Genetic Diagnosis of Hereditary Hemorrhagic Telangiectasia: Four Novel Pathogenic Variations in Turkish Patients

Aims: Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder characterized by telangiectasia, epistaxis, and vascular malformations. Pathogenic mutations were found in ENG, AVCRL1, SMAD4, and GDF genes. In this study, we present our database of patients with hereditary hemorrhagic telangiectasia regarding the phenotype-genotype relations and discuss two novel ENG gene pathogenic variations in two unrelated families. Methods: Next Generation Sequencing analysis was performed on the peripheral blood of nine patients with hereditary hemorrhagic telangiectasia in four unrelated families. All patients were diagnosed with hereditary hemorrhagic telangiectasia according to the Curaçao criteria. Data on treatment and screenings of visceral involvement were recorded from files. Results: We have found a pathogenic variation in either the ENG or ACVRL1 gene in each family. Two novel pathogenic variations in the ENG gene, including NM_000118.3 (ENG): c.416delC (p.P139fs*24) and NM_000118.3(ENG): c.1139dupT (p.Leu380PhefsTer16), were found in the same family. The NM_000020.2(ACVRL1): c.1298C>T (p.Pro433Leu) pathogenic variation in the ACVRL1 gene in our first family and a novel heterozygous likely pathogenic NM_000020.2(ACVRL1): c.95T>C (p.Val32Ala) variation was found in our second family. Seven of the nine patients were treated with thalidomide for controlling bleeding episodes. All patients responded to thalidomide. In one patient, the response to thalidomide was lost and switched to bevacizumab. Conclusion: In HHT certain type of mutations correlates with disease phenotypes and with next generation sequencing method, new pathogenic variations can be revealed which might help managing HHT patients.

Prognostic Impact of Canonical TGF-ß Signaling in Urothelial Bladder Cancer.

Background and objectives: Dysregulation of TGF-ß signaling plays multiple roles in cancer development and progression. In the canonical TGF-ß pathway, TGF-ß regulates the expression of hundreds of target genes via interaction with Smads, signal transducers and transcriptional modulators. We evaluated the association of TGF-ß1, Smad2, and Smad4, the key components of canonical TGFß pathway, with clinicopathologic characteristics of urothelial bladder cancer, and assessed their prognostic value in prediction of patients' outcome. Materials and Methods: Immunohistochemical analysis of TGF-ß1, Smad2, and Smad4 expression was performed on 404 urothelial bladder cancer samples, incorporated in tissue microarrays. Expression status was correlated with clinicopathological and follow-up data. The median follow-up was 61 months. Results: High expression of TGF-ß1, Smad2, and Smad4 was detected in 68.1%, 31.7% and 45.2% of the tumors, respectively. TGF-ß1 overexpression was significantly associated with high tumor grade, and advanced pathologic stage (p < 0.001, respectively). Conversely, high Smad2 and Smad4 expression was linked to low tumor grade (p = 0,003, p = 0.048, respectively), and low tumor stage (p < 0.001, p = 0.003, respectively). Smad2 showed an inverse correlation with variant morphology and divergent differentiation of urothelial tumors (p = 0.014). High TGF-ß1 correlated directly, while Smad2 and Smad4 correlated inversely to cancer-specific death (p = 0.043, p = 0.003, and p = 0.022, respectively). There was a strong relationship between Smad2 and Smad4 expression (p < 0.001). Survival analyses showed that high Smad2 and Smad4 expression was associated with longer overall survival (p = 0.003, p = 0.034, respectively), while in multivariate regression analysis TGF-ß1 manifested as an independent predictor of poor outcome. Conclusions: Unraveling the complex roles and significance of TGF-ß signaling in urothelial bladder cancer might have important implications for therapy of this disease. Assessment of TGF-ß pathway status in patients with urothelial bladder cancer may provide useful prognostic information, and identify patients that could have the most benefit from therapy targeting TGF-ß signaling cascade.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

AIMS: SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. METHODS AND RESULTS: Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. CONCLUSION: In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknown or uncertain primary site.

Frequent loss of heterozygosity of SMAD4 locus and prognostic impacts of SMAD4 immunohistochemistry in gastric adenocarcinoma with enteroblastic differentiation.

Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a rare variant of gastric adenocarcinoma. Clinicopathologically, GAED is known to be aggressive and is characterized by frequent vascular invasion, lymphatic invasion, and liver metastasis even in early stages. SMAD4 was identified as a frequently deleted gene in GAED by copy number variation analysis in our previous next-generation sequencing study; therefore, we examined the clinicopathological impacts of SMAD4 in 51 cases of GAEDs (early: 17, advanced: 34). We performed Sanger sequencing for SMAD4 mutations and loss of heterozygosity (LOH) analysis of the SMAD4 locus, in addition to immunohistochemistry for SMAD4, to determine its clinicopathological correlations and impacts on survival. The frequency of LOH at the SMAD4 locus was 45.1%, and it was significantly higher in GAED compared to in conventional gastric adenocarcinoma. SMAD4 mutations were not found in any case. Reduced SMAD4 expression was found in 60.8% of cases; it was significantly correlated with advanced stages and lymph node metastasis and showed trends of larger tumor size and lymphatic invasion. Reduced SMAD4 expression in metastatic lymph nodes was found in 21 of 36 cases. Survival analysis revealed that reduced SMAD4 expression significantly affected the patient's overall survival (OS) and recurrence-free survival (RFS), although multivariate analysis showed that only liver metastasis and lymphatic infiltration (Ly+) were independent prognostic factors for OS and RFS. The SMAD4 locus is one of the susceptibility genes in this tumor, although SMAD4 mutation was not detected. Furthermore, the inactivation of SMAD4 appeared to contribute to the aggressiveness of GAED.

Genetic assessment of recurrent pancreatic high-risk lesions in the remnant pancreas: Metachronous multifocal lesion or local recurrence?

BACKGROUND: It is difficult to determine whether a second high-risk lesion, including pancreatic ductal adenocarcinoma or high-grade pancreatic intraepithelial neoplasm, is a metachronous multifocal lesion or represents local recurrence after resection of the first high-risk lesion. This study attempts to clarify the characteristics of second high-risk lesions in the remnant pancreas using genetic analyses. METHODS: Clinicopathologic data were collected from 12 patients who underwent pancreatectomy for a second high-risk lesion in the remnant pancreas. We performed mutational and immunohistochemical analyses of 4 major genes-KRAS, TP53, CDKN2A, and SMAD4-associated with pancreatic ductal adenocarcinoma progression, as well as targeted next-generation sequencing. RESULTS: Mutations in the four genes in the second high-risk lesion were consistent with the first lesion in four patients but were inconsistent in the remaining eight patients, and thus we considered that the latter eight patients likely had metachronous multifocal high-risk lesions and the other four patients had local recurrence. The estimated cumulative recurrence rate after resection of the second high-risk lesion was greater in the local recurrence group compared with the metachronous multifocal group, and the estimated cumulative disease-specific survival rate was greater in the metachronous multifocal group. Targeted next-generation sequencing demonstrated that the second lesions in the metachronous multifocal high-risk lesion group showed differences in founder mutations compared with the first lesion. In the local recurrence group, the founder mutations in the second lesion were common with those in the first lesion. CONCLUSION: Genetic assessment might help discriminate metachronous multifocal high-risk lesions from local recurrence.

Cancerization of the Pancreatic Ducts: Demonstration of a Common and Under-recognized Process Using Immunolabeling of Paired Duct Lesions and Invasive Pancreatic Ductal Adenocarcinoma for p53 and Smad4 Expression.

Invasive pancreatic ductal adenocarcinoma (PDAC) can infiltrate back into and spread along preexisting pancreatic ducts and ductules in a process known as cancerization of ducts (COD). Histologically COD can mimic high-grade pancreatic intraepithelial neoplasia (HG-PanIN). We reviewed pancreatic resections from 100 patients with PDAC for the presence or absence of ducts with histologic features of COD. Features supporting COD included adjacent histologically similar invasive PDAC and an abrupt transition between markedly atypical intraductal epithelium and normal duct epithelium or circumferential involvement of a duct. As the TP53 and SMAD4 genes are frequently targeted in invasive PDAC but not HG-PanIN, paired PDAC and histologically suspected COD lesions were immunolabeled with antibodies to the p53 and Smad4 proteins. Suspected COD was identified on hematoxylin and eosin sections in 89 (89%) of the cases. Immunolabeling for p53 and Smad4 was performed in 68 (76%) of 89 cases. p53 was interpretable in 55 cases and all 55 (100%) cases showed concordant labeling between COD and invasive PDAC. There was matched aberrant p53 immunolabeling in 37 (67%) cases including overexpression in 30 (55%) cases and lack of expression in 7 (13%) cases. Smad4 immunolabeling was interpretable in 61 cases and 59 (97%) cases showed concordant labeling between COD and invasive PDAC. Matched loss of Smad4 was seen in 28 (46%) cases. The immunolabeling of invasive PDAC and COD for p53 and Smad4 supports the high prevalence of COD observed on hematoxylin and eosin and highlights the utility of p53 and Smad4 immunolabeling in differentiating COD and HG-PanIN.

Expression of inflammatory and fibrogenetic markers in acne hypertrophic scar formation: focusing on role of TGF-ß and IGF-1R.

Acne vulgaris is a universal skin disease and it may leave a scar when the original skin lesion disappears. These scars can cause cosmetic problems and psychological burden, leading to poor quality of life of patients. Acne scars are classified into atrophic scars and hypertrophic scars. As most of the acne scars are atrophic, many studies have been conducted focusing on the treatment of atrophic lesions. This study was conducted to investigate the underlying pathogenesis of acne hypertrophic scars by identifying roles of fibrogenetic and inflammatory markers. Skin biopsy samples were obtained from hypertrophic scars of face and back and from adjacent normal tissues as control group. Some samples from back were immature hypertrophic scars and the other samples were in mature stages. Immunohistochemistry staining and quantitative PCR were performed for fibrogenetic and inflammatory markers. Both in mature and immature hypertrophic scars, vimentin and α-SMA were increased. Production of TGF-ß3 protein as well as transcription of TGF-ß3 was also significantly elevated. In contrast, expression of TGF-ß1 showed no increase. Instead, expression levels of SMAD2 and SMAD4 were increased. Elevations of CD45RO, TNF-α and IL-4 and reduction of IL-10 were observed. In immature hypertrophic scars, IGF-1R and insulin-degrading enzyme expression were increased. Increased apoptosis was observed in immature stages of hypertrophic scars but not in mature stages. Elevations of TGF-ß3, SMAD2 and SMAD4 in hypertrophic scars and increase of IGF-1R in immature stages may give some clues for acne hypertrophic scar formation.

Smad4 and epithelial-mesenchymal transition proteins in colorectal carcinoma: an immunohistochemical study.

Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. During EMT, tumor cells acquire the capacity to migrate and invade the stroma. Activation of the transforming growth factor-b (TGF-b) signaling pathway is of major importance for the initiation of EMT. Smad4, an essential protein of this pathway, is known to complex with multiple transcription factors (e.g. Snail-1, Slug, Twist-1), in various types of cancer, promoting the repression or activation of target genes. The role of Smad4 in colorectal cancer (CRC) is not straightforward so far. In the present study forty eight resected CRC tumor specimens were immunohistochemically examined in order to assess the expression of Smad4 and its association with E-cadherin, Snail-1, Slug, Twist-1 protein expression and with various pathological parameters. Smad4 was found to be positively correlated with Snail-1, Slug and Twist-1 expression (p < 0.001). On the other hand it was negatively correlated with the expression of E-cadherin (p < 0.001). Furthermore, lymphatic invasion could be clearly associated with Smad4 expression, a finding complying with the metastatic ability of EMT cells. In conclusion, Smad4 could be considered as a central component of EMT transition in human colorectal cancer that combines with transcriptional factors to reduce E-cadherin and alter the expression of the epithelial phenotype.

Effect of genistein on myocardial fibrosis in diabetic rats and its mechanism.

The aim of the present study was to investigate the effects of genistein (GEN) on myocardial fibrosis in type 1 diabetic rats and explore the underlying mechanisms. Rats were divided into 4 groups: Normal control (N), diabetic control (D), low­dose GEN treatment (L) and high­dose GEN treatment (H) groups. Following 8 weeks, the ventricular hemodynamic parameters, fasting blood glucose (FBG), heart­weight to body­weight ratio (HW/BW), myocardial hydroxyproline (Hyp) content, serum creatine kinase MB isozyme (CK­MB), lactate dehydrogenase (LDH), tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß) and interleukin­6 (IL­6) levels were measured. The histomorphology and ultrastructure of the heart were observed. The protein expression of myocardial transforming growth factor­ß1 (TGF­ß1), mothers against decapentaplegic homolog (Smad)­3, phosphorylated (p)­Smad3, Smad4, collagen­I and collagen­III were estimated. Compared with the N group, while the cardiac function was decreased, the levels of FBG, HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 were increased in the D group. The myocardial histomorphological alterations and ultrastructure were damaged, and the protein expression of myocardial TGF­ß1, Smad3, p­Smad3, Smad4, collagen­I and collagen­III were increased in the D group. Compared with the D group, there were no differences in the ventricular hemodynamic parameters, FBG and p­Smad3 expression in the L group, while HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 levels were decreased. The myocardial histomorphological damage was alleviated and the protein expression of TGF­ß1, Smad3, Smad4, collagen­I and collagen­III was decreased in the L group. Compared with L group, excluding FBG, the aforementioned indices were improved in the H group. In conclusion, GEN can attenuate myocardial fibrosis in type 1 diabetic rats, and the underlying mechanisms may be associated with the reduction of CK­MB and LDH leakage, inhibition of the inflammatory reaction, and suppression of the TGF­ß1/Smad3 signaling pathway to regulate collagen expression.

Prognostic Value of c-MET Expression in Patients With Pancreatic Cancer Receiving Adjuvant and Neoadjuvant Chemoradiation Therapy.

PURPOSE: To determine the prognostic significance of c-MET expression and develop a predictor of distant failure in patients with resectable pancreatic cancer treated with chemoradiation. METHODS AND MATERIALS: We used a tissue microarray to study protein expression by immunohistochemistry in 102 patients treated surgically for pancreatic cancer. Two cores per patient were blindly scored from 0 (no staining) to 3 (strong staining) by a single pathologist. The Kaplan-Meier method was used to determine time to local and distant failure, overall survival, and progression-free survival. P values were calculated with the log-rank test. RESULTS: High tumor expression of c-MET was associated with a shorter time to distant failure in patients receiving neoadjuvant (n=23) or neoadjuvant therapy (n=73) (median 8.9 months vs 22.0 months, P=.0010). We then examined the ability of incorporating 2 known biomarkers, thymidylate synthase and DPC4 (SMAD4), with c-MET to risk-stratify patients. This multi-protein predictor divided our cohort into groups of similar numbers and was predictive of distant failure (median 13.4 months vs 24.2 months, P=.0094) but not of local control. CONCLUSION: c-MET is potentially predictive of distant failure. Using c-MET, DPC4, and thymidylate synthase, we developed a multi-protein predictor that could be used to risk-stratify patients and guide decisions regarding the sequencing of locoregional and systemic therapies in pancreatic cancer.

[The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins]. / Stepen' pokrytiia aminokislotnoi posledovatel'nosti pri ispol'zovanii razlichnykh metodov analiza mass-spektrometricheskikh dannykh, poluchennykh na model'nykh belkakh.

The aim of this study was to evaluate sequence coverage of five model proteins (CYB5A, SMAD4, RAB27B, FECH, and CXXC1) by means of shotgun proteomic data analysis employing different methods of data treatment including database-dependent search engines (MASCOT and X!Tandem) and de novo sequencing software ((PEAKS, Novor, and PepNovo+). In order to achieve maximal results, multiprotease hydrolysis including enzymes trypsin, LYS-C, ASPN and GluC was performed in solution and using the FASP method. High resolution mass spectrometry was carried out with a Q EXACTIVE HF hybrid mass spectrometer in the positive ionization mode; parent ions with the highest intensity and a charge range from +2 to +6 were fragmented in the HCD mode. 27 experiments were carried out (hydrolysis with each of 5 enzymes in solution, 4 for the FASP protocol, three technical repeats). Using parameters limiting false identification of peptides, the search engines and de novo sequencing software gave similar results. The degree of sequence coverage was not at least 40%, and in the best cases it reached 80-90%. The use of de novo sequencing software resulted in identification of the Y12H amino acid substitution in one model protein (CYB5A).

Significance of positive and inhibitory regulators in the TGF-ß signaling pathway in colorectal cancers.

Inactivation of genes in the transforming growth factor (TGF)-ß/SMAD signaling pathway is a well-known step for the progression of colorectal cancers (CRCs). Genetic mutations can occur in the precursors, and the combined prevalence of SMAD4, SMAD2, and SMAD3 mutations was seen in up to 50% of CRCs. High levels of serum TGF-ß1 were reported in patients with CRC and were associated with poor clinical outcome. PPM1A is an important inhibitory regulator in the TGF-ß signaling pathway and contributes to terminating the TGF-ß/SMAD signaling activity. We recently showed that PPM1A expression was lost in approximately 45% of pancreatic ductal adenocarcinomas and loss of PPM1A was associated with worse overall survival. Genome-wide analyses from The Cancer Genome Atlas revealed that abnormal TGF-ß signaling pathway is among the most common molecular changes in CRC. The complexity of the TGF-ß signaling pathway is its dual function as a tumor suppressor and tumor-promoting factor, depending on the cellular and molecular context. In this study, we simultaneously investigated the protein expression pattern of 3 regulators in the TGF-ß/SMAD signaling pathway, including SMAD4, PPM1A, and TGF-ß1, and their clinicopathological correlations in CRCs by immunohistochemistry. We observed that loss of SMAD4 and PPM1A was seen in 37.8% and 7.3% of CRCs, respectively. Loss of SMAD4, lymphovascular invasion, and distant metastasis were independently associated with worse overall survival in multivariate analysis. However, loss of PPM1A was associated with worse overall survival with less statistical strength. Our findings would provide new insights into the pathophysiological function of different components in the TGF-ß signaling pathway in CRC.

Statin Use After Diagnosis of Colon Cancer and Patient Survival.

BACKGROUND & AIMS: Statin use has been associated with a reduced incidence of colorectal cancer and might also affect survival of patients diagnosed with colon cancer. Statins are believed to inhibit Ras signaling and may also activate the bone morphogenetic protein (BMP) signaling pathway in colorectal cancer cells. We investigated the effects of statins on overall survival of patients with a diagnosis of colon cancer, and whether their effects were associated with changes in KRAS or the BMP signaling pathways. METHODS: Data were derived from the PHARMO database network (Netherlands) and linked to patients diagnosed with colon cancer from 2002 through 2007, listed in the Eindhoven Cancer Registry. We obtained information on causes of death from statistics Netherlands. We constructed a tissue microarray of 999 colon cancer specimens from patients who underwent surgical resection from 2002 through 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariable Poisson regression survival models and Cox analyses were used to study the effect of statins on survival. Tumor tissues were analyzed by immunohistochemistry for levels of SMAD4, BMPR1A, BMPR1B, and BMPR2 proteins. Tumor tissues were considered to have intact BMP signaling if they contained SMAD4 plus BMPR1A, BMPR1B, or BMPR2. DNA was isolated from tumor tissues and analyzed by quantitative polymerase chain reaction to detect mutations in KRAS. The primary outcome measures were overall mortality and cancer-specific mortality. RESULTS: In this cohort, 21.0% of the patients (210/999) were defined as statin users after diagnosis of colon cancer. Statin use after diagnosis was significantly associated with reduced risk of death from any cause (adjusted relative risk [RR], 0.67; 95% confidence interval [CI], 0.51-0.87; P = .003) and death from cancer (adjusted RR, 0.66; 95% CI, 0.49-0.89; P = .007). Statin use after diagnosis was associated with reduced risk of death from any cause or from cancer for patients whose tumors had intact BMP signaling (adjusted RR, 0.39; 95% CI, 0.22-0.68; P = .001), but not for patients whose tumors did not have BMP signaling (adjusted RR, 0.81; 95% CI, 0.55-1.21; P = .106; P < .0001 for the interaction). Statin use after diagnosis was not associated with reduced risk of death from any cause or from cancer for patients whose tumors did not contain KRAS mutations (adjusted RR, 0.81; 95% CI, 0.56-1.18; P = .273) or whose tumors did have KRAS mutations (adjusted RR, 0.59; 95% CI 0.35-1.03; P = .062; P = .90 for the interaction). CONCLUSIONS: In an analysis of 999 patients with a diagnosis of colon cancer, we associated statin with reduced risk of death from any cause or from cancer. The benefit of statin use is greater for patients whose tumors have intact BMP signaling, independent of KRAS mutation status. Randomized controlled trials are required to confirm these results.

The Impact of SMAD4 Loss on Outcome in Patients with Advanced Pancreatic Cancer Treated with Systemic Chemotherapy.

The role of the tumor suppressor mothers against decapentaplegic homolog 4 (SMAD4) has not yet been defined in patients (pts) with advanced pancreatic cancer (aPC). This translational research study was designed to evaluate the impact of tumoral SMAD4 loss on clinicopathological parameters and outcome in PC patients receiving palliative chemotherapy. Using immunohistochemistry, we examined SMAD4 expression in tumor tissue of 143 aPC pts treated within completed prospective clinical and biomarker trials. In uni- and multivariate analyses, SMAD4 expression status was correlated to clinicopathological patient characteristics and outcome. At chemotherapy initiation, 128 pts had metastatic PC; most pts (n = 99) received a gemcitabine-based regimen. SMAD4 loss was detected in 92 pts (64%); patient characteristics such as gender, age, tumor grading, disease stage or number of metastatic sites had no significant impact on tumoral SMAD4 status. In univariate analyses, SMAD4 loss had no impact on overall survival (hazard ratio (HR) 1.008, p = 0.656); however, we observed a prolonged progression-free survival (HR 1.565, p = 0.038) in pts with tumoral SMAD4 loss. This finding was confirmed in multivariate analyses (HR 1.790, p = 0.040), but only for gemcitabine-treated pts. In contrast to previous studies in resectable PC, loss of SMAD4 expression was not associated with a negative outcome in patients with advanced PC receiving systemic chemotherapy.

Protocol of the PANCALYZE trial: a multicenter, prospective study investigating the tumor biomarkers CXCR4, SMAD4, SOX9 and IFIT3 in patients with resected pancreatic adenocarcinoma to predict the pattern of recurrence of the disease.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies today with an urgent need for novel therapeutic strategies. Biomarker analysis helps to better understand tumor biology and might emerge as a tool to develop personalized therapies. The aim of the study is to investigate four promising biomarkers to predict the clinical course and particularly the pattern of tumor recurrence after surgical resection. DESIGN: Patients undergoing surgery for PDAC can be enrolled into the PANCALYZE trial. Biomarker expression of CXCR4, SMAD4, SOX9 and IFIT3 will be prospectively assessed by immunohistochemistry and verified by rt.-PCR from tumor and adjacent healthy pancreatic tissue of surgical specimen. Immunohistochemistry expression pattern of all four biomarkers will be combined into a single score. Beginning with the hospital stay clinical data from enrolled patients will be collected and followed. Different adjuvant chemotherapy protocols will be used to create subgroups. The combined biomarker expression score will be correlated with the further clinical course of the patients to test the hypothesis if CXCR4 positive, SMAD4 negative, SOX9 positive, IFIT3 positive tumors will predominantly develop metastatic spread. DISCUSSION: Pancreatic cancer is associated with different patterns of progression requiring personalized therapeutic strategies. Biomarker expression analysis might be a tool to predict the pattern of tumor recurrence and discriminate patients that develop systemic metastatic disease from those with tumors that rather develop local recurrence over time. This data might lead to personalized adjuvant treatment decisions as patients with tumors that stay localized might benefit from adjuvant local therapies like radiochemotherapy as compared to those with systemic recurrence who would benefit exclusively from chemotherapy. Moreover, the pattern of propagation might be a predefined characteristic of pancreatic cancer determined by the genetic signature of the tumor. In the future, biomarker expression analysis could be performed on tumor biopsies to develop personalized therapeutic pathways right after diagnosis of cancer. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00006179 .

Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms.

OBJECTIVE: Secretin-stimulated pancreatic juice contains DNA shed from cells lining the pancreatic ducts. Genetic analysis of this fluid may form a test to detect pancreatic ductal neoplasia. DESIGN: We employed digital next-generation sequencing ('digital NGS') to detect low-abundance mutations in secretin-stimulated juice samples collected from the duodenum of subjects enrolled in Cancer of the Pancreas Screening studies at Johns Hopkins Hospital. For each juice sample, digital NGS necessitated 96 NGS reactions sequencing nine genes. The study population included 115 subjects (53 discovery, 62 validation) (1) with pancreatic ductal adenocarcinoma (PDAC), (2) intraductal papillary mucinous neoplasm (IPMN), (3) controls with non-suspicious pancreata. RESULTS: Cases with PDAC and IPMN were more likely to have mutant DNA detected in pancreatic juice than controls (both p<0.0001); mutant DNA concentrations were higher in patients with PDAC than IPMN (p=0.003) or controls (p<0.001). TP53 and/or SMAD4 mutations were commonly detected in juice samples from patients with PDAC and were not detected in controls (p<0.0001); mutant TP53/SMAD4 concentrations could distinguish PDAC from IPMN cases with 32.4% sensitivity, 100% specificity (area under the curve, AUC 0.73, p=0.0002) and controls (AUC 0.82, p<0.0001). Two of four patients who developed pancreatic cancer despite close surveillance had SMAD4/TP53 mutations from their cancer detected in juice samples collected over 1 year prior to their pancreatic cancer diagnosis when no suspicious pancreatic lesions were detected by imaging. CONCLUSIONS: The detection in pancreatic juice of mutations important for the progression of low-grade dysplasia to high-grade dysplasia and invasive pancreatic cancer may improve the management of patients undergoing pancreatic screening and surveillance.

Overexpression of the BMP4/SMAD signaling pathway in skull base chordomas is associated with poor prognosis.

Chordomas are rare, locally invasive tumors with characteristic expression of the T-box transcription factor Brachyury. Little is yet known of the molecular events involved in the development of these tumors. Bone morphogenesis protein 4 (BMP4) signaling, which acts upstream of Brachyury in embryonic development, has been implicated in carcinogenesis in multiple malignancies. To explore the role of the canonical BMP4/SMAD signaling pathway in the pathogenesis of chordoma, we investigated, in 40 skull base chordomas, the expression of three major components of the signaling axis: BMP4, phospho-SMAD5 and SMAD4. Immunostaining revealed positive expression in 70%, 52.5% and 90% of cases, respectively. Eighteen (45%) of patients exhibited concurrent positive expression of these markers, which we defined as "high" expression of the BMP4/SMAD signaling pathway. Interestingly, when we compared the pattern of expression with clinicopathological parameters, we found that high expression of the pathway was more often observed in larger tumors (≥ 4 cm) than smaller ones (P = 0.010), and correlated significantly with dural invasion (P = 0.024). The Kaplan-Meier log-rank test showed that the 5-year overall survival rate for patients with high expression of the pathway was significantly lower than those with low expression (71.4% vs. 90.2%, P = 0.010). In conclusion, our results demonstrate for the first time that overexpression of the BMP4/SMAD signaling pathway could predict poor clinical outcome in skull base chordomas, suggesting activation of this pathway is involved in chordoma pathogenesis.

High expression of COUP-TF II cooperated with negative Smad4 expression predicts poor prognosis in patients with colorectal cancer.

OBJECTIVE: In order to evaluate whether the role of chicken ovalbumin upstream promoter transcription factor II (COUP-TF II) could sever as a predictor to stratify risk of human colorectal cancer (CRC) patients, and to elucidate the preliminary molecular mechanisms of COUP-TF II involved in the development and advancement of CRC reflected by investigating the relationship of COUP-TF II with PTEN, Smad4. METHODS: 112 cases tissue microarray and immunohistochemical SP method were used to detect the expression of COUP-TF II, PTEN and Smad4 in CRC tissues and adjacent non-tumorous tissues. The clinical relevance and prognosis of COUP-TF II, PTEN, Smad4 in CRC patients were analyzed. Furthermore, Cox proportional hazards model was performed to indicate the independent prognostic factors for CRC patients using various clinicopathological parameters and COUP-TF II, PTEN and Smad4. RESULTS: COUP-TF II proteins were positively expressed in 65.2% of CRC tissues and 15.5% paired non-CRC tissues, respectively. The expression of COUP-TF II was significantly correlated with TNM stage and lymph node metastasis and a negative correlation with Smad4 expression. Patients bearing higher levels of COUP-TF II expression showed lower DFS and OS. Most importantly, Cox proportional hazards regression analyses showed COUP-TF II positive/Smad4 negative status (DFS, P=0.001; OS, P=0.005) were independent prognostic factors for CRC patients. CONCLUSION: Positive COUP-TF II expression levels has significant value in determining CRC stage and metastasis and cooperates with negative Smad4 expression contributing to assess prognosis in patients with colorectal cancer, suggesting Smad4 may be involved in the above regulation progress probably.

TIF1γ interferes with TGFß1/SMAD4 signaling to promote poor outcome in operable breast cancer patients.

BACKGROUND: The Transforming growth factor ß (TGFß) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFß1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFß/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFß1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. METHODS: Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFß1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. RESULTS: 223 cases were assessable for TIF1γ, 204 for TGFß1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFß1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFß1 was not associated with any of the traditional prognostic factors. TGFß1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95% CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95 % CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFß1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFß1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95% CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95 % CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95 % CI: 1.4 to 5.4; p=0.005). CONCLUSION: There is a crosstalk between the TIF1γ and the TGFß1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.

MicroRNA-144 dysregulates the transforming growth factor-ß signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS. METHODS: Biopsy specimens were obtained from 20 lung transplant recipients with BOS((+)) and 19 without BOS((-)). Expression of miR-144 and its target, transforming growth factor-ß (TGF-ß)-induced factor homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting fibroblasts and determining the response to TGF-ß by analyzing Sma- and Mad-related family (Smads), fibroblast growth factor, TGF-ß, and vascular endothelial growth factor. Smooth muscle actin-α-positive stress fibers and F-actin filaments in lung fibroblasts were analyzed by immunofluorescence. RESULTS: Analysis of miR-144 in the biopsy specimens demonstrated 4.1 ± 0.8-fold increases in BOS(+) compared with BOS(-) patients, with a significant reduction in TGIF1 (3.6 ± 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-ß, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-α and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts. CONCLUSIONS: miR-144 is a critical regulator of the TGF-ß signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant.