-type mCRC, aged ≥20 years, were enrolled and received six cycles of modified FOLFOX6 (mFOLFOX6) plus panitumumab as induction therapy. Patients who completed induction therapy without progression were randomised to mFOLFOX6 plus panitumumab (group A) or to 5-FU/LV plus panitumumab (group B). The primary end-point was the progression-free survival (PFS) rate at 9 months after randomisation. The secondary end-points were PFS, overall survival (OS), time to treatment failure (TTF), response rate (RR) and safety. RESULTS: In total, 164 patients were enrolled; of whom, 113 patients were then randomised (group A, n = 56; group B, n = 57). The median follow-up after randomisation was 19.6 months. The PFS rates at 9 months and median PFS were 46.4% (80% confidence interval [CI], 38.1-54.9) and 9.1 months (95% CI, 8.6-11.1) in group A, compared with 47.4% (80% CI, 39.1-55.8) and 9.3 months (95% CI, 6.0-13.0) in group B, respectively. RR, OS and TTF were also similar in both groups. Grade ≥2 PN incidence was lower in group B (9.3%) than in group A (35.7%). CONCLUSION: Planned discontinuation of oxaliplatin after six cycles of mFOLFOX6 plus panitumumab is a potential treatment option in patients with mCRC, achieving similar efficacy while reducing oxaliplatin-associated PN compared with mFOLFOX6 plus panitumumab. TRIAL REGISTRATION NUMBER: NCT02337946.

Multicenter phase II trial of combination chemotherapy with weekly paclitaxel and 5-fluorouracil for the treatment of advanced or recurrent gastric carcinoma.

The aim of this study was to investigate the efficacy and safety of combination chemotherapy with weekly paclitaxel and 5-fluorouracil (5-FU) as first-line treatment in patients with advanced or recurrent gastric carcinoma. A total of 65 patients were treated with the following regimen, administered every 28 days; 5-FU 600 mg/m2 by 24-hour continuous infusion from days 1 through 5, and weekly paclitaxel 80 mg/m2 by 3-hour intravenous infusion on days 8, 14, and 21. A total of 272 cycles were conducted with a median of 4 (2-13) cycles per case. Out of 57 patients with measurable disease by RECIST criteria, there were 2 complete responses (3.5%), 20 partial responses (35.1%) and 25 cases with stable disease (43.9%). The overall response rate was 38.6% (95%CI: 26.0-51.2%). The median survival time and 1-year survival rates were 329 days and 47.4%, respectively. Both hematologic and non-hematologic toxicities were well tolerated.

Deletions on chromosome 8p22 may predict disease progression as well as pathological staging in prostate cancer.

PURPOSE: A recent report demonstrated that the deletion of chromosome 8p22 could predict disease progression in stage III (capsular penetrating) prostate cancer. We studied if the status of chromosomal deletions of 8p22 could reflect pathological stage as well as patient prognosis, thereby serving as a diagnostic tool to optimize the treatment strategy in prostate cancer. EXPERIMENTAL DESIGN: A total of 97 patients (41 Japanese and 56 Swedish) were studied by the fluorescence in situ hybridization technique. Seventy-seven patients (23 pT2, 18 pT3, and 36 pN+ tumors) underwent surgery (radical prostatectomy or lymph node dissection). The specimens were prepared by touch biopsy. From another 20 cases, fine-needle aspiration biopsies were obtained. RESULTS: 8p22 deletions were detected in 47 (61%) and 11 (55%) specimens of 77 touch biopsies and 20 fine-needle aspiration biopsies, respectively. No significant difference was found in the frequency of 8p22 deletion between different preparations of specimens, as well as between different races (Japanese versus Swedish). The frequency of 8p22 deletion was statistically higher in patients with pT3 or more than in those with pT2 (P < 0.01). Disease progression was evaluated in 57 patients. The Cox proportional hazards model revealed 8p22 deletion to be the strongest parameter to predict disease progression (hazards ratio = 5.75; P = 0.0001). CONCLUSIONS: Studies on chromosomal deletions of 8p22 by fluorescence in situ hybridization technique may serve as a genetic marker to optimize the treatment strategy in patients with prostate cancer to the optimal treatment.

Dax-1 as one of the target genes of Ad4BP/SF-1.

The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear hormone receptor superfamily. DAX-1 plays a critical role during gonadal and adrenal differentiation since mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. In recent studies, DAX-1 was reported to function as a transcriptional suppressor of Ad4BP/SF-1, a critical transcription factor in gonadal and adrenal differentiation. With respect to implication of Ad4BP/SF-1 in the transcriptional regulation of the DAX-1 gene, inconsistent findings have been previously reported. We investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site by transient transfection and electrophoretic mobility shift assays. In addition, immunohistochemical analyses with a specific antibody to Dax-1 indicated the presence of immunoreactive cells in steroidogenic tissues, pituitary gland, and hypothalamus. Although the distributions of Dax-1 and Ad4BP/SF-1 were very similar, they were not completely identical. The expression of Dax-1 was significantly impaired in knock-out mice of the Ftz-f1 gene, which encodes Ad4BP/ SF-1. Taken together, our findings indicate that Ad4BP/SF-1 controls the transcription of the Dax-1 gene.

Presence of amelanotic melanocytes within the outer root sheath in senile white hair.

In order to determine whether amelanotic melanocytes are present in senile white hair, we attempted to detect tyrosinase mRNA and its protein using in situ hybridization and immunohistochemical staining. Specifically stained cells containing tyrosinase mRNA or its protein were detected by each technique in 7/23 (30.4%) or 4/14 (28.6%) senile white hairs, respectively. These cells were located in the outer root sheath between the hair-bulb and the infundibulum. Silver staining was used to determine whether melanin was present within the outer root sheath of the senile white hair. Because none of the 21 white hairs tested showed specific staining within the outer root sheath, the absence of melanin was confirmed. Thus our results suggest the presence of amelanotic melanocytes within the outer root sheath of senile white hair.

Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor.

We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism.

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for the mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients' genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene.

[Phe21]big endothelin-1(18-34) and [Ala31]big endothelin-1(18-34) inhibit the human endothelin-converting enzyme-1 (ECE-1) expressed in CHO-K1 cells in a different fashion.

Endothelin-converting enzyme-1 (ECE-1) is one of the most important enzymes to convert big endothelin-1 (big ET-1) to ET-1. To identify the inhibitors of ECE-1, we examined the effects of variously substituted analogues of big ET-1 on ECE-1 activity using solubilized membranes prepared from human ECE-1-expressed CHO-K1 cells. Among the big ET-1 analogues tested, [Phe21]big ET-1(18-34) and [Ala31]big ET-1(18-34) exhibited a significant inhibition of ECE-1. A kinetic analysis revealed [Phe21]big ET-1(18-34) to be a competitive inhibitor (Ki=20.6 microM) and [Ala31]big ET-1(18-34) to be a noncompetitive inhibitor (Ki=35.6 microM). These results not only support the concept that ECE-1 recognizes big ET-1 both at the P1 position and at the C-terminal region but also revealed that these two regions are recognized by this enzyme in a different manner.

Detection of early gastric cancer by a real-time autofluorescence imaging system.

A light-induced fluorescence endoscopy in the gastrointestinal tract system was used in 52 patients with 54 lesions (33 early gastric cancers, 21 benign lesions) to assess its ability to detect early gastric cancer. Comparing the images with the histological findings, 21 of the 33 carcinomas appeared dark red, ten had a mixed pattern of dark red and white, and two could not be detected. Of the 21 benign lesions, 18 appeared light blue, as do normal mucosa, with this system. In 85% of the cancer lesions (28/33), cancer extension was correctly detected. The sensitivity and specificity were 94 and 86%, respectively. Real-time autofluorescence endoscopy is a useful adjunct to conventional white-light endoscopy for detecting early gastric cancer.

A high level of physical fitness during thirties is a negative risk factor for colonic polyps during fifties.

This case-control study examined the relationship between the occurrence of colonic polyps in men in their fifties and the level of their physical fitness while in their thirties. The subjects consisted of 51 male Japan Self-Defense Forces officials in their fifties who had colonic polyps, as diagnosed by colonoscopic examination, and 46 control individuals. As an indicator of physical fitness between 30 and 39, we selected the best time recorded for each individual during that decade of life for the 1,500 meter Physical Fitness Test run. We calculated the odds ratio for polyps according to selected risk factors (including physical fitness), and a logistic regression analysis was used to adjust for possible confounding variables. Odds ratio (95% confidence interval, p value) for colonic polyps with physical fitness in the thirties was 0.36 (0.16-0.82, p < 0.05). With adjustment for the subjects' maximum Body Mass Index in both their thirties and fifties, and serum total cholesterol, the odds ratio was 0.39 (0.15-0.99, p < 0.05). We suggest that the occurrence of colonic polyps in men in their fifties can be reduced by maintaining a high level of physical fitness while in their thirties.

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.

Purification and characterization of 3-hydroxy-3-methylglutaryl CoA reductase from potato tubers.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (NADPH) was solubilized by trypsin digestion from sliced potato tuber microsomes, and purified to apparent homogeneity in the absence of detergent with a recovery of 1.8%. The enzyme had a specific activity of 7,910 nmol of mevalonate formed per min per mg of protein. On molecular-sieving high-performance liquid chromatography, the activity was coincident with the single protein peak corresponding to a molecular weight of approximately 110 kDa. On SDS-polyacrylamide gel electrophoresis, the purified enzyme showed only one protein staining band corresponding to a molecular weight of approximately 55 kDa. The apparent Km value for S-HMG-CoA was 6.4 microM and that for NADPH was 25 microM.