TOWARDS PREDICTIVE PHARMACOGENETIC PROFILING FOR AZATHIOPRINE TREATMENT: CHARACTERIZATION OF SNPS IN RELEVANT DRUG METABOLISM GENES

Azathioprine (AZA) is one of the most widely used immunosuppressant drugs for the treatment of many inflammatory diseases, in particular autoimmune diseases where it is often administered as a steroid-sparing drug. It blocks T- and B-cell proliferation through incorporation of its active metabolites, 6-thioguanine nucleotides, into DNA. AZA is mainly catabolised through the thiopurine S-methyltransferase (TPMT) and xanthine oxidase (XO) pathways. While 70% of patients respond to AZA, 30% do not respond or show intolerance to the drug. Variants of genes coding for TPMT or XO, or other gene members of either pathway, which result or not in mutated enzymes, are believed to influence individual responses to AZA. With the aim of uncovering possible genotype-phenotype correlations that would help personalize AZA treatment through response-specific SNP profiles, we have analyzed TPMT and XDH (the gene coding for XO) for SNPs in 71 Italian patients in the context of their response to AZA (intolerant patients, n = 25; unresponsive patients, n = 16; and responsive patients, n = 30). We confirm the presence of known intronic and exonic TPMT and XDH polymorphisms not correlating with particular AZA responses. We identified two new intronic polymorphisms, G420-4A in TPMT and T652-21A in XDH, each in one intolerant patient. Whether or not intolerance is related to these mutations in the patients must be further elucidated. In XDH, we detected two novel non-synonymous mutations (c.G1004A, p.Arg335His; c.C2891T, p.Thr964Ile), one new synonymous mutation (c.G1194A, p.Leu398Leu), and one novel non-synonymous polymorphism (c.C1167T, p.Ala556Val). The missense mutation, p.Arg335His, is likely to have an effect on the structural conformation of the FAD-binding domain, thereby destabilizing the protein structure. This could therefore lead to a decreased XO activity, for example through increased protein degradation or through deficient binding of FAD, hence reduced efficiency of the enzyme function, and thereby intolerance to AZA as observed. The other non-synonymous mutation, p.Thr964Ile, occurred in the molybdopterin domain; it was found in one responsive patient, suggesting that this mutation did not modify the structure of the domain sufficiently to affect the enzymatic activity. The non-synonymous polymorphism, p.Ala556Val, causes a change from Ala to Val at position 556 in the connection segment between the FAD-binding and molybdopterin domains, which is unlikely to have a significant effect on the protein. This XDH polymorphism was found in one intolerant, one unresponsive, two responsive patients, and 3/100 healthy controls; it is therefore unlikely to be of significance in pharmacogenetic profiles predictive of AZA responses. The silent mutation, p.Leu398Leu, identified in an intolerant patient should not cause functional impairment of XO. Linkage disequilibrium (LD) of TPMT SNPs and haplotype analysis thereof demonstrated a new haplotype designated TPMT*3E; it comprises the previously reported mutations of the TPMT*3A allele associated with intolerance to AZA and the intronic T140+114A SNP. TPMT*3E was detected in four of the 25 AZA-intolerant patients and was not observed in unresponsive or responsive patients. The association of TPMT*3E with AZA intolerance, and its frequency, must be ascertained in larger, ethnically different cohorts. Nevertheless, in view of the highly significant association (Psim = 0.037) between TPMT*3E and AZA intolerance in our study, this haplotype should be taken into account when considering AZA treatment. LD analysis of XDH identified four different haplotype blocks, one of which was significantly associated with intolerance in our cohort (Psim = 0.017). This block includes five SNPs, one intronic and four located in the 3' untranslated regions (3' UTRs); these were previously described as single SNPs, but were not analyzed in the context of response to AZA. It is unclear how this haplotype results in intolerance to AZA. One possibility is that the SNPs affect the regulation of protein expression through alteration of the target sites for microRNAs that interact with the 3' UTRs to regulate the expression of mRNAs. This could therefore lead to AZA intolerance in our patients, through in an increase in down-regulation of XDH mRNA and thereby XO expression. In this work, we have demonstrated new haplotypes that should be taken into consideration in pharmacogenetic profiling for AZA. In particular, SNPs in the XDH have been poorly investigated thus far in the context of response to AZA; the new XDH haplotype is of major interest in the establishment of pharmacogenetic profiles that will permit prediction of the type of response to AZA, in particular to prevent life-threatening side effects. It should be further studied in the context of its association with other response-defining haplotypes or SNPs of TPMT and other AZA metabolism pathway genes

Cancer/Testis Antigen MAGE-C1/CT7: New Target for Multiple Myeloma Therapy

Cancer/Testis Antigens (CTAs) are a promising class of tumor antigens that have a limited expression in somatic tissues (testis, ovary, fetal, and placental cells). Aberrant expression of CTAs in cancer cells may lead to abnormal chromosome segregation and aneuploidy. CTAs are regulated by epigenetic mechanisms (DNA methylation and acetylation of histones) and are attractive targets for immunotherapy in cancer because the gonads are immune privileged organs and anti-CTA immune response can be tumor-specific. Multiple myeloma (MM) is an incurable hematological malignancy, and several CTAs have been detected in many MM cell lines and patients. Among CTAs expressed in MM we must highlight the MAGE-C1/CT7 located on the X chromosome and expressed specificity in the malignant plasma cells. MAGE-C1/CT7 seems to be related to disease progression and functional studies suggests that this CTA might play a role in cell cycle and mainly in survival of malignant plasma cells, protecting myeloma cells against spontaneous as well as drug-induced apoptosis

Studying pyrolysis products of bottom-of-the-barrel fuel with Py-GCxGC- TOF/CSD/FID

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A phase II study of primary dose-dense sequential doxorubicin plus cyclophosphamide and docetaxel in cT4 breast cancer

Background: Dose-dense chemotherapy with anthracyclines and taxanes has improved either disease free survival or overall survival in high risk patients with early breast cancer. Patients and Methods: The activity and safety of a dose-dense schedule (q14 days) of adriamycin 60 mg/sqm and cyclophosphamide 600 mg/sqm (AC) x 4 cycles followed by docetaxel 75 mg/sqm for 4 cycles with hematopoietic support in patients with stage IIIB breast cancer was explored. Patients with ER >= 10% tumors received concomitant endocrine therapy with 3-month triptorelin and letrozole. Results: Fifteen patients with histologically proven cT4b (three patients) and cT4d (twelve patients) MO breast cancer were enrolled. Median age was 48 years (range 25-66). Eight clinical responses including one pathological complete remission (pCR), three stable disease (including minor responses) and four progression of disease, one during AC and three during taxotere, were observed. Four patients had grade 3-4 non hematological toxicities and all except one discontinued treatment. Conclusion: Due to the high rate of progressive disease, this schedule should not represent a standard option in cT4 breast cancer

Effects of neoadjuvant trastuzumab, pertuzumab and palbociclib on Ki67 in HER2 and ER-positive breast cancer

The crosstalk between estrogen and HER2 receptors and cell-cycle regulation sustains resistance to endocrine therapy of HER2- and hormone receptor-positive breast cancer. We earlier reported that women with HER2 and ER-positive breast cancer receiving neoadjuvant dual HER2-block and palbociclib in the NA-PHER2 trial had Ki67 decrease and 27% pathological complete responses (pCR). We extended NA-PHER2 to Cohort B using dual HER2-block and palbociclib without fulvestrant and report here Ki67 drops at week-2 (mean change 1225.7), at surgery (after 16 weeks, mean change 129.5), high objective response (88.5%) and pCR (19.2%). In Cohort C [Ki67 > 20% and HER2low (IHC 1+/2+ without gene amplification)], women also received fulvestrant, had dramatic Ki67 drop at week 2 ( 1229.5) persisting at surgery ( 1219.3), and objective responses in 78.3%. In view of the favorable tolerability and of the efficacy-predictive value of Ki67 drop at week-2, the chemotherapy-free approach of NA-PHER2 deserves further investigation in HER2 and ER-positive breast cancer. The trial is registered with ClinicalTrials.gov, number NCT02530424

The discovery of the southernmost ultra-high-resolution Holocene paleoclimate sedimentary record in Antarctica

The response of the Antarctic ice sheet to climate warming is the main source of uncertainty regarding future global sea level rise, since little is known about its present and past dynamics. The last deglaciation is the most recent interval of large-scale climate warming, during which the Northern and Southern Hemisphere ice sheets retreated, and sea level rose globally, although at a non-uniform rate. Geologic records from the polar regions are fundamental in determining the factors that caused the major changes in ice sheets during the last deglacial under different boundary conditions. Here, we combine morpho-bathymetric and seismic data with sediment cores and oceanographic measurements to reconstruct the processes that influenced the deposition of the southernmost, most extensive, ultrahigh-resolution record of the Holocene in Edisto Inlet fjord (Ross Sea, Antarctica). We find that post-glacial sedimentation resulted in a layered diatom mud up to 110 m thick that was locally redistributed by bottom currents over confined drifts-moats in the central part of the fjord. After the Holocene climatic optimum, the fjord was not carved by ground ice, and there continued to be internal fjord water circulation associated with Ross Sea circulation. These results support a retreat of coastal glaciers by about 11 kiloyears ago (ka) from the continental shelf of North Victoria Land

Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26-27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70-80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p < 0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p < 0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p < 0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Laboratory of Molecular Biology and Genomics, Ludwig Institute for Cancer Research, São Paulo Branch, BrazilUniversidade Federal de São Paulo, Disciplina Hematol & Hemoterapia, São Paulo, BrazilLudwig Inst Canc Res, Lab Mol Biol & Genom, São Paulo, BrazilRecepta Biopharma, Ludwig Inst Canc Res, São Paulo, BrazilInCor, Fac Med, Setor Vetores Virais, Lab Genet & Cardiol Mol, São Paulo, BrazilJohns Hopkins Univ, Sch Med, Dept Neurosurg, Ludwig Collaborat Grp, Baltimore, MD 21205 USAUniv Med Ctr Hamburg Eppendorf, Dept Med 2, Hamburg, GermanyUniversidade Federal de São Paulo, Disciplina Hematol & Hemoterapia, São Paulo, BrazilWeb of Scienc

Molecular Insights into the Classification of Luminal Breast Cancers : the Genomic Heterogeneity of Progesterone-Negative Tumors

Estrogen receptor (ER)-positive progesterone receptor (PR)-negative breast cancers are infrequent but clinically challenging. Despite the volume of genomic data available on these tumors, their biology remains poorly understood. Here, we aimed to identify clinically relevant subclasses of ER+/PR- breast cancers based on their mutational landscape. The Cancer Genomics Data Server was interrogated for mutational and clinical data of all ER+ breast cancers with information on PR status from The Cancer Genome Atlas (TCGA), Memorial Sloan Kettering (MSK), and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) projects. Clustering analysis was performed using gplots, ggplot2, and ComplexHeatmap packages. Comparisons between groups were performed using the Student's t-test and the test of Equal or Given Proportions. Survival curves were built according to the Kaplan\u207bMeier method; differences in survival were assessed with the log-rank test. A total of 3570 ER+ breast cancers (PR- n = 959, 27%; PR+ n = 2611, 73%) were analyzed. Mutations in well-known cancer genes such as TP53, GATA3, CDH1, HER2, CDH1, and BRAF were private to or enriched for in PR- tumors. Mutual exclusivity analysis revealed the presence of four molecular clusters with significantly different prognosis on the basis of PIK3CA and TP53 status. ER+/PR- breast cancers are genetically heterogeneous and encompass a variety of distinct entities in terms of prognostic and predictive information

Oestrogen receptor status, pathological complete response and prognosis in patients receiving neoadjuvant chemotherapy for early breast cancer

The aim of this study was to ascertain if oestrogen receptor ( ER) status predicts for pathological complete response (pCR) to neoadjuvant chemotherapy in operable breast cancer, and the effects of pCR on survival. Using a single-institution database, 435 patients were identified, who received neoadjuvant chemotherapy for operable breast cancer and were eligible for the analysis. Patients whose tumours were ER negative were more likely to achieve a pCR than patients who were ER positive (21.6 vs 8.1%,