Phosphoproteomic analysis of neoadjuvant breast cancer suggests that increased sensitivity to paclitaxel is driven by CDK4 and filamin A.

Precision oncology research is challenging outside the contexts of oncogenic addiction and/or targeted therapies. We previously showed that phosphoproteomics is a powerful approach to reveal patient subsets of interest characterized by the activity of a few kinases where the underlying genomics is complex. Here, we conduct a phosphoproteomic screening of samples from HER2-negative female breast cancer receiving neoadjuvant paclitaxel (N = 130), aiming to find candidate biomarkers of paclitaxel sensitivity. Filtering 11 candidate biomarkers through 2 independent patient sets (N = 218) allowed the identification of a subgroup of patients characterized by high levels of CDK4 and filamin-A who had a 90% chance of achieving a pCR in response to paclitaxel. Mechanistically, CDK4 regulates filamin-A transcription, which in turn forms a complex with tubulin and CLIP-170, which elicits increased binding of paclitaxel to microtubules, microtubule acetylation and stabilization, and mitotic catastrophe. Thus, phosphoproteomics allows the identification of explainable factors for predicting response to paclitaxel.

Association of human papillomavirus viral load with HPV16 and high-grade intraepithelial lesion.

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other 'high-risk' viral types (HPV -18, -31, -33 or -51) and 'low-risk' types (P < 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p< 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.

Detection of c-erbB-2 gene amplification in cervical scrapes positive for human papillomavirus (HPV).

c-erbB-2 gene amplification has been described in a variety of human cancers, but it has been poorly studied in noncancerous cytological samples from genital specimens positive for human papillomavirus (HPV). Furthermore, the relationship between this genetic event and the presence of high-risk and low-risk HPV types is poorly studied. Eighty-four noncancerous cytological samples from exocervical specimens that were positive for HPV types 6, 16, and 18 were analyzed for c-erbB-2 gene amplification using the genomic differential polymerase chain reaction with the single copy reference gene. An association between c-erbB-2 gene amplification and the group corresponding to HPV type 6 was found. Within the low-risk HPV group, c-erbB-2 amplification was associated to cervical intraepithelial neoplasia of grade I (CIN I). Because in the samples analyzed, most of the CIN I stage was characterized by a koilocytotic pattern, c-erbB-2 amplification could be related to this kind of cellular alteration. It would be important to study c-erbB-2 gene amplification and also gene expression in different CIN stages in order to determine its role and significance in cervical cancer.

DNA damage by cadmium and arsenic salts assessed by the single cell gel electrophoresis assay.

Human exposure to metals is frequent due to their ubiquity, wide use in industry, and environmental persistence. Direct and indirect genotoxic effects of cadmium (Cd) and arsenic (As) were reported. However, the mechanisms of induction of genetic damage are not well known. The aim of the present work was to evaluate the degree of damage induced by Cd and As salts in a human lung fibroblasts cell line using the single cell gel electrophoresis assay (SCGE). MRC-5 cells were treated with cadmium chloride (CdCl(2)), cadmium sulfate (CdSO(4)), sodium arsenite (NaAsO(2)) and cacodylic acid (C(2)H(7)AsO(2)). A significant dose-dependent increment in the extent of DNA migration as well as in the percentage of cells with tails was observed (P<0.001) after treatment with CdSO(4) and NaAsO(2). Treatment with CdCl(2) induced a relatively low level of DNA strand breaks in comparison with that induced by CdSO(4). The increase migration observed with the three compounds could be originated either by the direct induction of DNA lesions or by the inhibition of excision repair mechanisms. On the other hand, cells treated with C(2)H(7)AsO(2) showed a decrease in the migration length with the three doses employed (P<0.001). The decrease in the rate of DNA migration could be a consequence of the induction of DNA cross-links by organic arsenicals.Cd and As salts induced DNA damage in fibroblast cells, detected as DNA migration in the single cell gel (SCG) assay. The distribution of DNA migration among cells as a function of dose revealed that the majority of exposed cells showed more DNA damage than cells obtained from control cultures. The potential for human exposure to both metals has been increased over the years due to the increment in their use. For this reason, elucidation of carcinogenic mechanisms is very important.

[Activation of K-ras and c-erbB-2 protooncogenes in human colonic adenocarcinomas]. / Activación de los protooncogenes K-ras y c-erbB-2 en adenocarcinomas colónicos humanos.

Although data on genetic alterations leading to development of colorectal cancer are abundant, no specific genetic alteration has been demonstrated for each class of tumor. The colorectal cancer phenotype is originated from an accumulation of different genetic alterations. The nature of these alterations, their order of appearance, and their associations vary greatly from one tumor to another, suggesting that the concept of a unique model of carcinogenesis is not applicable to these tumors. The aim of the present work was to study the association between K-ras and c-erbB-2 mutations and different clinicopathological variables in fifty-four samples from adenocarcinomas of the colon. The detection of K-ras activation was performed by specific enriched PCR. The genomic differential polymerase chain reaction with the single copy reference gene was employed for the detection of c-erbB-2 gene amplification. K-ras mutations were detected in 16 cases (29.63%) and c-erbB-2 amplifications in 1 sample (1.85%). Statistical analysis showed a significant association between K-ras codon 12 mutation frequency and Duke's stage B (p < 0.05). On the other hand, there was no association in relation to the other studied parameters. These results could indicate the occurrence of K-ras activation in early stages of the disease.

c-myc gene amplification detected in preinvasive intraepithelial cervical lesions.

The aim of the present work was to evaluate the c-myc gene amplification process in cervical samples and to analyze the relationship between the activation of this proto-oncogene and the cytologic and/or histologic status. Thirty-four normal cervical samples and 105 abnormal cervical tissue scrapes, previously used for PAP or histopathologic diagnosis, were analyzed for c-myc gene amplification. Detection of c-myc gene amplification was performed using a polymerase chain reaction (PCR)-based method known as target arbitrarily primed-PCR (TAP-PCR). For c-myc amplification, significant differences were found between normal samples and samples presenting different grades of lesions (P<0.001). A significant difference between high-grade squamous intraepithelial lesions (HG-SIL) and the other stages of cervical disease was also found (P<0.05). This study demonstrated that c-myc copy number increases according to the histologic grade of the lesion. These results could indicate that oncogene amplification takes place in preinvasive stages of cervical disease and could cooperate not only in tumor progression but also in cell transformation.

Activación de los protooncogenes K-ras y c-erbB-2 en adenocarcinomas colónicos humanos. / [Activation of K-ras and c-erbB-2 protooncogenes in human colonic adenocarcinomas]

Although data on genetic alterations leading to development of colorectal cancer are abundant, no specific genetic alteration has been demonstrated for each class of tumor. The colorectal cancer phenotype is originated from an accumulation of different genetic alterations. The nature of these alterations, their order of appearance, and their associations vary greatly from one tumor to another, suggesting that the concept of a unique model of carcinogenesis is not applicable to these tumors. The aim of the present work was to study the association between K-ras and c-erbB-2 mutations and different clinicopathological variables in fifty-four samples from adenocarcinomas of the colon. The detection of K-ras activation was performed by specific enriched PCR. The genomic differential polymerase chain reaction with the single copy reference gene was employed for the detection of c-erbB-2 gene amplification. K-ras mutations were detected in 16 cases (29.63

) and c-erbB-2 amplifications in 1 sample (1.85

). Statistical analysis showed a significant association between K-ras codon 12 mutation frequency and Dukes stage B (p < 0.05). On the other hand, there was no association in relation to the other studied parameters. These results could indicate the occurrence of K-ras activation in early stages of the disease.

Association between activated K-ras and c-erbB-2 oncogenes with "high-risk" and "low-risk" human papilloma virus types in preinvasive cervical lesions.

Clinical and epidemiological data have linked cervical cancer to the Human Papilloma Virus (HPV) infection. However, the presence of HPV infection alone is not enough to cause tumorigenesis, suggesting a role for additional host-cell genetic factors. The aim of the present work was to study the association of K-ras and c-erbB-2 mutations in cervical tissue samples with different grades of dysplasia and infected with HPV-6 ("low-risk" type) or HPV-16 and HPV-18 ("high-risk" types). Negative HPV-DNA samples were used as controls. The detection of K-ras and c-erbB-2 activation were performed by Artificial Refractory Mutation System (ARMS)-PCR and semiquantitative PCR, respectively. Statistical analysis showed a highly significant difference in K-ras codon 12 mutation frequency between high-risk and low-risk HPV-infected samples (p<0.05). On the other hand, amplification of the c-erbB-2 oncogene appeared associated to tissue samples infected with HPV-6 (p<0.003). Cervical carcinoma appears to arise from a series of well-characterized progressive histological changes, but the genetic alterations necessary for cervical tumorigenesis are not yet clear. These results raise the possibility for a role of certain proto-oncogenes and their activation in cervical neoplasia.

Differences in K-ras codon 12 mutation frequency between "high-risk" and "low-risk" HPV-infected samples.

OBJECTIVES: The current study was designed to determine the association between K-ras point mutations and the presence of high- and low-risk HPV types in noncancerous samples. METHODS: One hundred fifty-five noncancerous cytological samples from genital specimens positive for HPV-6, -16, or -18 were analyzed for codon 12 ras point mutations. DNA samples were subjected to nested PCR for the detection of HPV genome. Mutations at the first and second bases of K-ras codon 12 were detected using specific enriched PCR. RESULTS: Eleven percent of the HPV-positive samples analyzed showed mutation in K-ras codon 12 (17 of 155). Mutations were detected in 8 of 53 HPV-16- (15%) and 8 of 38 HPV-18-positive samples (21%). Nevertheless, when HPV-6-positive samples were screened for K-ras mutations only 1 of 64 samples was found mutated (1.56%). Statistical analysis using the chi(2) test showed a highly significant difference (chi(2) = 9.865, P < 0.01) when the rate of mutation for samples positive for high-risk HPV types (16 and 18) was compared with the frequency found in low-risk-infected samples (HPV-6). CONCLUSION: These results could be considered an indicator of the association between K-ras codon 12 mutations and the presence of high-risk HPV types.

[Analysis of prevalence of point mutations in codon 12 of oncogene K-ras from non-cancerous samples of cervical cytology positive for type 16 or 18 PVH]. / Análisis de la prevalencia de mutaciones puntuales en el codón 12 del oncogen K-ras en muestras no cancerosas de citología exfoliativa cervical positivas para VPH tipo 16 o 18.

Ninety-one non cancerous samples from genital specimens positives for VPH 16 or 18 and 27 non-infected samples as controls were studied. Mutations at codon 12 in K-ras gene was analyzed using enriched alelic PCR technique. Among the samples studied 17.58% showed mutations in this codon. Significant differences were observed between the control group (negative DNA-HPV) and positives DNA-HPV samples (p < 0.01). No differences were found between both viral types in relation to the mutation frequency. The presence of mutations in the K-ras gene in non cancerous cytological samples point out new questions about the role of mutations in proto-oncogenes and the development of cervical cancer.