Clinical features and mutation analysis of class 1/2/3 BRAF mutation colorectal cancer.

BACKGROUND: BRAF (B-Raf proto-oncogene, serine/threonine kinase)-mutated colorectal cancer (CRC) still has poor prognostic. The efficacy of BRAF inhibitor is unpredictable just that intrinsic genetic complexity, immune microenvironment and partially unknown reason. Understanding the co-mutation mechanism can help improve treatment and follow-up strategies. METHODS: We retrospectively analyzed 35 (BRAF-mutated/BRAF wild-type) Chinese CRC and 125 Western CRC who underwent next-generation sequencing (NGS). Co-occurrence mutation analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was enabled in this study. RESULTS: Thirty-five (10.32%) patients were BRAF-mutated, with 17 patients were BRAF V600E in Beijing Hospital. Patients with BRAF mutation had significant association with high tumor mutational burden (TMB-H) (P=0.0004) and high microsatellite instability (MSI-H) (P=0.0003) than those with BRAF wild-type. In 125 BRAF-mutated Western CRC patients, the frequency of age at diagnosis, gender, sample type, Tumor-Node-Metastasis (TNM), MSI, TMB, and BRAF mutation type was consistent with Chinese data. However, the primary tumor location showed significant statistical differences (P<0.0001). Class 1 were more likely to occur in elder and female. Western cohort was consistent with above in Chinese cohort. Other clinicopathological features were not significantly associated with mutation type. However, Western cohort showed class 1 exhibited primary sample type predominance in both class 1 vs. others (P<0.05) and class 1 vs. class 3 (P<0.05). Meanwhile, the data showed TMB-H (57.69% vs. 11.76%, P<0.001) and MSI-H (28.21% vs. 0%, P<0.05) of the class 1 BRAF mutation proportion were significantly higher, compared with class 3 BRAF mutation. In concurrent oncogenic mutations, compared with non-class 1 BRAF mutation, class 1 are more likely to co-occur with passenger mutation. Data from Western populations showed similar results. We also found that the class 1 mutation was mutually exclusive with co-KRAS (Kirsten rat sarcoma viral oncogene homologue) mutation in CRC, and co-APC (APC regulator of WNT signaling pathway) mutation appeared more frequently in non-class 1 BRAF mutation. KEGG pathway showed that fewer proto-cancer signaling pathways were enriched in the class 1, which further confirmed that this type had stronger tumorigenicity. GO enrichment also proved that class 1 had stronger tumorigenicity. Finally, prognostic analysis showed median overall survival (mOS) of 19.43 months in class 1 vs. 47.57 months in non-class 1 (P=0.0002). Further study showed that the mOS of class 1, class 2, class 3 and class NA (unknown) was 19.43, 28.50, 47.57 months and not reached (P=0.0001), respectively. CONCLUSIONS: This study showed class 1/non-class 1 BRAF mutation in CRC had significantly differences in co-mutation features, genomic markers and prognostic. Understanding BRAF mutation types and co-mutation mechanism will contribute to accurately grasping treatment and follow-up strategies and promoting the development of precision therapy for CRC in the future.

Optic disc detection based on fully convolutional network and weighted matrix recovery model.

Eye diseases often affect human health. Accurate detection of the optic disc contour is one of the important steps in diagnosing and treating eye diseases. However, the structure of fundus images is complex, and the optic disc region is often disturbed by blood vessels. Considering that the optic disc is usually a saliency region in fundus images, we propose a weakly-supervised optic disc detection method based on the fully convolution neural network (FCN) combined with the weighted low-rank matrix recovery model (WLRR). Firstly, we extract the low-level features of the fundus image and cluster the pixels using the Simple Linear Iterative Clustering (SLIC) algorithm to generate the feature matrix. Secondly, the top-down semantic prior information provided by FCN and bottom-up background prior information of the optic disc region are used to jointly construct the prior information weighting matrix, which more accurately guides the decomposition of the feature matrix into a sparse matrix representing the optic disc and a low-rank matrix representing the background. Experimental results on the DRISHTI-GS dataset and IDRiD dataset show that our method can segment the optic disc region accurately, and its performance is better than existing weakly-supervised optic disc segmentation methods. Graphical abstract of optic disc segmentation.

Small extrachromosomal circular DNAs as biomarkers for multi-cancer diagnosis and monitoring.

BACKGROUND: Small extrachromosomal circular DNAs (eccDNAs) have the potential to be cancer biomarkers. However, the formation mechanisms and functions of small eccDNAs selected in carcinogenesis are not clear, and whether the small eccDNA profile in the plasma of cancer patients represents that in cancer tissues remains to be elucidated. METHODS: A novel sequencing workflow based on the nanopore sequencing platform was used to sequence naturally existing full-length small eccDNAs in tissues and plasma collected from 25 cancer patients (including prostate cancer, hepatocellular carcinoma and colorectal cancer), and from an independent validation cohort (including 7 cancer plasma and 14 healthy plasma). RESULTS: Compared with those in non-cancer tissues, small eccDNAs detected in cancer tissues had a significantly larger number and size (P = 0.040 and 2.2e-16, respectively), along with more even distribution and different formation mechanisms. Although small eccDNAs had different general characteristics and genomic annotation between cancer tissues and the paired plasma, they had similar formation mechanisms and cancer-related functions. Small eccDNAs originated from some specific genes had great multi-cancer diagnostic value in tissues (AUC ≥ 0.8) and plasma (AUC > 0.9), especially increasing the accuracy of multi-cancer prediction of CEA/CA19-9 levels. The high multi-cancer diagnostic value of small eccDNAs originated from ALK&ETV6 could be extrapolated from tissues (AUC = 0.804) to plasma and showed high positive predictive value (100%) and negative predictive value (82.35%) in a validation cohort. CONCLUSIONS: As independent and stable circular DNA molecules, small eccDNAs in both tissues and plasma can be used as ideal biomarkers for cost-effective multi-cancer diagnosis and monitoring.

Case report: Submucosal cavernous lymphangioma causing jejuno-jejunal intussusception in an adult.

Cavernous lymphangioma often occurs in the head, neck, trunk, and extremities of infants and children, and it is rare to cause a small intestine intussusception in adults. In this case, a 32-year-old woman presented with abdominal pain, vomiting, and a 5 cm × 5 cm abdominal mass on the left side of the abdomen. Laboratory tests showed anemia and CT showed small intestinal intussusception. After conservative treatments, her symptoms disappeared. However, 18F-FDG PET/CT suggested malignancy and her symptoms reappeared after eating something. Segmental jejunal resection was performed and pathology showed submucosal cavernous lymphangioma. At the 1-year follow-up, the patient was asymptomatic. Then this paper reviewed the literature on small intestinal cavernous lymphangioma in adults and found that this is the first English case report of intussusception caused by a jejunal submucosal cavernous lymphangioma in an adult. Current problem is that adult intussusception and intestinal lymphangioma are difficult to diagnose preoperatively. Imaging techniques such as tomography and PET/CT aid in the diagnosis of these benign lesions. Surgical resection was considered to be the required treatment and seems to have had no recurrence in adults according to the literature.

Clearance of circulating tumor DNA in a high-risk stage-IV rectal carcinoma patient with synchronous liver metastases after conversion surgery is correlated with pathologic complete response.

Colorectal cancer is the third most common cancer worldwide, and its incidence continues to grow. Approximately one-third of patients with colorectal cancer develop liver metastases during the natural course of disease. Complete surgical resection is associated with very low mortality in colorectal liver metastasis patients, but only a small fraction of colorectal liver metastasis patients fulfill the selection criteria for surgical treatment. We herein describe a high-risk stage-IV rectal carcinoma patient who was initially unresectable according to the National Comprehensive Cancer Network guidelines with a clinical risk score of 4 but received conversion surgery combined with systemic chemotherapy and achieved a favorable long-term clinical outcome (pathologic complete response) of approximately 28 months. Furthermore, serial circulating tumor DNA monitoring using next-generation sequencing provided a comprehensive view of the patient's clinical and pathologic status for better clinical decision support over the course of the disease. The absence of circulating tumor DNA/cells after conversion surgery was correlated with pathologic complete response. This case study not only demonstrated that a curative oncosurgical approach could be considered for high-risk colorectal liver metastasis patients under specific circumstances but also highlighted the role of circulating tumor DNA monitoring to gain further insight into the evolution of a patient's response over time.

Serial Circulating Tumor DNA in Predicting and Monitoring the Effect of Neoadjuvant Chemoradiotherapy in Patients with Rectal Cancer: A Prospective Multicenter Study.

PURPOSE: We investigated the value of circulating tumor DNA (ctDNA) in predicting tumor response to neoadjuvant chemoradiotherapy (nCRT), monitoring tumor burden, and prognosing survival in patients with locally advanced rectal cancer (LARC). EXPERIMENTAL DESIGN: This prospective multicenter trial recruited 106 patients with LARC for treatment with nCRT followed by surgery. Serial ctDNAs were analyzed by next-generation sequencing at four timepoints: at baseline, during nCRT, presurgery, and postsurgery. RESULTS: In total, 1,098 mutations were identified in tumor tissues of the 104 patients being analyzed (median, seven mutations/patient). ctDNA was detected in 75%, 15.6%, 10.5%, and 6.7% of cases at the four timepoints, respectively. None of the 29 patients with pathologic complete response (ypCR) had preoperative ctDNA detected. The preoperative ctDNA-positive rate was significantly lower in the well-responded patients with pathologic tumor regression grade of ypCAP 0-1 than ypCAP 2-3 group (P < 0.001), lower in ypCR than non-ypCR group (P = 0.02), and lower in pathologic T stage (ypT) 0-2 than ypT 3-4 group (P = 0.002). With a median follow-up of 18.8 months, 13 patients (12.5%) experienced distant metastasis. ctDNA positivity at all four timepoints was associated with a shorter metastasis-free survival (MFS; P < 0.05). Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in baseline ctDNA was a strong independent predictor of MFS (HR, 1.27; P < 0.001). CONCLUSIONS: We show that ctDNA is a real-time monitoring indicator that can accurately reflect the tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of MFS.

Transgenic expression of GFP-LC3 perturbs autophagy in exocrine pancreas and acute pancreatitis responses in mice.

Pancreatitis is a common, sometimes fatal, disease of exocrine pancreas, initiated by damaged acinar cells. Recent studies implicate disordered macroautophagy/autophagy in pancreatitis pathogenesis. ATG8/LC3 protein is critical for autophagosome formation and a widely used marker of autophagic vacuoles. Transgenic GFP-LC3 mice are a valuable tool to investigate autophagy ; however, comparison of homeostatic and disease responses between GFP-LC3 and wild-type (WT) mice has not been done. We examined the effects of GFP-LC3 expression on autophagy, acinar cell function, and experimental pancreatitis. Unexpectedly, GFP-LC3 expression markedly increased endogenous LC3-II level in pancreas, caused by downregulation of ATG4B, the protease that deconjugates/delipidates LC3-II. By contrast, GFP-LC3 expression had lesser or no effect on autophagy in liver, lung and spleen. Autophagic flux analysis showed that autophagosome formation in GFP-LC3 acinar cells increased 3-fold but was not fully counterbalanced by increased autophagic degradation. Acinar cell (ex vivo) pancreatitis inhibited autophagic flux in WT and essentially blocked it in GFP-LC3 cells. In vivo pancreatitis caused autophagy impairment in WT mice, manifest by upregulation of LC3-II and SQSTM1/p62, increased number and size of autophagic vacuoles, and decreased level of TFEB, all of which were exacerbated in GFP-LC3 mice. GFP-LC3 expression affected key pancreatitis responses; most dramatically, it worsened increases in serum AMY (amylase), a diagnostic marker of acute pancreatitis, in several mouse models. The results emphasize physiological importance of autophagy for acinar cell function, demonstrate organ-specific effects of GFP-LC3 expression, and indicate that application of GFP-LC3 mice in disease models should be done with caution.Abbreviations: AP: acute pancreatitis; Arg-AP: L-arginine-induced acute pancreatitis; ATG: autophagy-related (protein); AVs: autophagic vacuoles; CCK: cholecystokinin-8; CDE: choline-deficient, D,L-ethionine supplemented diet; CER: caerulein (ortholog of CCK); CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ER: endoplasmic reticulum; LAMP: lysosomal-associated membrane protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; TEM: transmission electron microscopy; TFEB: transcription factor EB; ZG: zymogen granule(s).

MicroRNA-423-5p inhibits colon cancer growth by promoting caspase-dependent apoptosis.

Plasma microRNA (miR)-423-5p is a potential biomarker for the detection of colon cancer. However, the expression and biological role of miR-423-5p in colon tumorigenesis remains unclear. In the current study, reverse transcription-quantitative polymerase chain reaction was used to determine miR-423-5p expression in malignant colon tissues and plasma from patients with colon cancer. Cell viability, colony formation and apoptosis assays, as well as western blotting, were performed to investigate the biological role and regulatory mechanisms of miR-423-5p in colon cancer. The results demonstrated that miR-423-5p expression was downregulated in tumor tissues and plasma from patients with colon cancer, as well as in colon cancer cell lines. Furthermore, overexpression of miR-423-5p promoted colon cancer cell apoptosis and resulted in the inhibition of cell proliferation and colony formation. Mechanistically, miR-423-5p induced the expression of caspases 3, 8 and 9, as well as p53 in colon cancer. The effect of z-VAD treatment indicated that the miR-423-5p-mediated colon cancer cell apoptosis is caspase-dependent. These results suggest that miR-423-5p is a tumor suppressor in colon cancer and a potential diagnostic target to enable the early detection of colon cancer.

Bioinformatic Analysis of GLI1 and Related Signaling Pathways in Chemosensitivity of Gastric Cancer.

BACKGROUND This study assessed the prognostic value of GLI1 in gastric cancer and analyzed the possible GLI1-related signaling network in chemosensitivity. MATERIAL AND METHODS Bioinformatic data mining was performed by using data in the TCGA-Stomach Cancer (TCGA-STAD) and the Kaplan-Meier plotter. GLI1 co-expressed genes in TCGA-STAD were subjected to KEGG pathway analysis. The genes enriched in the KEGG pathways were further subjected to Protein-Protein Interaction (PPI) analysis. RESULTS In TCGA-STAD, high GLI1 gene/exon expression was associated with significantly worse survival (p=0.016 and 0.0023 respectively). In the Kaplan-Meier plotter, high GLI1 expression was associated with unfavorable overall survival (OS) (HR: 1.68, 95%CI: 1.42-2, p<0.0001) and first progression-free survival (FPS) (HR: 1.72, 95%CI: 1.4-2.11, p<0.0001). In TCGA-STAD, 600 GLI1 co-expressed genes were identified (absolute Pearson's r ≥0.5). The most significant pathways were pathways in cancer (p=230.0E-12) and the Hedgehog signaling pathway (p=6.9E-9). PI3K-AKT pathway (p=17.0E-9) has the largest proportion of gene enrichment. Some GLI1 co-expressed genes in the PI3K-AKT pathway are central nodes in the PPI network and also play important roles in chemosensitivity of gastric cancer. Nevertheless, the mechanisms underlying their co-expression are still largely unexplored. CONCLUSIONS High GLI1 expression is associated with unfavorable OS and FPS in patients with gastric cancer. As a member of the Hedgehog signaling pathway, GLI1 co-expressed genes are also largely enriched in PI3K/AKT pathway in gastric cancer, which is closely related to chemoresistance. The underlying mechanisms are still largely unexplored and need further study.

Clinical effect of DAPK promoter methylation in gastric cancer: A systematic meta-analysis.

BACKGROUND: The loss of death-associated protein kinase (DAPK) gene expression through promoter methylation is involved in many tumors. However, the relationship between DAPK promoter methylation and clinicopathological features of gastric cancer (GC) remains to be done. Therefore, we performed a meta-analysis to assess the role of DAPK promoter methylation in GC. METHODS: Literature databases were searched to retrieve eligible studies. The pooled odds ratios (ORs) with its 95% confidence intervals (CIs) were calculated using the Stata 12.0 software. RESULTS: Final 22 available studies with 1606 GC patients and 1508 nonmalignant controls were analyzed. A significant correlation was found between DAPK promoter methylation and GC (OR = 3.23, 95% CI = 1.70-6.14, P < 0.001), but we did not find any significant association in Caucasian population, and in blood samples in subgroup analyses. DAPK promoter methylation was associated with tumor stage and lymph node status (OR = 0.69, 95% CI = 0.49-0.96, P = 0.03; OR = 1.50, 95% CI = 1.12-2.01, P = 0.007; respectively). However, we did not find that DAPK promoter methylation was associated with gender status and tumor histology. CONCLUSION: Our findings suggested that DAPK promoter methylation may play a key role in the carcinogenesis and progression of GC. In addition, methylated DAPK was a susceptible gene for Asian population. However, more studies with larger subjects should be done to further evaluate the effect of DAPK promoter methylation in GC patients, especially in blood and Caucasian population subgroup.

[Comparative analysis of postoperative complications on elderly colorectal cancer patients over 65 years with and without comorbid cardiovascular diseases].

OBJECTIVE: To investigate the risk of postoperative complications in elderly colorectal cancer patients over 65 years with comorbid cardiovascular diseases. METHODS: A total of 381 elderly colorectal cancer patients over 65 years were pathologically diagnosed as colorectal adenocarcinoma and underwent the first surgery in Beijing Hospital during January 2013 and December 2014. Patients were divided into comorbid cardiovascular disease group (258 cases) and non-cardiovascular disease group (123 cases) according to the existence of comorbid cardiovascular disease. The morbidity of postoperative complication was compared between two groups. RESULTS: There was no significant difference in the morbidity of postoperative complication between two groups [27.9%(72/258) vs. 29.3%(36/123), P>0.05]. According to the Clavien-Dindo classification of postoperative complications, the morbidities of complication at all levels between two groups were not significantly different(all P>0.05). But in terms of cardiovascular complications, the morbidity of comorbid cardiovascular disease group was significantly higher than that of non-cardiovascular disease group [7.4%(19/258) vs. 0.8%(1/123), χ2=6.678, P=0.010], while no significant differences in pulmonary and abdominal complications were found between two groups(all P>0.05). The morbidities of other complications (deep vein thrombosis, urinary tract infection and renal complications, etc.) of comorbid cardiovascular disease group were lower than those in non-cardiovascular disease group [2.7%(7/258) vs. 8.1%(10/123), χ2=5.733, P=0.017]. Different types of cardiovascular diseases, different levels of cardiac risk index and American Society of Anesthesiologists(ASA) rating were not significantly related to the patient's occurrence of postoperative complications(all P>0.05). CONCLUSIONS: Surgery treatment for elderly colorectal cancer patients over 65 years with comorbid cardiovascular diseases is safe. However, strict cardiovascular monitoring should be performed and necessary measures should be carried out in time.

MicroRNA-190 regulates FOXP2 genes in human gastric cancer.

OBJECTIVE: To investigate how microRNA-190 (miR-190) regulates FOXP2 genes in gastric cancer (GC) cell line SGC7901. METHODS: We identified that miR-190 could target FOXP2 genes by using dual luciferase enzyme assay. Precursor fragment transfection of miR-190 was performed with GC cell line SGC7901 and human gastric mucosal cell line GES-1. miR-190 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and FOXP2 protein expression was measured by Western blotting. RESULTS: FOXP2-3'-untranslated region (UTR) in miR-190 transfection group was significantly decreased as compared with other groups. There were no significant differences in fluorescence signals of FOXP2mut-3'-UTR in each group. Therefore, it was assumed that miR-190 can target FOXP2 genes. Through RT-PCR verification, it was observed that the expression level of miR-190 was significantly higher in GC cell line SGC7901 than in human gastric mucosa cell line GES-1 after transfection with miR-190 mimics. The expression level of miR-190 was significantly higher in GES-1 cells than in SGC7901 cells after transfection with miR-190 inhibitors. Western blotting results showed the expression level of FOXP2 was significantly lower in GC cell line SGC7901 than in GES-1 cells. Compared with blank, mimics control, and inhibitors control groups, the miR-190 mimics group showed significantly enhanced proliferation, migration, and invasion abilities, while miR-190 inhibitors group showed decreased abilities toward proliferation, migration, and invasion (P<0.05). The transcription level of miR-190 and the expression level of FOXP2 in tumor tissues and adjacent normal tissues in GC patients were verified to be consistent with those of cell line experiments. CONCLUSION: Upregulation of miR-190 can lead to downregulation of FOXP2 protein expression. miR-190 may serve as a potential target for GC diagnosis.

Genetic inhibition of protein kinase Cε attenuates necrosis in experimental pancreatitis.

Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis.

[Laparoscopic with extralevator abdominoperineal excision for low rectal carcinoma].

OBJECTIVE: To explore the feasibility of laparoscopic with extralevator abdominoperineal excision (LELAPE group) for low rectal cancer. METHODS: From June 2011 to January 2013, 35 patients with low rectal cancer undergoing laparoscopic abdominoperineal excision at the Department of Gastroenterological Surgery, Beijing Hospital were analyzed retrospectively. Among them, 20 received laparoscopic abdominoperineal excision (LAPE group). There were 12 males and 8 females with an average age of (63 ± 6) years old. Another 15 patients underwent laparoscopic extralevator abdominoperineal excision (LELAPE group). There were 10 males and 5 females with an average age of (61 ± 7) years old. Operative duration, blood loss volume, time of postoperative out-of-bed activity, recovery of gastrointestinal function, removal time of drainage tube, edge of perineal position take out stitches time, postoperative hospital stay and complication rates were relative analyzed. RESULTS: There was no significant difference in operative time, time of postoperative out-of-bed activity, recovery of gastrointestinal function, removal time of perineal stitches, postoperative hospital stay and complication rates between 2 groups ((259 ± 52) vs (246 ± 55) min, (35 ± 13) vs (33 ± 9) d,(61 ± 25) vs (63 ± 20) h, (15.7 ± 2.5 ) vs (16.8 ± 2.9) d, (12 ± 3) vs (15 ± 4) d, 2/15 vs 3/20, all P > 0.05). Blood loss volume of perineal position in LELAPE group was less than those in LAPE group ((76 ± 31) vs (148 ± 36) ml, P < 0.05). Removal of perineal drainage tube in LELAPE group was earlier than that in LAPE group ((6.2 ± 1.6) vs (10.3 ± 1.8) d, P < 0.05). CONCLUSION: LELAPE is a safe and feasible surgical approach for low rectal cancer.

Impaired autophagy and organellar dysfunction in pancreatitis.

Recent findings from our group, obtained on experimental in vivo and ex vivo models of pancreatitis, reveal that this disease causes a profound dysfunction of key cellular organelles, lysosomes and mitochondria. We found that autophagy, the main cellular degradative, lysosome-driven process, is activated but also impaired in acute pancreatitis because of its' inefficient progression/resolution (flux) resulting from defective function of lysosomes. One mechanism underlying the lysosomal dysfunction in pancreatitis is abnormal processing (maturation) and activation of cathepsins, major lysosomal hydrolases; another is a decrease in pancreatic levels of key lysosomal membrane proteins LAMP-1 and LAMP-2. Our data indicate that lysosomal dysfunction plays an important initiating role in pancreatitis pathobiology. The impaired autophagy mediates vacuole accumulation in acinar cells; furthermore, the abnormal maturation and activation of cathepsins leads to increase in intra-acinar trypsin, the hallmark of pancreatitis; and LAMP-2 deficiency causes inflammation and acinar cell necrosis. Thus, the autophagic and lysosomal dysfunctions mediate key pathologic responses of pancreatitis. On the other hand, we showed that pancreatitis causes acinar cell mitochondria depolarization, mediated by the permeability transition pore (PTP). Genetic (via deletion of cyclophilin D) inactivation of PTP prevents mitochondrial depolarization and greatly ameliorates the pathologic responses of pancreatitis. Further, our data suggest that mitochondrial damage, by stimulating autophagy, increases the demand for efficient lysosomal degradation and therefore aggravates the pathologic consequences of lysosomal dysfunction. Thus, the combined autophagic, lysosomal and mitochondrial dysfunctions are key to the pathogenesis of pancreatitis.