Polymorphism of alpha-1-antitrypsin in hematological malignancies

Alpha-1-antitrypsin (AAT) or serine protease inhibitor A1 (SERPINA1) is an important serine protease inhibitor in humans. The main physiological role of AAT is to inhibit neutrophil elastase (NE) released from triggered neutrophils, with an additional lesser role in the defense against damage inflicted by other serine proteases, such as cathepsin G and proteinase 3. Although there is a reported association between AAT polymorphism and different types of cancer, this association with hematological malignancies (HM) is, as yet, unknown. We identified AAT phenotypes by isoelectric focusing (in the pH 4.2-4.9 range) in 151 serum samples from patients with HM (Hodgkins lymphomas, non-Hodgkins lymphomas and malignant monoclonal gammopathies). Healthy blood-donors constituted the control group (n = 272). The evaluated population of patients as well as the control group, were at Hardy-Weinberg equilibrium for the AAT gene (χ2 = 4.42, d.f.11, p = 0.96 and χ2 = 4.71, d.f.11, p = 0.97, respectively). There was no difference in the frequency of deficient AAT alleles (Pi Z and Pi S) between patients and control. However, we found a significantly higher frequency of PiM1M1 homozygote and PiM1 allele in HM patients than in control (for phenotype: f = 0.5166 and 0.4118 respectively, p = 0.037; for allele: f = 0.7020 and 0.6360 respectively, p = 0.05). In addition, PiM homozygotes in HM-patients were more numerous than in controls (59% and 48%, respectively, p = 0.044). PiM1 alleles and PiM1 homozygotes are both associated with hematological malignancies, although this is considered a functionally normal AAT variant

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2h. Treatments with 200ng/ml cisplatin or 400ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400 respectively) that showed low level (approximately 2-fold) resistance after 8 treatments. Treatments with 1000ng/ml cisplatin or 2000ng/ml oxaliplatin for 2h also produced sublines, however these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that "regrowth resistance" initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulfoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggests the taxanes may be useful in the treatment of platinum resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC

Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil

To identify mechanisms underlying oxaliplatin resistance, a subline of the human gastric adenocarcinoma TSGH cell line, S3, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations. Compared with the parental TSGH cells, the S3 subline showed 58-fold resistance to oxaliplatin; it also displayed 11-, 2-, and 4.7-fold resistance to cis-diammine-dichloroplatinum (II) (CDDP), copper sulphate, and arsenic trioxide, respectively. Interestingly, S3 cells were fourfold more susceptible to 5-fluorouracil-induced cytotoxicity due to downregulation of thymidylate synthase. Despite elevated glutathione levels in S3 cells, there was no alteration of resistant phenotype to oxaliplatin or CDDP when cells were co-treated with glutathione-depleting agent, l-buthionine-(S,R)-sulphoximine. Cellular CDDP and oxaliplatin accumulation was decreased in S3 cells. In addition, amounts of oxaliplatin- and CDDP–DNA adducts in S3 cells were about 15 and 40% of those seen with TSGH cells, respectively. Western blot analysis showed increased the expression level of copper transporter ATP7A in S3 cells compared with TSGH cells. Partial reversal of the resistance of S3 cells to oxaliplatin and CDDP was observed by treating cell with ATP7A-targeted siRNA oligonucleotides or P-type ATPase-inhibitor sodium orthovanadate. Besides, host reactivation assay revealed enhanced repair of oxaliplatin- or CDDP-damaged DNA in S3 cells compared with TSGH cells. Together, our results show that the mechanism responsible for oxaliplatin and CDDP resistance in S3 cells is the combination of increased DNA repair and overexpression of ATP7A. Downregulation of thymidylate synthase in S3 cells renders them more susceptible to 5-fluorouracil-induced cytotoxicity. These findings could pave ways for future efforts to overcome oxaliplatin resistance

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands