Erythropoietin treatment in chemotherapy-induced anemia in previously untreated advanced esophagogastric cancer patients.

BACKGROUND: The impact of erythropoiesis-stimulating agents in chemotherapy-induced anemia has been a constant topic of debate over recent years. We prospectively assessed the efficacy of epoetin beta (Epo-b) in improving hemoglobin (Hb) levels and outcome in patients within an open label, randomized clinical phase II trial with advanced or metastatic gastric/esophagogastric cancer. METHODS: Previously untreated patients were randomized to receive 3-weekly cycles of capecitabine (1000 mg/m(2) bid) for 14 days plus on day 1 either irinotecan 250 mg/m(2) or cisplatin 80 mg/m(2). Epo-b (30000 IU once weekly) was initiated in patients with Hb <11 g/dl and continued until Hb ≥12 g/dl was reached. If after 4 weeks the Hb increase was <0.5 g/dl, Epo-b was increased to 30000 IU, twice weekly. RESULTS: Of 118 patients enrolled, 32 received Epo-b treatment; of these, 65 % achieved an increase in Hb levels of at least 2 g/dl, with 74 % achieving the target Hb of ≥12 g/dl. Within the study population, patients receiving Epo-b showed better overall survival (median 14.5 vs. 8.0 months, P = 0.056) as well as a significantly improved disease control rate (78 vs. 55 %, P = 0.025). Patients in the irinotecan group profited significantly (P < 0.05) in terms of progression-free survival and overall survival under Epo-b treatment (median 6.5 vs 4.1 months and median 15.4 vs 8.4 months, respectively). CONCLUSIONS: Epo-b was effective in raising Hb levels in patients with advanced esophagogastric cancer. Patients receiving Epo-b had a significantly increased response to chemotherapy and a clear trend to improved survival.

MUTYH gene expression and alternative splicing in controls and polyposis patients.

Mutational loss of the human DNA repair gene MUTYH in the germline predisposes for colorectal polyposis and cancer, a recessively heritable disease called MUTYH-associated polyposis. The MUTYH gene shows heavy alternative splicing, but the transcripts relevant for biological function and cancer prevention have not been determined. This knowledge is required to assess the consequences that germline variants of unknown functional significance may have. We therefore quantified expression and investigated patterns of alternative splicing in control individuals, tissue samples, and carriers of two frequent germline alterations. MUTYH expression differed organ dependently, correlating with proliferative activity. Alternative first exons were used tissue specifically; transcripts for mitochondrial proteins predominated in muscle tissues, while ascending colon and testes showed the highest fractions of transcripts for nuclear proteins. Colon cancer cell lines produced predominant transcripts for nuclear protein. Exon skipping was frequent and governed by splice-site quality. Five transcripts were found to encode the biologically relevant products of the MUTYH gene. Carriers of the disease-causing mutation c.1187G>A (p.Gly396Asp) showed normal transcript composition, but the frequent single-nucleotide polymorphism rs3219468:G>C largely reduced one transcript species of MUTYH. Since this alteration decreases protein production of the gene, an increased cancer risk for compound heterozygous carriers is possible.

Growth differentiation factor 15--an early marker of abnormal function of the Fontan circuit in patients with univentricular hearts.

BACKGROUND: In patients after the Fontan procedure, assessment of ventricular function is difficult and amino-terminal pro-B-type natriuretic peptide levels failed to be directly related to echocardiographic measures of systolic ventricular function. The aim of the study was to evaluate growth differentiation factor 15 (GDF-15), a marker of various stress pathways in the heart and extracardiac tissues. METHODS: Plasma GDF-15 levels were measured in 38 consecutive patients after the Fontan procedure and compared to clinical, echocardiographic, and laboratory data; liver tissue stiffness; and venous hepatic flow velocities. RESULTS: Mean GDF-15 levels were 987.2±440.5 pg/mL in patients with an ejection fraction (EF)<50% as compared to 520.2±143.1 pg/mL in those with an EF≥50% (P<.001). Growth differentiation factor 15 levels were significantly related to the EF of the single ventricle (r=-0.66, P<.001), New York Heart Association functional class (r=0.43, P=.008), and γGT levels (r=0.50, P=.002) but weakly to liver tissue stiffness. According to receiver operating characteristic curve analysis, an EF<50% was best predicted by GDF-15 levels (area under the curve [AUC] 0.90, P<.001), peak venous hepatic flow at deep inspiration (AUC 0.89, P=.002), and age at Fontan operation (AUC 0.86, P=.001). Growth differentiation factor 15 and age at Fontan operation proved to be independent predictors in the multivariate analysis. The optimal cutoff of GDF-15 for the prediction of an EF<50% was calculated to be 613 pg/mL with a sensitivity of 90.0% and specificity of 85.7%. CONCLUSIONS: Growth differentiation factor 15 might be helpful in detecting early abnormal function of the Fontan circuit in patients with univentricular hearts. In patients with GDF-15 levels exceeding 613 pg/mL, further cardiac evaluation should be considered because impaired systolic function of the single ventricle may be present.

Adenoma development in a patient with MUTYH-associated polyposis (MAP): new insights into the natural course of polyp development.

PURPOSE: Biallelic germ-line mutations in MUTYH have recently been found to predispose for MUTYH-associated polyposis (MAP). Affected patients present with a wide range of clinical phenotypes at the time of diagnosis, but there is little precise information about the natural course of this disease. RESULTS: Fourteen years of colonoscopic surveillance of an MAP patient (compound heterozygous p.Y165C/p.G382D) showed that adenoma development was slow after initial diagnosis of a single colorectal carcinoma at the age of 44, but then the annual number of new adenomas increased substantially in the patient's early fifties. CONCLUSION: This course of the disease, with a strong subsequent acceleration of polyp development, may explain the wide range of polyp numbers counted in newly diagnosed MAP patients as a result of the time of observation. Therefore, MAP should also be considered in younger patients (35-55 years) with only few adenomas or colorectal cancer. The high frequency of medium and severe dysplasia in the patient's preferential small adenomas suggests accelerated progression from adenoma to carcinoma in MAP, but this observation must be confirmed by further studies.

Evaluation of the MLH1 I219V alteration in DNA mismatch repair activity and ulcerative colitis.

BACKGROUND: Inflammatory bowel diseases (IBDs; ulcerative colitis, UC, and Crohn's disease, CD) show familial clustering suggestive of a genetic background. A linkage susceptibility region for these diseases (IBD9) lies on chromosome 3p and includes the DNA mismatch repair gene MLH1. Loss of MLH1 confers the characteristic microsatellite instability (MSI) phenotype which is also frequently found in the mucosa of IBD patients. A common germline alteration of MLH1 (655A>G) results in the amino acid exchange MLH1 I219V. Conflicting data exist on its effect on the function of the protein and it has recently been reported to cosegregate with refractory UC, suggesting that this alteration may impair mismatch repair activity and thereby contribute to certain forms of UC. METHODS: We analyzed the MLH1 I219V alteration using in silico and biochemical analyses and assessed its appearance in 67 well-classified UC patients in comparison to 40 healthy individuals. RESULTS: The analyses showed that I219 is a conserved, buried hydrophobic residue, and that I219V is unlikely to abolish MLH1 function but may modulate it. Quantitative biochemical evaluation showed identical stability and activity of the protein. Furthermore, the alteration occurred equally frequently in analyzed patients and healthy volunteers. CONCLUSIONS: The MLH1 I219V alteration does not directly contribute to the etiology of UC through an impairment of mismatch repair. A putative linkage disequilibrium of MLH1 I219V with the causative gene(s) of the IBD9 locus is rather distant.

Validity of N-terminal propeptide of the brain natriuretic peptide in predicting left ventricular diastolic dysfunction diagnosed by tissue Doppler imaging in patients with chronic liver disease.

BACKGROUND AND AIMS: Left ventricular diastolic dysfunction has been reported in patients with liver cirrhosis. Although conventional Doppler echocardiography has been used to assess diastolic filling dynamics, this technique is limited in diagnosing left ventricular diastolic dysfunction. The aim of the study was to validate the N-terminal propeptide of the brain natriuretic peptide (NT-proBNP) in predicting left ventricular diastolic dysfunction diagnosed by tissue Doppler imaging in patients with chronic liver disease. METHODS: In 64 patients, left ventricular diastolic dysfunction was classified using tissue Doppler imaging and serum levels of NT-proBNP were measured. RESULTS: Left ventricular diastolic dysfunction was found in 25 of 31 (80.6%) patients with severe liver fibrosis/cirrhosis versus 2 of 8 (25.0%) patients with moderate and 6 of 25 (24.0%) patients with mild liver fibrosis (P<0.001). Mean NT-proBNP levels were 407.1+/-553.4 pg/ml in patients with severe fibrosis/cirrhosis as compared with 60.8+/-54.9 pg/ml and 55.4+/-41.4 pg/ml in patients with mild and moderate fibrosis (P=0.001). NT-proBNP was most accurate in predicting advanced left ventricular diastolic dysfunction with an area under the receiver-operating characteristic curve of 0.90 (95% confidence interval, 0.77-1.0; P<0.001). A cutoff value of greater than 290 pg/ml was highly predictive of advanced left ventricular diastolic dysfunction. CONCLUSION: NT-proBNP is a useful marker in detecting advanced left ventricular diastolic dysfunction in patients with chronic liver disease. Patients with severe liver fibrosis/cirrhosis and NT-proBNP levels exceeding 290 pg/ml should undergo further cardiac evaluation.

Hypothesis: Possible role of retinoic acid therapy in patients with biallelic mismatch repair gene defects.

A boy showing symptoms of a Turcot-like childhood cancer syndrome together with stigmata of neurofibromatosis type I is reported. His brother suffers from an infantile myofibromatosis, and a sister died of glioblastoma at age 7. Another 7-year-old brother is so far clinically unaffected. The parents are consanguineous. Molecular diagnosis in the index patient revealed a constitutional homozygous mutation of the mismatch repair gene PMS2. The patient was in remission of his glioblastoma (WHO grade IV) after multimodal treatment followed by retinoic acid chemoprevention for 7 years. After discontinuation of retinoic acid medication, he developed a relapse of his brain tumour together with the simultaneous occurrence of three other different HNPCC-related carcinomas. We think that retinoic acid might have provided an effective chemoprevention in this patient with homozygous mismatch repair gene defect. We propose to take a retinoic acid chemoprevention into account in children with proven biallelic PMS2 mismatch repair mutations being at highest risk concerning the development of a malignancy.

Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair.

MutLalpha, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSalpha and assembles and controls further repair enzymes. We tested if the interaction of MutLalpha with DNA-bound MutSalpha is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLalpha for MutSalpha is located on the edge of an extensive beta-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein-protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL-MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLalpha-MutSalpha interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.

Panel of three novel serum markers predicts liver stiffness and fibrosis stages in patients with chronic liver disease.

Latest data suggest that placental growth factor (PLGF), growth differentiation factor-15 (GDF-15) and hepatic growth factor (HGF) are involved in hepatic fibrogenesis. Diagnostic performance of these markers for non-invasive liver fibrosis prediction was evaluated based on liver histology and stiffness. In total 834 patients were recruited. Receiver-operating-characteristics were used to define cut-offs for markers correlating to fibrosis stages. Odds-ratios were calculated for the presence/absence of fibrosis/cirrhosis and confirmed in the sub-group of patients phenotyped by elastography only. Logistic and uni- and multivariate regression analyses were used to test for association of markers with liver fibrosis stages and for independent prediction of liver histology and stiffness. Marker concentrations correlated significantly (P<0.001) with histology and stiffness. Cut-offs for liver fibrosis (≥F2) were PLGF = 20.20 pg/ml, GDF15 = 1582.76 pg/ml and HGF = 2598.00 pg/ml. Logistic regression confirmed an increase of ORs from 3.6 over 33.0 to 108.4 with incremental (1-3) markers positive for increased liver stiffness (≥12.8kPa; all P<0.05). Subgroup analysis revealed associations with advanced fibrosis for HCV (three markers positive: OR = 59.9, CI 23.4-153.4, P<0.001) and non-HCV patients (three markers positive: OR = 144, CI 59-3383, P<0.001). Overall, serum markers identified additional 50% of patients at risk for advanced fibrosis presenting with low elastography results. In conclusion, this novel combination of markers reflects the presence of significant liver fibrosis detected by elastography and histology and may also identify patients at risk presenting with low elastography values.

Correction: Panel of three novel serum markers predicts liver stiffness and fibrosis stages in patients with chronic liver disease.

[This corrects the article DOI: 10.1371/journal.pone.0173506.].

A randomized multi-center phase II trial of the angiogenesis inhibitor Cilengitide (EMD 121974) and gemcitabine compared with gemcitabine alone in advanced unresectable pancreatic cancer.

BACKGROUND: Anti-angiogenic treatment is believed to have at least cystostatic effects in highly vascularized tumours like pancreatic cancer. In this study, the treatment effects of the angiogenesis inhibitor Cilengitide and gemcitabine were compared with gemcitabine alone in patients with advanced unresectable pancreatic cancer. METHODS: A multi-national, open-label, controlled, randomized, parallel-group, phase II pilot study was conducted in 20 centers in 7 countries. Cilengitide was administered at 600 mg/m2 twice weekly for 4 weeks per cycle and gemcitabine at 1000 mg/m2 for 3 weeks followed by a week of rest per cycle. The planned treatment period was 6 four-week cycles. The primary endpoint of the study was overall survival and the secondary endpoints were progression-free survival (PFS), response rate, quality of life (QoL), effects on biological markers of disease (CA 19.9) and angiogenesis (vascular endothelial growth factor and basic fibroblast growth factor), and safety. An ancillary study investigated the pharmacokinetics of both drugs in a subset of patients. RESULTS: Eighty-nine patients were randomized. The median overall survival was 6.7 months for Cilengitide and gemcitabine and 7.7 months for gemcitabine alone. The median PFS times were 3.6 months and 3.8 months, respectively. The overall response rates were 17% and 14%, and the tumor growth control rates were 54% and 56%, respectively. Changes in the levels of CA 19.9 went in line with the clinical course of the disease, but no apparent relationships were seen with the biological markers of angiogenesis. QoL and safety evaluations were comparable between treatment groups. Pharmacokinetic studies showed no influence of gemcitabine on the pharmacokinetic parameters of Cilengitide and vice versa. CONCLUSION: There were no clinically important differences observed regarding efficacy, safety and QoL between the groups. The observations lay in the range of other clinical studies in this setting. The combination regimen was well tolerated with no adverse effects on the safety, tolerability and pharmacokinetics of either agent.

DNA mismatch repair and Lynch syndrome.

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2-5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.

N-terminus of hMLH1 confers interaction of hMutLalpha and hMutLbeta with hMutSalpha.

Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSalpha (hMSH2-hMSH6) and hMutLalpha (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSalpha recruits hMutLalpha, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLalpha as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSalpha using a novel interaction assay. We show that, although the interaction requires ATP, hMutLalpha does not need to hydrolyse this nucleotide to join hMutSalpha on DNA, suggesting that ATP hydrolysis by hMutLalpha happens downstream of complex formation. The analysis of the individual subunits of hMutLalpha demonstrated that the hMutSalpha-hMutLalpha interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N-terminus of hMLH1 confers this interaction. In contrast, only the C-terminus stabilised and co-immunoprecipitated hPMS2 when both proteins were co-expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C-terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLbeta (hMLH1-hPMS1). We show that hMutLbeta interacts as efficiently with hMutSalpha as hMutLalpha, and that it predominantly binds to hMutSalpha via hMLH1 as well.

hMutSalpha forms an ATP-dependent complex with hMutLalpha and hMutLbeta on DNA.

The DNA binding properties of hMutSalpha and hMutLalpha and complex formation of hMutSalpha with hMutLalpha and hMutLbeta were investigated using binding experiments on magnetic bead-coupled DNA substrates with nuclear extracts as well as purified proteins. hMutSalpha binding to homoduplex DNA was disrupted by lower NaCl concentrations than hMutSalpha binding to a mismatch. ATP markedly reduced the salt resistance of hMutSalpha binding but hMutSalpha still retained affinity for heteroduplexes. hMutSalpha formed a complex with hMutLalpha and hMutLbeta on DNA in the presence of ATP. This complex only formed on 81mer and not 32mer DNA substrates. Complex formation was enhanced by a mismatch in the DNA substrate, and hMutLalpha and hMutLbeta were shown to enter the complex at different ATP concentrations. Purified hMutLalpha showed an intrinsic affinity for DNA, with a preference for single-stranded over double-stranded DNA.

Detection of microsatellite instability from archival, hematoxylin-eosin-stained colorectal cancer specimen.

Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer (HNPCC). Owing to early onset of colorectal cancer before the age of 50 years and/or familial clustering of HNPCC-related malignancies, the diagnosis of HNPCC was suspected in 2 patients. Because no paraffin-embedded tumor tissue was available, we used archival 5-microm, hematoxylin-eosin-stained tumor specimen slides for direct MSI analysis. Tissue was microdissected and cells were lysed using 1% Triton. Fluorescence polymerase chain reaction amplification of a panel of 7 microsatellite markers, including all markers of the current international reference panel (BAT-25, BAT-26, D2S123, D5S346, and D17S250), demonstrated MSI in one patient and excluded MSI in the other. In conclusion, this study demonstrates the feasibility of MSI analysis by direct fluorescence polymerase chain reaction amplification using hematoxylin-eosin-stained tissue specimens without the need for prior DNA extraction.

Malignant fibrous histiocytoma is a rare Lynch syndrome-associated tumor in two German families.

Lynch syndrome is characterized by germline mutations of the DNA mismatch repair genes MLH1 and MSH2. The tumor spectrum includes early onset colorectal, urogenital and other cancers. Soft tissue sarcomas have been anecdotally reported in patients with Lynch syndrome, but coincidental manifestation could not be excluded. In this report, we screened a cohort of Lynch syndrome families for tumors outside the established tumor spectrum. We identified two patients with Lynch syndrome and a malignant fibrous histiocytoma (MFH). In both families a causative MSH2 germline mutation (MSH2 c.2038C ≥ T or MSH2 c.942 ± 3A ≥ T) could be detected. Archival tumor material from both resected MFH was analyzed for microsatellite instability expression of MLH1 and MSH2. A mutator phenotype was detected in both MFH with loss of MSH2 protein expression. Subsequently, the causative MSH2 germline mutation was confirmed in both patients. Of note, both tumors were diagnosed at a local advanced stage but could be curatively resected 21 and 11 year ago, respectively. Both patients are alive without local or distant recurrence. In conclusion, our data further support that patients with Lynch syndrome are at increased risk for rare tumors such as MFH. However, the prognosis compared to sporadic MFH seems to be favorable.

Analysis of the human MutLalpha.MutSalpha complex.

Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.