Feasibility of comprehensive genomic profiling using endoscopic ultrasound-guided tissue acquisition with a 22-gauge Franseen needle.

Aim: Comprehensive genomic profiling (CGP) test for solid tumors is now increasingly utilized in clinical practice, especially in pancreatobiliary cancer, and specimens obtained by endoscopic ultrasound-guided tissue acquisition (EUS-TA) are often submitted for tissue-based CGP test. In this study, we evaluated the feasibility of EUS-TA using a 22-gauge Franseen needle for the CGP test. Methods: Consecutive patients with solid tumors who underwent EUS-TA using a 22-gauge Franseen needle, and whose tissue samples were pre-checked for suitability for CGP test, were included in this single-center, retrospective analysis. The success rates of appropriate sample collection for CGP evaluated by pathologists (1st quality control) and CGP test (2nd quality control) were evaluated. In addition, The EUS-TA slides were evaluated for the tissue area and tumor area content, using the image software. Results: A total of 50 cases, with 78% of pancreatic cancer, were included in the analysis. A median of 3 passes of EUS-TA were performed with an adverse event rate of 4%. The success rates for 1st and 2nd quality control for CGP tests were 86% and 76%, respectively. The image analyses suggested EUS-TA specimen did not always fulfill CGP test criteria, with 18% of tissue area ≥16 mm2 and 38% of tumor area content ≥20%, even in cases with successful CGP tests. The suction method yielded a significantly larger amount of DNA but without a significant difference in the multivariate analysis. Conclusions: The present study demonstrated the feasibility of EUS-TA using a 22-gauge Franseen needle for CGP test.

Intraductal Papillary Mucinous Neoplasm Surveillance Leads to Early Diagnosis and Better Outcomes of Concomitant Cancer.

OBJECTIVE: To examine whether long-term surveillance of intraductal papillary mucinous neoplasms (IPMNs) leads to early diagnosis and better clinical outcomes of pancreatic ductal adenocarcinomas (PDACs) developing concomitantly with IPMNs. SUMMARY BACKGROUND DATA: Long-term image-based surveillance is recommended for patients with low-risk IPMNs. However, it is unknown whether the surveillance can improve surgical and survival outcomes of patients with concomitant PDACs. METHODS: Using a prospective single-institutional cohort of 4,620 patients with pancreatic cysts including 3,638 IPMN patients, we identified 63 patients who developed concomitant PDAC during long-term surveillance. We compared overall survival (OS) of 46 cases with concomitant PDAC to that of 460 matched cases diagnosed with non-IPMN-associated PDAC at the same institution. Multivariable hazard ratios and 95% confidence intervals (CIs) for overall mortality were computed using the Cox regression model with adjustment for potential confounders. RESULTS: Concomitant PDACs were identified at an earlier cancer stage compared to non-IPMN-associated PDACs with 67% and 38% cases identified at stage 2 or earlier, respectively (P<0.001) and 57% and 21% cases with R0 resection, respectively (P<0.001). Compared to non-IPMN-associated PDACs, concomitant PDACs were associated with longer OS (P=0.034) with a multivariable hazard ratio of 0.61 (95% CI, 0.39-0.96). The 5-year survival rate of patients with concomitant PDAC was higher compared to patients with non-IPMN-associated PDAC (34% vs. 18%, respectively; P=0.018). CONCLUSIONS: The surveillance for patients with IPMNs was associated with early identification of concomitant PDACs and longer survival of patients diagnosed with this malignancy.

Balloon endoscopy-assisted endoscopic retrograde cholangiopancreatography for hepatolithiasis in patients with hepaticojejunostomy.

BACKGROUND AND AIM: Balloon endoscopy-assisted endoscopic retrograde cholangiopancreatography (BE-ERCP) is an emerging procedure for pancreatobiliary diseases in patients with surgically altered anatomy. However, data on BE-ERCP for hepatolithiasis after hepaticojejunostomy (HJS) are still limited. METHODS: Stone removal success, adverse events and recurrence were retrospectively studied in consecutive patients who underwent BE-ERCP for hepatolithiasis after HJS between January 2011 and October 2022. Subgroup analysis was performed to compare clinical outcomes between patients who had undergone HJS over 10 years before (past HJS group) and within 10 years (recent HJS group). RESULTS: A total of 131 patients were included; 39% had undergone HJS for malignancy and 32% for congenital biliary dilation. Scope insertion and complete stone removal were successful in 89% and 73%, respectively. Early adverse events were observed in 9.9%. Four patients (3.1%) developed gastrointestinal perforation but could be managed conservatively. Hepatolithiasis recurrence rate was 17%, 20% and 31% in 1-year, 3-year, and 5-year after complete stone removal. The past HJS group was the only risk factor for failed stone removal (odds ratio 10.4, 95% confidence interval 2.99-36.5) in the multivariable analysis. Failed scope insertion (20%) and failed guidewire or device insertion to the bile duct (22%) were two major reasons for failed stone removal in the past HJS group. CONCLUSIONS: BE-ERCP for hepatolithiasis was effective and safe in cases with HJS but the complete stone removal rate was low in the past HJS group. Recurrent hepatolithiasis was common and careful follow up study is needed even after complete stone removal.

Skeletal muscle status and survival among patients with advanced biliary tract cancer.

BACKGROUND: Studies have demonstrated a prognostic role of sarcopenia (i.e., loss of skeletal muscle volume and functionality) in patients with various cancer types. In patients with biliary tract cancer, the quantity and quality of skeletal muscles and their serial changes have not been fully investigated in relation to survival outcomes. METHODS: We identified 386 patients with unresectable or recurrent biliary tract cancer and calculated skeletal muscle index (SMI) and skeletal muscle density (SMD) to estimate muscular quantity and quality, respectively, based on computed tomography images. Using the Cox regression model with adjustment for potential confounders, we calculated hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) and overall survival (OS) according to skeletal muscle status and its serial change. RESULTS: Compared to patients without sarcopenia, patients with sarcopenia were associated with shorter PFS (multivariable HR, 1.60; 95% CI, 1.15-2.22; P = 0.005), but not with OS (P = 0.027) at the adjusted α level of 0.013. SMD at baseline was associated with OS (multivariable HR comparing the extreme quartiles, 1.52; 95% CI, 1.07-2.14; Ptrend = 0.012), but not with PFS (Ptrend = 0.13). A reduction in SMI rather than that in SMD was associated with OS. Progressive disease was a risk factor for reductions in SMI and SMD. CONCLUSIONS: Skeletal muscle quantity and quality and their serial changes were associated with survival outcomes in patients with advanced biliary tract cancer. Our data highlight the importance of designing nutritional and physical interventions for improvements in skeletal muscle status.

Retrospective comparative study of new slim-delivery and conventional large-cell stents for stent-in-stent methods for hilar malignant biliary obstruction.

OBJECTIVES: Endoscopic management of unresectable hilar malignant biliary obstruction (HMBO) is technically challenging, and effectiveness of stent-in-stent using large-cell, metal stents was reported. A new, large-cell stent with a 6F tapered delivery system was recently developed. The aim of this study was to compare clinical outcomes of slim-delivery and conventional large-cell stents. METHODS: This was a multicenter retrospective comparative study of stent-in-stent methods using slim-delivery stents (Niti-S Large Cell SR Slim Delivery [LC slim-delivery]) and conventional stents (Niti-S large-cell D-type; LCD) for unresectable HMBO. RESULTS: Eighty-three patients with HMBO were included; 31 LC slim-delivery and 52 LCD. Overall technical and clinical success rates were 100% and 90% in LC slim-delivery group and 98% and 88% in LCD group. Use of the LC slim-delivery was associated with shorter stent placement time in the multiple regression analysis, with a stent placement time of 18 and 23 min in LC slim-delivery and LCD groups, respectively. The early adverse event (AE) rate of LC slim-delivery was 10%, with no cholangitis or cholecystitis as compared to 23% in the LCD group. Recurrent biliary obstruction (RBO) rates and time to RBO were comparable between the two groups: 35% and 44%, and 8.5 and 8.0 months in LC slim-delivery and LCD groups, respectively. The major cause of RBO was tumor ingrowth (82%) in the LC slim-delivery group and sludge (43%) and ingrowth (48%) in LCD group. CONCLUSION: Stent-in-stent methods using LC slim-delivery shortened stent placement time with low early AE rates and comparable time to RBO in patients with HMBO.

Development and external validation of a nomogram for prediction of post-endoscopic retrograde cholangiopancreatography pancreatitis.

BACKGROUND AND AIMS: Endoscopic retrograde cholangiopancreatography (ERCP) is widely performed for management of pancreatobiliary diseases; however, post-ERCP pancreatitis (PEP) remains as an unsolved problem. Although various risk factors for PEP have been reported, the prediction of PEP remains controversial. This study aimed to develop a predictive model for PEP. METHODS: Consecutive patients undergoing ERCP for biliary indications at two centers were retrospectively studied. Using data from a training cohort, we utilized a multivariable model to select five variables to construct a nomogram. The predictive model was internally and externally validated. Based on the nomogram, the patients were categorized into low-, moderate-, and high-risk groups. RESULTS: Using the data of 2224 patients in the training cohort, five variables were selected to generate a nomogram: 1) sex, 2) indication for ERCP, 3) difficult cannulation, 4) guidewire insertion into the pancreatic duct, and 5) endoscopic sphincterotomy or sphincteroplasty. The most significant risk factor was endoscopic papillary balloon dilation such as endoscopic sphincterotomy or sphincteroplasty. The bias-corrected concordance index was 0.72 in the training cohort and 0.72 in the validation cohort. Calibration curves for both cohorts demonstrated good agreement between the predicted and observed frequencies of the actual outcome. In the validation cohort, PEP developed in 5.0% and 14% of patients in the moderate- and high-risk groups, respectively. CONCLUSIONS: We successfully developed a good predictive model for PEP. The prevention of PEP in high risk patients should be investigated further.

Second Covered and Uncovered Self-Expandable Metal Stents for Recurrent Gastric Outlet Obstruction: A Retrospective Comparative Study.

Background: Endoscopic self-expandable metal stent (SEMS) placement is a current mainstay for malignant gastric outlet obstruction (GOO), but symptomatic recurrence due to initial SEMS dysfunction commonly occurs. We aimed to compare the safety and effectiveness of second SEMS for recurrent GOO (RGOO). Methods: Between April 2006 and December 2022, a total of 95 cases with malignant RGOO undergoing second endoscopic SEMS placement were enrolled. Technical and clinical success rates, RGOO, time to RGOO (TRGOO), stent patency rate, adverse events (AE), and overall survival (OS) were retrospectively compared between covered and uncovered SEMS (cSEMS/uSEMS) groups. Risk factors for TRGOO were also explored. Results: Baseline characteristics were well balanced between cSEMS (n = 48) and uSEMS (n = 47) groups, except for the causes of the initial SEMS dysfunction. High technical and clinical success rates with a similar incidence of AE (15% vs. 17%, p = 0.78) and OS (median of 101 vs. 102 days, p = 0.68) were achieved in both groups. There were no statistical differences in cumulative incidence of RGOO (19% vs. 13%, p = 0.58), TRGOO (median, not reached in both groups, p = 0.57), and stent patency rates at 1, 2, and 3 months between the groups (60%, 47% and 26%, respectively vs. 70%, 55% and 38%, respectively). However, TRGOO tended to be longer in cSEMS in cases with RGOO due to tumor ingrowth (median, not reached vs. 111 days, p = 0.19). A Cox regression analysis demonstrated that chemotherapy after second SEMS placement was significantly associated with an improved TRGOO (the hazard ratio of 0.27 [95% confidence interval, 0.08-0.93], p = 0.03). Conclusions: Regardless of the type of SEMS, second SEMS placement was similarly safe and effective for RGOO. The type of second SEMS might be considered based on the cause of initial SEMS dysfunction.

Clinical trajectory of intraductal papillary mucinous neoplasms progressing to pancreatic carcinomas during long-term surveillance: a prospective series of 100 carcinoma cases.

BACKGROUND: Trajectories of serological and morphological signatures have not been documented in pancreatic carcinogenesis related to intraductal papillary mucinous neoplasms (IPMNs). METHODS: Using a prospective cohort of 3437 IPMN patients, we identified 100 IPMN patients who developed pancreatic carcinomas during long-term surveillance. We examined serial changes of blood markers (carbohydrate antigen 19-9 [CA19-9], hemoglobin A1c [HbA1c], and pancreatic enzymes) and morphological features (worrisome features and high-risk stigmata) during the prediagnostic period of pancreatic carcinomas, overall and by carcinoma types (IPMN-derived vs. concomitant pancreatic carcinomas). RESULTS: CA19-9 elevation was observed in 39 patients and was associated with a metastatic stage. Compared to IPMN-derived carcinomas, concomitant carcinomas were more likely to represent CA19-9 elevation (60% vs. 30%, respectively; P = 0.005). HbA1c levels elevated only in 3 patients. Pancreatic enzyme elevation was observed in 18 patients with no differences in frequencies between the carcinoma types. All patients with elevated levels of blood markers had positive findings on cross-sectional imaging. High-risk stigmata or worrisome features were observed in all patients but one with concomitant carcinoma. The most common types of worrisome features were the main pancreatic duct dilatation and CA19-9 elevation in IPMN-derived and concomitant carcinomas, respectively. Compared to IPMN-derived carcinomas, concomitant carcinomas were less likely to harbor high-risk stigmata (16% vs. 86%, respectively; P < 0.001). CONCLUSIONS: The usefulness of currently available blood biomarkers was limited in early detection of pancreatic carcinomas related to IPMNs. Morphological alterations were well correlated with long-term risk of IPMN-derived carcinomas, but not with that of concomitant carcinomas.

Three Autopsy Cases of Non-Meningococcal Waterhouse-Friderichsen Syndrome with Hypoplastic Spleen or Post-Splenectomy Status.

We report three cases of Waterhouse-Friderichsen syndrome (WFS) that were confirmed during forensic autopsies. Case 1 involved a man in his 50s post-splenectomy. Bacteriological examination revealed Streptococcus pneumoniae (S. pneumonia). The patient was considered to have died of asphyxiation after aspirating vomit. Case 2 involved a man in his 40s. Bacteriological examination again revealed S. pneumoniae. Histopathological examination showed hypoplasia of the spleen. This patient was considered to have died of multiple-organ failure due to sepsis, disseminated intravascular coagulation, and WFS. Case 3 involved a post-splenectomy woman in her 60s with a history of systemic lupus erythematosus. Bacteriological examination revealed Streptococcus oralis. This patient was considered to have died of multiple-organ failure due to sepsis, disseminated intravascular coagulation, and WFS. These three cases were included among forensic autopsies conducted in the last 5 years. WFS has been considered a rare disease, but may be more frequent than previously assumed. If a mildly ill patient displays a sudden change in status and dies within a short period of time, we consider it necessary to perform not only bacteriological examinations, but also histopathological examination of the spleen during autopsy.

Pancreatic cancer secondary to intraductal papillary mucinous neoplasm with collision between gastric cancer and B-cell lymphoma: A case report.

BACKGROUND: Patients with intraductal papillary mucinous neoplasm (IPMN) have an increased risk of pancreatic and extrapancreatic malignancies. Lymphomas are rare extrapancreatic malignancies, and in situ collisions of early gastric cancer and diffuse large B-cell lymphoma (DLBCL) are even rarer. Here, we report the first case of pancreatic cancer comorbid with in situ collision of extrapancreatic malignancies (early gastric cancer and DLBCL) in a follow-up IPMN patient. Furthermore, we have made innovations in the treatment of such cases. CASE SUMMARY: An 81-year-old Japanese female diagnosed with IPMN developed elevated carbohydrate antigen (CA) 19-9 levels during follow-up. Because her CA19-9 levels continued to rise, endoscopic ultrasound (EUS) was performed and revealed a suspicious lesion at the pancreatic tail. However, lesions in the pancreas were not found by computed tomography, magnetic resonance imaging, or endoscopic retrograde cholangiopancreatography. To make an exact patho-logical diagnosis, EUS-guided fine needle aspiration was performed. To our supprise, early gastric cancer was found in preoperative gastroscopy. The gastric cancer was completely resected through endoscopic submucosal dissection before postoperative pathology identified early adenocarcinoma collided with DLBCL. Subsequent EUS-guided fine needle aspiration provided pathological support for the pancreatic cancer diagnosis, and then laparoscopic distal pancreatectomy and splenectomy were performed. CA19-9 levels returned to normal postoperatively. CONCLUSION: Endoscopic submucosal dissection is appropriate for submucosal lymphomas in patients intoleratant of chemotherapy. EUS can detect small IPMN-related pancreatic tumors.

Rapidly declining trend of signet ring cell cancer of the stomach may parallel the infection rate of Helicobacter pylori.

BACKGROUND: Studies indicate that gastric cancer (GC) incidence has decreased, whereas signet ring cell carcinoma (SRC) incidence has increased. However, recent trends in GC incidence are unclear. We used our hospital cancer registry to evaluate the changes in the incidence of GC, SRC, and non-SRC (NSRC) over time in comparison to changes in the H. pylori infection rates over time. METHODS: We identified 2532 patients with GC enrolled in our registry between January 2007 and December 2018 and statistically analyzed SRC and NSRC incidence. The H. pylori infection rate in patients with SRC was determined by serum anti-H. pylori antibody testing, urea breath test, biopsy specimen culture, and immunohistochemical analysis (IHC) of gastric tissue. Additionally, genomic detection of H. pylori was performed in SRCs by extracting DNA from formalin-fixed paraffin-embedded gastric tissue and targeting 16S ribosomal RNA of H. pylori. RESULTS: Overall, 211 patients had SRC (8.3%). Compared with patients with NSRC, those with SRC were younger (P <  0.001) and more likely to be female (P <  0.001). Time series analysis using an autoregressive integrated moving average model revealed a significant decrease in SRC (P <  0.001) incidence; NSRC incidence showed no decline. There was no difference in H. pylori infection prevalence between the SRC and NSRC groups. IHC and genomic methods detected H. pylori in 30 of 37 (81.1%) SRCs. CONCLUSIONS: Reduction in H. pylori infection prevalence may be associated with the decrease in the incidence of SRC, which was higher than that of NSRC.

Validation of Tokyo Guideline 2013 as Treatment of Acute Cholecystitis by Real World Data.

BACKGROUND: The Tokyo Guidelines (TG; 2013) indicated that emergency cholecystectomy is an important early treatment option for acute cholecystitis; however, surgical intervention is not necessarily indicated in patients with advanced age. We evaluated percutaneous transhepatic gallbladder aspiration (PTGBA), percutaneous transhepatic gallbladder drainage (PTGBD), and the administration of antibiotics alone as treatment options for acute -cholecystitis. METHODS: From January 2010 to December 2017, 159 patients with acute cholecystitis were treated at our institution. The data from these patients were retrospectively analyzed. RESULTS: Of these 159 cases, 109 underwent PTGBA, 28 underwent PTGBD, and 22 were administered antibiotics alone. None of the 159 patients needed urgent (early) cholecystectomy, and all patients were discharged without mortality. PTGBA was unsuccessful in only 6 of 109 patients; PTGBD was performed in these 6 cases. Long-term follow-up was conducted in all cases. Of the 159 patients, 146 had gallbladder stones initially, while 13 had none at the time of presentation. Of these 146 patients with gallbladder stones, 84 underwent elective cholecystectomy, while 62 did not. Of the 84 patients who underwent elective cholecystectomy, 2 developed choledocholithiasis; of the 62 patients who did not undergo elective cholecystectomy, 5 developed choledocholithiasis and 2 developed acute cholecystitis. The incidences of choledocholithiasis and acute cholecystitis did not significantly differ between the 2 groups (p = 0.06). CONCLUSIONS: Despite the recommendations in the TG (2013), emergency cholecystectomy was not needed in any of the present patients with acute cholecystitis. Acute cholecystitis can be successfully treated with -PTGBA or PTGBD, which are simple procedures with good short- and long-term safety. These procedures are highly recommended for patients with acute cholecystitis, especially in the elderly population.

Deficiency of mismatch repair genes is less frequently observed in signet ring cell compared with non-signet ring cell gastric cancer.

Signet ring cell (SRC) gastric cancer at advanced stage has poor prognosis. While a recent study reported nearly one-third of SRC cases contain tumors with deficient mismatch repair (MMR) genes, other studies in SRC have been inconclusive. To re-analyze the results, we performed immunohistochemical staining of MLH1, MSH2, MSH6 and PMS2 proteins in 38 SRC gastric tumors compared with 109 non-SRC (NSRC) tumors from 94 patients. In contrast to the previous study, all SRC gastric tumors normally expressed MMR proteins, whereas 22 of 109 of NSRC (20%) showed deficient MMR proteins. To reinforce our results, we referred to the Cancer Genome Atlas (TCGA) genomic database and found that only 6 (6%) of 99 samples with diffuse gastric tumors showed deficient MMR, whereas 64 (21%) of 304 in intestinal gastric tumors showed deficient MMR. Our results as well as the TCGA database indicated that MMR genes are infrequently inactivated in SRC gastric cancer. These findings indicate that SRC patients may not be the best candidates for immuno-oncology therapy.

Molecular subtype switching in early-stage gastric cancers with multiple occurrences.

BACKGROUND: Multiple gastric cancers at the same time (synchronous) or recurrence after 1 year (metachronous) are frequently encountered. Since their genetic profiles were not well elucidated, we molecularly subtyped the genetic events of synchronous and metachronous early-stage gastric cancers. METHODS: We studied mismatch repair (MMR) genes in 84 tumors from 31 patients (15 synchronous and 16 metachronous) by immunohistochemistry. We performed microsatellite instability analysis and targeted sequencing of 58 significantly mutated genes (SMGs) in 35 tumors from thirteen patients. Genomic data from TCGA were used for comparisons with advanced-stage cancers. RESULTS: Among the 31 patients, at least one deficient-MMR (dMMR) tumor was observed in eight (26%). Of eight patients, seven showed a mixture of proficient-MMR (pMMR) and dMMR tumors. The one case with only dMMR had six recurrent tumors within 2 years. To further subtype, we sequenced 58 SMGs in 35 samples (25 pMMR and 10 dMMR) from thirteen patients. In 35 samples, 163 mutations were identified, but none matched in almost cases, strongly indicating different clonal origins, whether synchronous or metachronous occurrences. Of the 25 pMMR cases, 1 belonged to Epstein-Barr virus (EBV), 24 belonged to chromosomal instability (CIN) subtypes. Of the thirteen cases, repetitive CIN, a mixture of CIN and MSI, a mixture of CIN and EBV, and repetitive MSI were observed in nine (70%), two (15%), one (8%) and one (8%), respectively. CONCLUSIONS: Despite multiple tumors occurring in the same patient simultaneously or several years apart, clonal origin was totally different. 'Switching' or 'mixing' of dMMR and pMMR, EBV or CIN occurred, which had clinical relevance with regard to immunotherapy.

Influence of NUDT15 variants on hematological pictures of patients with inflammatory bowel disease treated with thiopurines.

AIM: The single nucleotide polymorphism (SNP) c.415C>T in exon 3 of NUDT15 affects thiopurine-induced leukopenia in Asian patients with Crohn's disease. Meanwhile, three additional genetic variants of NUDT15 were reported in patients with acute lymphoblastic leukemia. We evaluated the effects of these additional genetic variants of NUDT15 in patients with inflammatory bowel disease (IBD) treated with thiopurines. METHODS: Ninety-six Japanese patients with IBD were enrolled. Genotyping for the NUDT15 and TPMT genes was performed using Custom TaqMan SNP genotyping assays or Sanger sequencing. The changes in white blood cell (WBC) count, mean corpuscular volume (MCV), platelet count, hemoglobin, CRP, amylase, albumin, AST, ALT, and ESR were evaluated. RESULTS: Genetic variants of exon 1 and exon 3 of NUDT15 were identified in 24 of 96 patients (25.0%). C.52G > A and c.36_37insGGAGTC in exon 1 were found in three patients each. All three patients with c.36_37insGGAGTC in exon 1 were heterozygotes of p.Arg139Cys in exon 3. Eighteen patients had p.Arg139Cys in exon 3 alone. The WBC count gradually decreased after initiation of thiopurine treatment in the mutated cases (n = 24), and was significantly lower at 6, 8, 10, and 16 wk (P = 0.0271, 0.0037, 0.0051, and 0.0185, respectively). The WBC counts were also evaluated in patients with and without prednisolone treatment. In the patients with prednisolone treatment, the WBC count tended to show a greater decrease in the mutated cases, with significant differences at 8 and 10 wk (P = 0.012 and 0.029, respectively). In the patients without prednisolone treatment, the WBC count was significantly lower at 2, 4, 8, and 14 wk in mutated cases (P = 0.0196, 0.0182, 0.0237 and 0.0241, respectively). MCV increased after starting thiopurine treatment in the mutated cases, and was significantly higher at 10 wk (P = 0.0085). Platelet count, hemoglobin, CRP, amylase, albumin, AST, ALT and ESR did not differ significantly between the wild-type and mutated cases. TPMT mutations were not found in any of the patients. CONCLUSION: Mutations in exon 1 of NUDT15 also affect thiopurine-induced leukopenia in patients with IBD. To discuss thiopurine-induced leukopenia in more detail, investigation of SNPs in both exon 1 and exon 3 of NUDT15 is needed.

Complex regulation of secondary metabolism controlling pathogenicity in the phytopathogenic fungus Alternaria alternata.

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes), which produce different host-selective toxins and cause disease on different plants. The Japanese pear, strawberry and tangerine pathotypes produce AK-toxin, AF-toxin and ACT-toxin, respectively, which have a common structural moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid (EDA). Here, we identified a new gene, AKT7 (AK-toxin biosynthetic gene 7), from the Japanese pear pathotype, which encodes a cytochrome P450 monooxygenase and functions to limit AK-toxin production. AKT7 homologs were found in the strawberry pathotype, but not the tangerine pathotype. However, the strawberry pathotype homolog appeared to include a premature stop codon. Although the Japanese pear pathotype strain has multiple copies of AKT7, a single-copy disruption resulted in mutants with increased production of AK-toxin and EDA. AKT7 overexpression in the three pathotypes caused marked reductions of toxin and EDA production, suggesting that Akt7 catalyzes a side reaction of EDA or its precursor. AKT7 overexpression caused reduced virulence in these pathotypes. We also found that AKT7 transcripts predominantly include misspliced mRNAs, which have premature stop codons. Our observations suggest that the AK-toxin production required for full virulence is regulated in a complex way by the copy number and intron information content of AKT7.