Array-based pharmacogenomics of molecular-targeted therapies in oncology.

The advent of microarrays over the past decade has transformed the way genome-wide studies are designed and conducted, leading to an unprecedented speed of acquisition and amount of new knowledge. Microarray data have led to the identification of molecular subclasses of solid tumors characterized by distinct oncogenic pathways, as well as the development of multigene prognostic or predictive models equivalent or superior to those of established clinical parameters. In the field of molecular-targeted therapy for cancer, in particular, the application of array-based methodologies has enabled the identification of molecular targets with 'key' roles in neoplastic transformation or tumor progression and the subsequent development of targeted agents, which are most likely to be active in the specific molecular setting. Herein, we present a summary of the main applications of whole-genome expression microarrays in the field of molecular-targeted therapies for solid tumors and we discuss their potential in the clinical setting. An emphasis is given on deciphering the molecular mechanisms of drug action, identifying novel therapeutic targets and suitable agents to target them with, and discovering molecular markers/signatures that predict response to therapy or optimal drug dose for each patient.

Mutational analysis of the cell cycle inhibitor Kip1/p27 in childhood leukemia.

BACKGROUND: Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. METHODS: Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. RESULTS: Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. CONCLUSIONS: Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia.

Genomic rearrangements on VCAM1, SELE, APEG1and AIF1 loci in atherosclerosis.

The inflammatory nature of atherosclerosis has been well established. However, the initial steps that trigger this response in the arterial intima remain obscure. Previous studies reported a significant rate of genomic alterations in human atheromas. The accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells may be important for disease development. To address this issue, 78 post-mortem obtained aortic atheromas were screened for microsatellite DNA alterations versus correspondent venous blood. To evaluate the significance of these observations, 33 additional histologically normal aortic specimens from age and sex-matched cases were examined. Loss of heterozygosity (LOH) was found in 47,4% of the cases and in 18,2% of controls in at least one locus. The LOH occurrence in aortic tissue is associated to atherosclerosis risk (OR 4,06, 95% CI 1,50 to 10,93). Significant genomic alterations were found on 1p32-p31, 1q22-q25, 2q35 and 6p21.3 where VCAM1, SELE, APEG1 and AIF1 genes have been mapped respectively. Our data implicate somatic DNA rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition.

Detection of point mutations at codon 12 of KI-ras in ophthalmic pterygia.

AIMS: Ophthalmic pterygium is a potentially vision-threatening lesion of unknown etiology, related to an exposure to solar light. Mutations to the ras genes are frequently observed in lesions related to an exposure to solar light. The present study aims at screening pterygia for mutations at codons 12 and 13 of the ras genes. METHODS: In all, 50 pterygia were examined, together with respective blood samples and specimens of normal conjunctiva. A PCR reaction was performed to amplify sequences containing codons 12 and 13 of Ki-ras, H-ras, and N-ras. An RFLP analysis was then performed to detect point mutations at codon 12. The mutational status at codons 12 and 13 was further explored with sequencing of PCR products. RESULTS: RFLP analysis revealed Ki-ras mutations at codon 12 in five (10%) of pterygia, whereas H-ras or N-ras mutations were not observed. Sequencing confirmed Ki-ras mutations at codon 12 and revealed absence of mutations at codon 13. The presence of Ki-ras mutations was significantly correlated with postoperative recurrence (P=0.02) and young age (P=0.04). Mutations were not observed in specimens of blood or normal conjunctiva for any of the genes examined. CONCLUSIONS: The absence of N-ras mutations is in agreement with previous reports concerning mucosal lesions. The detection of Ki-ras mutations and the association with postoperative recurrence implies a possible role of Ki-ras in the clinical profile of pterygium. The mechanism of Ki-ras mutations is unclear and could be independent of the action of UV light.

The transcobalamin codon 259 polymorphism influences the risk of human spontaneous abortion.

BACKGROUND: The remethylation cycle of methionine is folate and vitamin B(12) (cobalamin) dependent and appears to be crucial for embryonic development, probably through effects on synthesis of DNA, proteins and polyamines. Transcobalamin (TC) transports vitamin B(12) to the tissues. The objective of the present investigation was to explore the putative association between the major TC genetic polymorphism (Pro259Arg) and human spontaneous abortion. METHODS: The prevalence of the TC Pro259Arg polymorphism was determined in DNA samples from embryos that had been spontaneously aborted between the 6th and 20th week after conception, and adult controls using solid-phase minisequencing technique. RESULTS: The 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57.0% respectively; P = 0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2% respectively; P < 0.001). CONCLUSIONS: The 259-Pro allele seems to have beneficial influences during embryogenesis, conceivably through its positive effect on vitamin B(12) intracellular bioavailability. Our results warrant additional investigations addressing the question if vitamin B(12) supplementation in addition to folic acid supplementation may prevent spontaneous abortion in women planning a pregnancy.

MYCL1, FHIT, SPARC, p16(INK4) and TP53 genes associated to lung cancer in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic interstitial pneumonia limited to the lung and characterized by a fibroproliferative response with only minor signs of inflammation, which almost always causes rapid fibrotic destruction of the lung. In this study, we investigated genomic instability in IPF, using microsatellite DNA analysis, aiming to detect any specific genetic alterations for this disease. We used 40 highly polymorphic microsatellite DNA markers, in multiplex PCR assays, to examine 52 sputum specimens from IPF patients versus correspondent venous blood. Loss of heterozygosity (LOH) was found in 20 (38.5%) patients in at least one locus. These alterations were found on markers previously associated with lung cancer located on 1p34.3, 3p21.32-p21.1, 5q32-q33.1, 9p21 and 17p13.1 where MYCL1, FHIT, SPARC, p16(Ink4) and TP53 genes have been mapped respectively. These data provide new insights into IPF pathogenesis and a new perspective for its correlation with lung cancer.

Genomic instability on hMSH2, hMLH1, CD48 and IRF4 loci in pulmonary sarcoidosis.

Pulmonary sarcoidosis shares certain features with immune disease and neoplasia, and microsatellite DNA alterations are detectable in sputum specimens of pulmonary sarcoidosis patients. The biological basis and significance of these findings remain obscure, while information regarding the genetic basis of the disease is limited. Using multiplex PCR-based microsatellite analysis, we investigated 40 markers located on 1p, 1q, 2p, 2q, 3p, 5q, 6p, 7p, 9p, 11q, 14q and 17p in 38 sputum specimens of pulmonary sarcoidosis patients. Loss of heterozygosity (LOH) was found in 13 of 38 (34.2%) patients in at least one locus. These alterations occurred in the subset of markers located in or close to DNA mismatch repair (MMR) genes, hMSH2 (2p22.3-p16.1) and hMLH1 (3p2l.32-p21.1), as well as in CD48 (1q21-q23) and IRF4 (6p23-p25), genes associated with lymphocyte activation. Microsatellite instability (MIN) was observed in five cases (13.2%) in at least one locus. Our data suggest that genomic instability in pulmonary sarcoidosis could be due to MMR defects, while alterations of lymphocyte-specific agents could account for granuloma formation.

Low-penetrance genes are associated with increased susceptibility to endometriosis.

OBJECTIVE: To investigate whether genetic polymorphisms of CYP1A1, GSTM1, and GSTT1 are associated with endometriosis. DESIGN: Genetic polymorphism analysis. SETTING: University department. PATIENT(S): A family with four women in two generations who had endometriosis and one member with suspected endometriosis in the third generation were compared with a group of fertile women. INTERVENTION(S): Laparoscopic examination. MAIN OUTCOME MEASURE(S): Blood specimens were obtained from fertile females and available affected female family members. Multiplex polymerase chain reaction (PCR) and restriction fragment length polymorphism PCR was done to determine each participant's genotype. RESULT(S): All affected family members had genotype CYP1A1 wt/m1 and GSTM1 null deletion. The frequency of this genotype in 54 fertile women was 13%. A 17-year-old family member with suspected endometriosis had the same genotype. One affected member was also a carrier of a GSTT1 null deletion. This combination was not found in any of the fertile participants. The most frequent genotypes in the sample were CYP1A1 wt/wt, with GSTM1 null deletion and at least one functional allele of GSTT1, and CYP1A1 wt/wt, with at least one functional allele of GSTM1 and GSTT1 (33% and 31%, respectively). CONCLUSION(S): The combination of CYP1A1 m1 polymorphism and GSTM1 null deletion is closely associated with penetration of the endometriosis phenotype, whereas GSTT1 null deletion may add to the penetration of this trait.

Genetic polymorphisms and transcriptional pattern analysis of CYP1A1, AhR, GSTM1, GSTP1 and GSTT1 genes in breast cancer.

In order to detect the contribution of cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), glutathione S-transferases M1 (GSTM1), P1 (GSTP1), and T1 (GSTT1) genes in breast cancer, genetic analysis was performed, as well as transcriptional analysis in sporadic primary tumours and corresponding adjacent normal tissues from the same patient. CYP1A1 3'-untranslated region (3'-UTR) termed as m1 (MspI) polymorphism and the null(-) deletions of both GSTM1 and GSTT1 genes were examined in genomic DNA from blood samples of 207 female breast cancer patients and 171 age and sex matched controls. The frequencies of the m1 genotype of the CYP1A1 gene in cases and controls were 0.13 and 0.15, while the frequencies of homozygotes with GSTM1(-) were 0.52, in each, and for homozygotes with GSTT1(-) were 0.14 and 0.10, respectively. Statistical analysis of these genotypes in combinations did not reveal any significant difference between the breast cancer population and the control group. Expression of mRNA levels of CYP1A1, GSTM1, GSTP1, GSTT1 and AhR genes in 31 breast cancer patients, revealed inter-individual variation in an independent manner to patient age, genotype, or tumour stage. Eighty-seven percent of the tumour specimens tested were deregulated, compared to their normal counterparts, in at least one locus. Up-regulation of CYP1A1 was observed only when one of the GSTM1 or GSTP1 was down-regulated while the other remained constant. Genotyping analysis did not show any correlation to breast cancer risk. However, RT-PCR analysis provided evidence that CYP1A1, AhR, GSTM1, GSTP1 and GSTT1 genes are frequently deregulated in breast cancer and could be used as molecular biomarkers for better clinical management of such patients, with respect to chemotherapy.

Microsatellite DNA assays reveal an allelic imbalance in p16(Ink4), GALT, p53, and APOA2 loci in patients with endometriosis.

OBJECTIVE: To detect allelic imbalance on specific genetic loci occurring in endometriosis. DESIGN: Microsatellite analysis. SETTING: Paraffin-embedded tissues histologically confirmed as endometriotic or normal endometrium. PATIENT(S): Premenopausal women undergoing laparoscopy for suspected endometriosis. INTERVENTION(S): Laparoscopic excision of specimens. MAIN OUTCOME MEASURE(S): Allelic imbalance and alterations of intensity of microsatellite alleles. RESULT(S): Five of 17 microsatellite DNA markers (29.4%) showed allelic imbalance. Eight samples (36.4%) showed allelic imbalance in at least one locus. Loci 9p21, 1q21, and 17p13.1 exhibited imbalance in 27.3%, 4.5%, and 4.5%, respectively. A 3-fold increase of the fractional allelic loss was observed from disease stage II to III and IV, whereas only 1.3-fold was found between patients of 41-50 and 20-40 years. CONCLUSION(S): We found that loss of heterozygosity on p16(Ink4), GALT, and p53, as well as on APOA2, a region frequently lost in ovarian cancer, occurs in endometriosis, even in stage II of the disease. The occurrence of such genomic alterations may represent important events in the development of endometriosis. The 9p21 locus may contain a gene associated with the pathogenesis of the disease, and therefore its loss may be a prognostic marker of the disease.

Loss of heterozygosity in adenomyosis on hMSH2, hMLH1, p16Ink4 and GALT loci.

Adenomyosis is a gynecological condition in which tissue histologically similar to that in endometrium is found within the myometrium in the uterus. Although, lesions of both adenomyosis and endometriosis are identical to their sources with respect to structure and function, they are generally regarded as separate and distinct nosologic processes. In this study, we used 17 microsatellite markers, in four tetraplex and one single PCR assay, to determine the incidence of loss of heterozygosity (LOH) in 31 cases of adenomyosis. The markers used are located close to tumor suppressor genes, DNA repair genes, and genes which are thought to be involved in endometriosis. Moreover, the markers were involved in regions frequently lost in ovarian cancer, on chromosomal arms 1p, 1q, 2p, 2q, 3p, 9p, 9q, 17p and 17q. Nine samples (29.0%) showed LOH in at least one locus. Loci 2p22.3-p16.1, 3p24.2-p22 and 9p21 exhibited imbalance (19.4%, 9.7% and 6.5% respectively). This is the first report, that LOH occurs in adenomyosis. The regional chromosomal losses were detectable early during the development of this condition. In addition, hMSH2, hMLH1, p16Ink4 and GALT genes were associated for the first time with adenomyosis and its pathogenesis.

Loss of heterozygosity in DNA mismatch repair genes in human atherosclerotic plaques.

To detect the incidence of loss of heterozygosity (LOH) in DNA mismatch repair genes (MMR) occurring in atherosclerosis, fifty human autopsy cases of atherosclerosis were examined for LOH using 19 microsatellite markers, in three single and four tetraplex microsatellite assays. The markers used are located on or close to MMR genes. Fourteen specimens (28%) showed allelic imbalance in at least one locus. Loci hMSH2 (2p22.3-p16.1), hPMS1 (2q24.1-q32.1), and hMLH1 (3p21.32-p21.1) exhibited LOH (10, 10, and 12% respectively). We found that loss of heterozygosity on hMSH2, hPMS1, and hMLH1, occurs in atherosclerosis. The occurrence of such genomic alterations may represent important events in the development of atherosclerosis.

Low incidence of H-, K- and N-ras oncogene mutations in cytological specimens of laryngeal tumours.

Laryngeal cancer is a rare type of neoplasia, constituting approximately 2% of all human cancers. Mutations of the ras gene family is one of the main activating mechanisms in human cancer. Their involvement in head and neck cancer has been mainly demonstrated at the level of the overexpression whereas ras mutations in these cancers are rare in the Western world. In the present study we explored the incidence of codon 12-point mutation in the H-, K- and N-ras genes, in 41 laryngeal cytological specimens. These specimens corresponded to 19 benign and 22 malignant lesions of the larynx. Only two specimens carried a codon 12-point mutation in the K-ras gene (4.8%) while no mutation was detected in the H- and N-ras genes. K-ras mutations were detected in one benign and one malignant specimen. These results indicate low incidence of ras oncogene mutations in laryngeal cytological specimens.