MITO END-3: efficacy of avelumab immunotherapy according to molecular profiling in first-line endometrial cancer therapy.

BACKGROUND: Immunotherapy combined with chemotherapy significantly improves progression-free survival (PFS) compared to first-line chemotherapy alone in advanced endometrial cancer (EC), with a much larger effect size in microsatellite instability-high (MSI-H) cases. New biomarkers might help to select patients who may have benefit among those with a microsatellite-stable (MSS) tumor. PATIENTS AND METHODS: In a pre-planned translational analysis of the MITO END-3 trial, we assessed the significance of genomic abnormalities in patients randomized to standard carboplatin/paclitaxel without or with avelumab. RESULTS: Out of 125 randomized patients, 109 had samples eligible for next-generation sequencing analysis, and 102 had MSI tested. According to The Cancer Genome Atlas (TCGA), there were 29 cases with MSI-H, 26 with MSS TP53 wild type (wt), 47 with MSS TP53 mutated (mut), and 1 case with POLE mutation. Four mutated genes were present in >30% of cases: TP53, PIK3CA, ARID1A, and PTEN. Eleven patients (10%) had a BRCA1/2 mutation (five in MSI-H and six in MSS). High tumor mutational burden (≥10 muts/Mb) was observed in all MSI-H patients, in 4 out of 47 MSS/TP53 mut, and no case in the MSS/TP53 wt category. The effect of avelumab on PFS significantly varied according to TCGA categories, being favorable in MSI-H and worst in MSS/TP53 mut (P interaction = 0.003); a similar non-significant trend was seen in survival analysis. ARID1A and PTEN also showed a statistically significant interaction with treatment effect, which was better in the presence of the mutation (ARID1A P interaction = 0.01; PTEN P interaction = 0.002). CONCLUSION: The MITO END-3 trial results suggest that TP53 mutation is associated with a poor effect of avelumab, while mutations of PTEN and ARID1A are related to a positive effect of the drug in patients with advanced EC.

Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas.

The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the São Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.

Analysis of the polymorphisms XRCC1Arg194Trp and XRCC1Arg399Gln in gliomas.

XRCC genes (X-ray cross-complementing group) were discovered mainly for their roles in protecting mammalian cells against damage caused by ionizing radiation. Studies determined that these genes are important in the genetic stability of DNA. Although the loss of some of these genes does not necessarily confer high levels of sensitivity to radiation, they have been found to represent important components of various pathways of DNA repair. To ensure the integrity of the genome, a complex system of DNA repair was developed. Base excision repair is the first defense mechanism of cells against DNA damage and a major event in preventing mutagenesis. Repair genes may play an important role in maintaining genomic stability through different pathways that are mediated by base excision. In the present study, we examined XRCC1Arg194Trp and XRCC1Arg399Gln polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the allele Trp of the XRCC1Arg194Trp polymorphism had an increased risk of tumor development (OR = 8.80; confidence interval at 95% (95%CI) = 4.37-17.70; P < 0.001), as did the allele Gln of XRCC1Arg399Gln (OR = 1.01; 95%CI = 0.53-1.93; P = 0.971). Comparison of overall survival of patients did not show significant differences. We suggest that XRCC1Arg194Trp and XRCC1Arg399Gln polymorphisms are involved in susceptibility for developing astrocytomas and glioblastomas.

GSTP1 Ile105Val polymorphism in astrocytomas and glioblastomas.

Glutathione S-transferases (GSTs) constitute a superfamily of ubiquitous multifunctional enzymes that are involved in the cellular detoxification of a large number of endogenous and exogenous chemical agents that have electrophilic functional groups. People who have deficiencies in this family of genes are at increased risk of developing some types of tumors. We examined GSTP1 Ile105Val polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the Val allele of the GSTP1 Ile105Val polymorphism had an increased risk of tumor development (odds ratio = 8.60; 95% confidence interval = 4.74-17.87; P < 0.001). Overall survival of patients did not differ significantly. We suggest that GSTP1 Ile105Val polymorphisms are involved in susceptibility to developing astrocytomas and glioblastomas.

Real-world outcomes according to treatment strategies in ALK-rearranged non-small-cell lung cancer (NSCLC) patients: an Italian retrospective study.

PURPOSE: Anaplastic lymphoma kinase (ALK) rearrangement confers sensitivity to ALK inhibitors (ALKis) in non-small-cell lung cancer (NSCLC). Although several drugs provided an impressive outcome benefit, the most effective sequential strategy is still unknown. We describe outcomes of real-life patients according to the treatment strategy received. PATIENTS: We retrospectively collected 290 ALK rearranged advanced NSCLC diagnosed between 2011 and 2017 in 23 Italian institutions. RESULTS: After a median follow-up of 26 months, PFS for crizotinib and a new generation ALKis were 9.4 [CI 95% 7.9-11.2] and 11.1 months [CI 95% 9.2-13.8], respectively, while TTF were 10.2 [CI 95% 8.5-12.6] and 11.9 months [CI 95% 9.7-17.4], respectively, being consistent across the different settings. The composed outcomes (the sum of PFS or TTF) in patients treated with crizotinib followed by a new generation ALKis were 27.8 months [CI 95% 24.3-33.7] in PFS and 30.4 months [CI 95% 24.7-34.9] in TTF. The median OS from the diagnosis of advanced disease was 39 months [CI 95% 31.8-54.5]. Patients receiving crizotinib followed by a new generation ALKis showed a higher median OS [57 months (CI 95% 42.0-73.8)] compared to those that did not receive crizotinib [38 months (CI 95% 18.6-NR)] and those who performed only crizotinib as target agent [15 months (CI 95% 11.3-34.0)] (P < 0.0001). CONCLUSION: The sequential administration of crizotinib and a new generation ALKis provided a remarkable clinical benefit in this real-life population, being an interesting option to consider in selected patients.

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors.

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development (odds ratio, OR = 3.23; confidence interval at 95%, 95%CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95%CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

Concurrent sequence variation of TP53 and TP73 genes in anaplastic astrocytoma.

Disruption or loss of tumor suppressor gene TP53 is implicated in the development or progression of almost all different types of human malignancies. Other members of the p53 family have been identified. One member, p73, not only shares a high degree of similarity with p53 in its primary sequence, but also has similar functions. Like p53, p73 can bind to DNA and activate transcription. Using PCR-SSCP and gene sequencing, we analyzed the TP53 and TP73 genes in a case of a grade III anaplastic astrocytoma that progressed to glioblastoma. We found a deletion of AAG at position 595-597 of TP53 (exon 6), resulting in the deletion of Glu 199 in the protein and a genomic polymorphism of TP73, identified as an A-to-G change, at position E8/+15 at intron 8 (IVS8-15A>G). The mutation found at exon 6 of the gene TP53 could be associated with the rapid tumoral progression found in this case, since the mutated p53 may inactivate the wild-type p53 and the p73alpha protein, which was conserved here, leading to an increase in cellular instability.

[Identification of genetic alterations by multiple ligation-dependent probe amplification (MLPA) analysis in oligodendrogliomas]. / Identificación de alteraciones genéticas en oligodendrogliomas mediante amplificación dependiente de ligasa de múltiples sondas (MLPA) (multiple ligation-dependent probe amplification).

Concurrent deletion at 1p/19q is a common signature of oligodendrogliomas, and it may be identified in low-grade tumours (grade II) suggesting it represents an early event in the development of these brain neoplasms. Additional non-random changes primarily involve CDKN2A, PTEN and EGFR. Identification of all of these genetic changes has become an additional parameter in the evaluation of the clinical patients' prognosis, including good response to conventional chemotherapy. Multiple ligation-dependent probe amplification (MLPA) analysis is a new methodology that allows an easy identification of the oligodendrogliomas' abnormalities in a single step. No need of the respective constitutional DNA from each patient is another advantage of this method. We used MLPA kits P088 and P105 to determine the molecular characteristics of a series of 40 oligodendrogliomas. Deletions at l p and 19q were identified in 45% and 65% of cases, respectively. Alterations of EGFR, CDKN2A, ERBB2, PTEN and TP53 were also identified in variable frequencies among 7% to 35% of tumours. These findings demonstrate that MLPA is a reliable technique to the detection of molecular genetic changes in oligodendrogliomas.

Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil.

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors.

Cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

DAPK1 promoter hypermethylaiton in brain metastases and peripheral blood.

The DAPK1 gene works as a regulator of apoptosis and is frequently inactivated in cancer by aberrant promoter hypermethylation. Loss of DAPK1 expression is associated with a selective advantage for tumor cells to resist apoptotic stimuli, allowing them to separate from the original tumor; from this point of view, DAPK1 could be considered a tumor metastases inhibitor gene. To verify the participation of DAPK1 silencing in cerebral invasion, we analyzed its promoter methylation status in a series of 28 samples from cerebral metastases using MSP and sequencing of the MSP-product. We have found hypermethylation in 53.6% (15/28) metastatic tumor samples as well as in 27.8% (5/18) of its peripheral blood samples. Our data suggest an important role of DAPK1 for silencing through promoter CpG island hypermethylation in the development of brain metastases from solid tumors. The detection of aberrant hypermethylation on DAPK1 promoter from peripheral blood samples has potential clinical implications as a tumor prognosis marker.

Mutation analysis of gene PAX6 in human gliomas.

Gliomas are the most common tumors of the central nervous system. In spite of the marked advances in the characterization of the molecular pathogenesis of gliomas, these tumors remain incurable and, in most of the cases, resistant to treatments, due to their molecular heterogeneity. Gene PAX6, which encodes a transcription factor that plays an important role in the development of the central nervous system, was recently recognized as a tumor suppressor in gliomas. The objective of the present study was to analyze the mutational status of the coding and regulating regions of PAX6 in 94 gliomas: 81 astrocytomas (11 grade I, 23 grade II, 8 grade III, and 39 grade IV glioblastomas), 5 oligodendrogliomas (3 grade II, and 2 grade III), and 8 ependymomas (5 grade II, and 3 grade III). Two regulating regions (SX250 and EIE) and the 11 coding regions (exons 4-13, plus exon 5a resulting from alternative splicing) of gene PAX6 were analyzed and no mutation was found. Therefore, we conclude that the tumor suppressor role of PAX6, reported in previous studies on gliomas, is not due to mutation in its coding and regulating regions, suggesting the involvement of epigenetic mechanisms in the silencing of PAX6 in these tumors.

Investigation of chromosome 21 aneuploidies in breast fibroadenomas by fluorescence in situ hybridisation.

Fibroadenoma (FA) is a benign breast tumour that occurs in about 25% of women. Cytogenetic studies suggest that numerical chromosomal aberrations may contribute to tumorigenesis, but chromosomal instability is still poorly characterised in breast cancer. The aim of this study was to investigate numerical alterations of chromosome 21 in 15 breast FAs. All samples were analysed by classical cytogenetics and by fluorescence in situ hybridisation (FISH) for chromosome 21 DNA sequences. Classical cytogenetics analysis showed that all cells were diploidies with modal number varying between 43 and 47 chromosomes, and clonal chromosome alterations in 46.7% of tumours. Clonal numerical alterations involved, preferentially, chromosomes 8, 10, 12, 16 and 21. FISH analysis showed a statistically significant difference for chromosome 21 monosomy between seven samples and control group. This monosomy varied from 24.5% to 43.5% of analysed cells. The presence of chromosomal alterations in FAs may be a consequence of the proliferation process and is probably not related to the aetiology of this type of lesion. The study of benign proliferations and comparison with chromosome alterations in their malignant counterparts should result in an understanding of the genes acting in cell proliferation alone and those that cause these cells to both undergo malignant transformation and become invasive.

Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach.

Ten permanent clones derived from a single biopsy specimen of an untreated human adenocarcinoma of the stomach were established and characterized in vitro. Tissue culture growth properties, doubling times, plating efficiencies, growth fractions, cell cycle phase distributions, DNA indices, modal chromosome numbers, and ploidies were determined. Growth fractions were nearly 100%, and doubling times ranged from 23 to 37 hr. The plating efficiencies were generally high for tumor cells in culture, ranging up to 70%. Modal chromosome numbers varied from 45 to 48, with a wider range of variability in about 25% of the cells studied in each clone. In addition, the parent cell line (from which the clones were isolated) was shown to grow in athymic mice and to have the same histochemical and cytological characteristics as the specimen taken from the patient. It is important to characterize human tumor cells in vitro in this detailed manner, since they serve as excellent model systems for other studies involving the heterogeneous responses to drugs and radiation. The identification of mechanisms of drug sensitivity and resistance and the testing of drug and radiation combination treatment schedules in such in vitro systems can provide valuable insight into the design of clinical protocols for treatment of stomach cancer in humans.

Molecular study of the tumour suppressor gene PTEN in gastric adenocarcinoma in Brazil.

Among all tumours diagnosed worldwide, gastric adenocarcinoma is the second most frequent type of malignancy. In Brazil, it is estimated to be the fifth most frequent type of neoplasia. According to the classification of Laurén, these tumours are divided into well differentiated and ill differentiated gastric adenocarcinomas. There are studies suggesting that the first type develops through remodulation of genes involved in the suppressor pathway and the second through remodulation of genes belonging to the mutational pathway. The gene PTEN is located in region 10q23 and is altered in several human tumours. In gastric cancer, this gene is thought to take part in the suppressor pathway. In our study, DNA was obtained from 48 gastric adenocarcinoma samples, amplified, screened for all exons of the PTEN gene by PCR-SSCP and then confirmed by sequencing. There was only one sample that presented an alteration and that was a transversion. Our results corroborate the hypothesis that somatic alterations in the PTEN gene are rare events in gastric cancer.

Polymorphisms of the TP53 codon 72 and WRN codon 1367 in individuals from Northern Brazil with gastric adenocarcinoma.

Gastric cancer is the second most frequent type of neoplasia and also the second most common cause of death in the world. TP53 codon 72, which produces variant proteins with an arginine (Arg) or proline (Pro), has been reported to be associated with cancers of the lung, oesophagus, stomach and cervix. Werner's syndrome (WS) is a premature ageing disease caused by a mutation in the WRN gene. The WRN protein acts as a DNA helicase and as an exonuclease. WRN codon 1367 produces variant proteins with an Arg or cysteine (Cys). This polymorphism has been studied, in order to understand the clinical impact of the molecular variants in WS and in age-related disorders. In the present study, the TP53 codon 72 and the WRN codon 1367 polymorphisms were investigated in 54 gastric adenocarcinoma patients (31 diffuse-type and 25 intestinal-type) and 54 controls. DNA samples were extracted, and PCR-RFLP was utilised for genotyping TP53 codon 72 and WRN codon 1367. The allele frequencies of the TP53 polymorphism were: Arg=0.74 and Pro=0.26. The allele frequencies of the WRN polymorphism were: Cys=0.73 and Arg=0.27. The crude genotypic frequencies in gastric cancer patients were similar to those of the controls, but in the WRN codon 1367 polymorphisms the mean age tended to be higher in the Arg/Arg genotypes. There also was an association, although not statistically significant, between the presence of Helicobacter pylori and the genotypes Cys/Cys and Cys/Arg and a higher percentage of cardia cancer among the Arg/Arg genotypes, and of non-cardia cancer among genotypes Cys/Cys and Cys/Arg. These findings may be a reflection of differences in the interaction between WRN codon 1367 polymorphisms and local factors in the stomach. To our knowledge, this is the first study to examine a genetic polymorphism of the WRN gene in cancer. The precise mechanisms of action of the TP53 and WRN polymorphisms involved in the aetiopathogeny of this disease need further investigation.

Aberrant methylation of multiple genes in neuroblastic tumours. relationship with MYCN amplification and allelic status at 1p.

Aberrant hypermethylation occurs in tumour cell CpG islands and is an important pathway for the repression of gene transcription in cancers. We investigated aberrant hypermethylation of 11 genes by methylation-specific polymerase chain reaction (PCR), after treatment of the DNA with bisulphite, and correlated the findings with MYCN amplification and allelic status at 1p in a series of 44 neuroblastic tumours. This tumour series includes five ganglioneuromas (G), one ganglioneuroblastoma (GN) and 38 neuroblastomas (six stage 1 tumours; five stage 2 tumours; six stage 3 cases; 19 stage 4 tumours, and two stage 4S cases). Aberrant methylation of at least one of the 11 genes studied was detected in 95% (42 of 44) of the cases. The frequencies of aberrant methylation were: 64% for thrombospondin-1 (THBS1); 30% for tissue inhibitor of metalloproteinase 3 (TIMP-3); 27% for O6-methylguanine-DNA methyltransferase (MGMT); 25% for p73; 18% for RB1; 14% for death-associated protein kinase (DAPK), p14ARF, p16INK4a and caspase 8, and 0% for TP53 and glutathione S-transferase P1 (GSTP1). No aberrant methylation was observed in four control normal tissue samples (brain and adrenal medulla). MYCN amplification was found in 11 cases (all stage 4 neuroblastomas), whereas allelic loss at 1p was identified in 16 samples (13 stage 4 and two stage 3 neuroblastomas, and one ganglioneuroma). All but one case with caspase 8 methylation also displayed MYCN amplification. Our results suggest that promoter hypermethylation is a frequent epigenetic event in the tumorigenesis of neuroblastic tumours, but no specific pattern of hypermethylated genes could be demonstrated.

Pathological and karyotypic abnormalities in advanced gastric carcinomas.

Twenty samples of stomach cancers were analyzed by conventional cytogenetic and histopathological techniques. Nineteen tumors were diagnosed as adenocarcinomas and one as an adenosquamous carcinoma. Multiple and complex chromosomal abnormalities were found in the cases evaluated cytogenetically. This heterogeneity of chromosomal changes appears to indicate a certain correlation with tumor progression. Histological analysis showed a distinctive growth pattern of gastric cancer samples and a potential for invasiveness and recurrence for all tumors as well as a poor prognosis.

Karyotypic evolution of human meningioma. Progression through malignancy.

Cytogenetic analysis of an untreated sarcomatous meningioma from a patient submitted to two surgeries separated by 1 year are reported. The material from the first surgery was mostly hypodiploid, with a modal chromosome number of 42. Alterations of chromosome 22 were found in 80.6% of the cells. Four chromosome markers were found involving chromosomes 1, 2, 6, and 22, and numerical alterations involving chromosomes 8, 9, 10, 13, 14, 15, 18, 19, 20, 21, and Y. Although the modal chromosome number of the material from the second surgery was 45 (17.9% of the cells), 43.1% of its cells were hyperdiploid, 73% of these being in the triploid-tetraploid range. Dicentric and ring chromosomes were very frequent. Alterations involving chromosome 22 were still present. There was a recurrent trisomy of chromosome 3. To our knowledge, this is the first cytogenetic description, with banding techniques, of a malignant meningioma.