Diego D'Ávila Paskulin

ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intratumoral Heterogeneity for Molecular Drivers

Activated anaplastic lymphoma kinase (ALK) and ROS1 tyrosine kinases, through gene fusions, have been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma specimens using a 4-target, 4-color break-apart FISH assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical colorectal cancer specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intratumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in colorectal cancer. Implications: ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection. Mol Cancer Res; 12(1); 111–8. ©2013 AACR.

TNF -308G > A promoter polymorphism (rs1800629) and outcome from critical illness

BACKGROUND The susceptibility to adverse outcome from critical illness (occurrence of sepsis, septic shock, organ dysfunction/failure, and mortality) varies dramatically due to different degrees of inflammatory response. An over expression of tumor necrosis factor alpha (TNF-α) can lead to the progression of the inflammatory condition. OBJECTIVE We assessed the relationship of the genotype distribution of -308G >A TNF-α polymorphism with regard to the development of sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients. METHODS Observational, hospital-based cohort study of 520 critically ill Caucasian patients from southern Brazil admitted to the general ICU of São Lucas Hospital, Porto Alegre, Brazil. Patients were monitored daily from the ICU admission day to hospital discharge or death, measuring SOFA score, sepsis, and septic shock occurrences. The -308G >A TNF-α SNP effect was analyzed in the entire patient group, in patients with sepsis (349/520), and in those who developed septic shock (248/520). RESULTS The genotypic and allelic frequencies were -308GG = 0.72; -308GA = 0.27; -308AA = 0.01; -308G = 0.85; -308A = 0.15. No associations were found with sepsis, septic shock, organ dysfunction, and/or mortality rates among the TNF-α genotypes. Our results reveal that the -308G >A TNF-α SNP alone was not predictive of severe outcomes in critically ill patients. CONCLUSION The principal novel input of this study was the larger sample size in an investigation with -308G > A TNF-α SNP. The presence of -308A allele is not associated with sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients.

The TP53 fertility network

The TP53 gene, first described in 1979, was identified as a tumor suppressor gene in 1989, when it became clear that its product, the p53 nuclear phosphoprotein, was frequently inactivated in many different forms of cancers. Nicknamed “guardian of the genome”, TP53 occupies a central node in stress response networks. The p53 protein has a key role as transcription factor in limiting oncogenesis through several growth suppressive functions, such as initiating apoptosis, senescence, or cell cycle arrest. The p53 protein is directly inactivated in about 50% of all tumors as a result of somatic gene mutations or deletions, and over 80% of tumors demonstrate dysfunctional p53 signaling. Beyond the undeniable importance of p53 as a tumor suppressor, an increasing number of new functions for p53 have been reported, including its ability to regulate energy metabolism, to control autophagy, and to participate in various aspects of differentiation and development. Recently, studies on genetic variations in TP53 among different populations have led to the notion that the p53 protein might play an important role in regulating fertility. This review summarizes current knowledge on the basic functions of different genes of the TP53 family and TP53 pathway with respect to fertility. We also provide original analyses based on genomic and genotype databases, providing further insights into the possible roles of the TP53 pathway in human reproduction.

Interaction between TP63 and MDM2 genes and the risk of recurrent pregnancy loss

OBJECTIVE Recent studies have investigated the role of the p53 gene family in reproductive processes. Each member of the gene family acts through different mechanisms: p53 is involved in genomic stability and regulation of blastocyst implantation; p63 acts as a regulator of the quality and maturation of oocytes; and p73 controls the meiotic spindle. Polymorphisms in the genes of the p53 family have been associated with female infertility. One polymorphism in MDM2, the main regulator of the p53 family, has also been associated with this condition. Although polymorphisms in the TP53 gene have been related to recurrent pregnancy loss (RPL), there have been no studies associating polymorphisms in p63 and p73 with RPL. Therefore, the aim of this study was to evaluate the role of polymorphisms in the TP63 (rs17506395), TP73 (rs2273953, rs1801173), and MDM2 (SNP309, rs2279744) genes as risk factors for RPL. STUDY DESIGN A case-control study was conducted in 153 women with RPL and 143 fertile women with at least two living children and no history of pregnancy loss. Molecular analysis was performed by TaqMan Allelic Discrimination assay. The statistical analysis was performed using SPSS software version 20.0 and the chi-square test, Students t-test, Mann-Whitney test and logistic regression to compare the evaluated characteristics between both groups and RPL outcome. RESULTS The allelic and genotypic frequencies did not differ between the groups when analyzed separately, however, the interaction between the TP63 TT and MDM2 TT genotypes was shown to increase the risk of RPL (OR=2.19, CI 95%: 1.28-3.75, p=0.004), even when adjusted for alcohol consumption, smoking, number of pregnancies and ethnicity (OR=1.97, CI 95%: 1.27-3.58, p=0.025). CONCLUSIONS Our results suggest that genes from the p53 family proteins, evaluated here, have an influence on the risk of RPL.

Effects of lifestyle modification after breast cancer treatment: a systematic review protocol

BackgroundThere is no consensus in the literature regarding the effectiveness of lifestyle modification interventions, including recommendations about specific diet or exercise program for patients with breast cancer. Diet interventions and regular physical activity may reduce the risk of breast cancer and its recurrence. The primary aim of our study is to evaluate the effects of different lifestyle modification interventions (diet and physical activity) in the survival of patients with stages I to III breast cancer after treatment.Methods/designThis review will be conducted according to the Cochrane Handbook for Systematic Reviews of Intervention and will be reported following the PRISMA statement recommendations. CENTRAL, MEDLINE and EMBASE databases will be searched for peer-reviewed literature. Randomized controlled trials of diet, exercise, or both, compared with usual care, after treatment of breast cancer stage I to III will be included in the systematic review. Two authors will independently screen titles and abstracts of studies for potential eligibility. Data will be combined using random-effect meta-analysis models with restricted maximum-likelihood as variance estimator, and will be presented as relative risk or standardized mean difference with 95% CI. The quality of evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework and summary of findings tables will be presented for patient important outcomes.DiscussionOur study may improve the current understanding of the role that lifestyle-modifiable factors can play in saving or prolonging the lives of women who have been treated for breast cancer, and also on modifying their quality of life.Systematic review registrationThe review has been registered with PROSPERO (registration number CRD42014008743).

Progressive disseminated histoplasmosis: a systematic review on the performance of non-culture-based diagnostic tests

The diagnosis of progressive disseminated histoplasmosis is often a challenge to clinicians, especially due to the low sensitivity and long turnaround time of the classic diagnostic methods. In recent years, studies involving a variety of non-culture-based diagnostic tests have been published in the literature. We performed a systematic review by selecting studies evaluating non-culture-based diagnostic methods for progressive disseminated histoplasmosis. We searched for articles evaluating detection of antibody, antigens, as well as DNA-based diagnostic methods. A comprehensive PUBMED, Web of Science, and Cochrane Library search was performed between the years 1956 and 2016. Case reports, review articles, non-human models and series involving less than 10 patients were excluded. We found 278 articles and after initial review 18 articles were included: (12) involved antigen detection methods, (4) molecular methods, and (2) antibody detection methods. Here we demonstrate that the pursuit of new technologies is ultimately required for the early and accurate diagnosis of disseminated histoplasmosis. In particular, urinary antigen detection was the most accurate tool when compared with other diagnostic techniques.

ESR1 rs9340799 Is Associated with Endometriosis-Related Infertility and In Vitro Fertilization Failure

Estrogen receptor alpha has a central role in human fertility by regulating estrogen action in all human reproductive tissues. Leukemia inhibitory factor (LIF) expression, a cytokine critical for blastocyst implantation, is mediated by estrogen signaling, so we hypothesized that ESR1 gene polymorphisms might be candidate risk markers for endometriosis-related infertility and in vitro fertilization (IVF) failure. We included 98 infertile women with endometriosis, 115 infertile women with at least one IVF failure and also 134 fertile women as controls. TaqMan SNP assays were used for genotyping LIF (rs929271), MDM2 (rs2279744), MDM4 (rs1563828), USP7 (rs1529916), and ESR1 (rs9340799 and rs2234693) polymorphisms. The SNP ESR1 rs9340799 was associated with endometriosis-related infertility (P < 0.001) and also with IVF failure (P = 0.018). After controlling for age, infertile women with ESR1 rs9340799 GG genotype presented 4-fold increased risk of endometriosis (OR 4.67, 95% CI 1.84–11.83, P = 0.001) and 3-fold increased risk of IVF failure (OR 3.33, 95% CI 1.38–8.03, P = 0.007). Our results demonstrate an association between ESR1 rs9340799 polymorphism and infertile women with endometriosis and also with women who were submitted to IVF procedures and had no blastocyst implantation.

Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His, R337H) Founder Mutation: Clues from Haplotyping of Short Tandem Repeats on Chromosome 17p.

Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial history. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Iberic origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenerational distance) and thus pre-dating European migration to Brazil. So far, the founder p.Arg337His haplotype has not been detected outside Brazil, with the exception of two residents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.

Detecção quantitativa de vírus BK em receptores de transplante renal: um estudo prospectivo de validação

Abstract Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Abstract P4-15-22: The ASCO/CAP guideline update for HER2 testing increases the number of breast cancer patients eligible for HER2-targeted therapy

Background: The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) published, in Nov/2013, the interpretive guidelines for HER2 testing of breast cancer patients. This aimed to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. The former version of these criteria was written in 2007. Objectives: to compare the HER2 immunohistochemical (IHC) analysis using the 2007 versus 2013 algorithms in a cohort of breast cancer cases diagnosed in a single institution in Southern Brazil. The cases were previously classified as HER2 1+ or 2+, using the 2007 criteria. Methods: the sample included 100 invasive breast cancer cases. The HercepTest (Dako, Denmark) was used for determination of HER2 expression. The HER2 testing was analyzed independently by two pathologists. The FISH analysis was done using a HER2/D17Z1 probe set. Preliminary Results: The HER2 IHC interpretation changed in 11/69 (15.9%) cases: 8.7% negative or equivocal cases by the 2007 guidelines were positive by the 2013 classification and 7.2% of HER2 1+ cases became equivocal (p Citation Format: Juliana Giacomazzi, Carolina R Hartmann, Diego D Paskulin, Luis Fernando Rivero, Maira Caleffi, Alessandro C Pasqualotto, Marcia S Graudenz, Daniela D Rosa. The ASCO/CAP guideline update for HER2 testing increases the number of breast cancer patients eligible for HER2-targeted therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-22.