Circulating Tumor DNA Detection in the Management of Anti-EGFR Therapy for Advanced Colorectal Cancer

Background: Anti-EGFR antibodies are a standard care for advanced KRAS-wild type colorectal cancers. Circulating tumor DNA (ctDNA) monitoring during therapy can detect emergence of KRAS mutant clones and early resistance to therapy.Case Presentation: We describe a 61-years-old man presenting a metastatic and recurrent rectal cancer treated with different chemotherapy regimens. His tumor was KRAS wild-type based on tissue analysis and he was treated sequentially with cetuximab-based chemotherapy, chemotherapy alone and panitumumab-based chemotherapy. We performed sequential analysis of ctDNA using droplet digital PCR (ddPCR) and a commercial assay designed for the detection of frequent KRAS mutations during his clinical follow-up. Prior to the first cetuximab-based chemotherapy ctDNA analysis demonstrated an absence of KRAS mutations. Emergence of KRAS mutations in ctDNA occurred ~3 months after treatment initiation and preceded clinical and imaging progression in about 2 months. Fractional abundance of KRAS mutation rapidly increased to 70.7% immediately before a chemotherapy alone regimen was initiated. Interestingly, KRAS mutation abundance decreased significantly during the first two months of chemotherapy, reaching a fractional abundance of 3.0%, despite minimal clinical benefit with this therapy. Re-challenge with a different anti-EGFR antibody was attempted as later line, but high levels of KRAS mutations in ctDNA before therapy correlated with an absence of clinical benefit.Conclusions: The monitoring of resistance mutations in KRAS using ctDNA during the treatment of KRAS wild-type advanced colorectal cancers can detect the emergence of resistant clones prior to clinical progression. Dynamics of resistant clones may alter during periods on and off anti-EGFR antibodies, detecting window of opportunities for a re-challenge with these therapies

Association between self-care and quality of life in chronic kidney disease patients

Our study assessed the capacity for self-care and the association with quality of life of people living with Chronic Kidney Disease. We performed a cross-sectional and correlational study. One hundred individuals in hemodialytic treatment in a substitutive kidney therapy unit in the inner state of São Paulo composed the sample. We collected data through personal interview, using a questionnaire for sociodemographic characteristics, the Appraisal of Self-Care Agency Scale – Revised (ASAS-R) and, the Kidney Disease and Quality of Life Short – Form (KDQOL-SF). The mean ASAS-R score was 60.64 (±8.24) indicating good self-care capacity. We found a positive moderate and statistically significant relationship between the capacity for self-care and the KDQOL-SF domains: symptoms/problems, cognitive function, physical function, emotional wellbeing and energy/fatigue. We concluded that self-care is related to the quality of life in hemodialytic patients with chronic kidney disease

Relação entre autocuidado e sintomas depressivos e ansiosos de indivíduos em tratamento hemodialítico

Objetivo avaliar a capacidade para o autocuidado e sua relação com os sintomas depressivos e ansiosos de pacientes em hemodiálise.Métodos estudo correlacional, com corte transversal, realizado com 100 participantes. Foram utilizados os instrumentos: Escala Revisada para a Avaliação da Agência de Autocuidado, que indica a capacidade para o autocuidado dos pacientes, e a Escala Hospitalar de Ansiedade e Depressão, que avalia a presença ou não de sintomas depressivos e ansiosos.Resultados os participantes apresentaram um escore médio de 60,64 (±8,24), na Escala Revisada para a Avaliação da Agência de Autocuidado, indicando bom nível de agenciamento para o autocuidado. Verificou-se correlação negativa, de moderada magnitude, entre a capacidade de autocuidado e os sintomas ansiosos (r=-0,328) e também com os sintomas depressivos (r=-0,387).Conclusão indivíduos em tratamento hemodialítico apresentaram bom agenciamento para o autocuidado e aqueles com sintomas depressivos e/ou ansiosos mostraram menor capacidade para o autocuidado

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G > T:A and C:G > A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G > T:A and C:G > A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct

Identification of FAM46D as a novel cancer/testis antigen using EST data and serological analysis

Cancer/testis Antigens (CTAs) are immunogenic proteins with a restricted expression pattern in normal tissues and aberrant expression in different types of tumors being considered promising candidates for immunotherapy. We used the alignment between EST sequences and the human genome sequence to identify novel CT genes. By examining the EST tissue composition of known CT clusters we defined parameters for the selection of 1184 EST clusters corresponding to putative CT genes. The expression pattern of 70 CT gene candidates was evaluated by RT-PCR in 21 normal tissues, 17 tumor cell lines and 160 primary tumors. We were able to identify 4 CT genes expressed in different types of tumors. The presence of antibodies against the protein encoded by 1 of these 4 CT genes (FAM46D) was exclusively detected in plasma samples from cancer patients. Due to its restricted expression pattern and immunogenicity FAM46D represents a novel target for cancer immunotherapy. (c) 2009 Elsevier Inc. All rights reserved.Ludwig Institute for Cancer Research, Sao Paulo, BrazilFAPESP Fundacao de Amparo a Pesquisa do Estado de Sao PauloCNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoFAPESP Fundaicao de Amparo a Pesquisa do Estado de Sao PauloCAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues