Citations

Thiopurine methyltransferase (TPMT) activity exhibits a monogenic codominant inheritance and catabolizes thiopurines. TPMT variant alleles are associated with low enzyme activity and pronounced pharmacologic effects of thiopurines. Loss‐of‐function alleles in the NUDT15 gene are common in Asians and Hispanics and reduce the degradation of active thiopurine nucleotide metabolites, also predisposing to myelosuppression. We provide recommendations for adjusting starting doses of azathioprine, mercaptopurine, and thioguanine based on TPMT and NUDT15 genotypes (updates on www.cpicpgx.org ).

Abstract Objective: Response to menopausal hormone therapy (MHT) shows individual variation. SLCO1B1 encodes the OATP1B1 transporter expressed in the liver that transports many endogenous substances, including estrone sulfate, from the blood into hepatocytes. This study evaluated the relationship between genetic variation in SLCO1B1 and response to MHT in women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic, Rochester, MN. Methods: KEEPS participants were randomized to oral conjugated equine estrogen (n = 33, oCEE), transdermal 17β-estradiol (n = 33, tE2), or placebo (n = 34) for 48 months. Menopausal symptoms (hot flashes, night sweats, insomnia, palpitations) were self-reported before treatment and at 48 months. Estrone (E1), E2, and sulfated conjugates (E1S, E2S) were measured using high-performance liquid chromatography-tandem mass spectrometry. SLCO1B1 rs4149056 (c.521T>C, p.Val174Ala) was genotyped using a TaqMan assay. Results: After adjusting for treatment, there was a significant association between the SLCO1B1 rs4149056 TT genotype (encoding normal function transporter) and lower E1S, E1S/E1, and E2S (P = 0.032, 0.010, and 0.008, respectively) compared with women who were heterozygous (TC) or homozygous (CC) for the reduced function allele. The interactions between genotype, treatment, and E2S concentration were stronger in women assigned to tE2 (P = 0.013) than the women taking oCEE (P = 0.056). Among women assigned to active treatment, women with the CT genotype showed a significantly greater decrease in night sweats (P = 0.041) than those with the TT genotype. Conclusions: Individual variation in sulfated estrogens is explained, in part, by genetic variation in SLCO1B1. Bioavailability of sulfated estrogens may contribute to relief of night sweats.

Abstract Hot flashes have typically been classified as “symptoms of menopause” that should be tolerated or treated until they resolve. However, mounting evidence points to hot flashes as a manifestation of one or several underlying pathophysiological processes. Associations exist between the presence, timing of onset, severity, and duration of hot flashes, and the risk of several neurological (affecting sleep, mood, and cognition) and cardiovascular conditions. In addition, four consistent patterns of vasomotor disturbances have been identified across different countries, making it unlikely that these patterns are solely explained by socioeconomic or cultural factors. The changing hormonal environment of menopause may unmask differences in the autonomic neurovascular control mechanisms that put an individual woman at risk for chronic conditions of aging. These differences may have a genetic basis or may be acquired across the life span and are consistent with the variability of the clinical manifestations of aging observed in women after bilateral oophorectomy. It is time to investigate the pathophysiological mechanisms underlying the four patterns of vasomotor symptoms more closely, and to shift from describing hot flashes as symptoms to be tolerated to manifestations of an underlying autonomic neurovascular dysregulation that need to be addressed.

Smoking is a major risk factor for failure of coronary artery bypass grafts (CABG). Experiments were designed to determine effects of transdermal nicotine, independent of smoking, on structure and function of CABG. Saphenous veins were placed as CABG in untreated dogs (control) or in dogs treated with transdermal nicotine (one 11-mg or two 22-mg patches/day) for 5 wk. Serum nicotine and plasma nitric oxide were measured. Grafts were removed and prepared for organ chamber studies and histology. Serum nicotine averaged 12.1 and 118.7 ng/ml in the 11 mg/day and 44 mg/day groups, respectively. Plasma nitric oxide was higher in dogs treated with 11 mg/day doses compared with controls. In organ chamber studies, endothelium-dependent relaxations to thrombin and A-23187 and endothelium-independent relaxations to nitric oxide were greatest in grafts from dogs treated with 11 mg/day doses. Intimal thickness of the grafts were similar among groups. However, staining for bone sialoprotein was increased in the media of grafts from the 11 mg/day treatment group. These data suggest that transdermal nicotine in doses comparable and double to those used for conventional smoking cessation treatment in humans does not adversely affect early patency of canine CABG up to 4 wk postoperatively. Transdermal nicotine, however, may increase production of and response to nitric oxide in bypass grafts.

A 28‐year‐old female with PIK3CA‐related segmental overgrowth presented with headaches. She also had a unilateral vestibular schwannoma (VS), as well as 3 small (<2 cm) meningiomas, which according to the Manchester consensus diagnostic criteria for neurofibromatosis 2 (NF2) is sufficient for a clinical diagnosis. Analysis of blood revealed a mosaic PIK3CA c.2740G>A (p.Gly914Arg) mutation, confirming the diagnosis of PIK3CA‐related overgrowth, but no mutations in NF2 were detected. Although VS has not previously been reported in PIK3CA‐related segmental overgrowth, meningiomas have, raising the question of whether this patient's VS and meningiomas represent coincidental NF2 or phenotypic extension of her overgrowth syndrome. Genetic analysis of the VS revealed a heterozygous NF2 mutation c.784C>T (p.Arg262Ter) and loss of a portion of 22q, including NF2, SMARCB1, and LZTR1 genes. These results suggest that the patient has 2 different mosaic disorders, NF2 and PIK3CA‐related overgrowth. The PIK3CA mutation was also present in the VS. Confirmation of the clinical diagnosis of mosaic NF2 in this patient has implications for monitoring and highlights the possibility of co‐occurrence of mosaicism for multiple rare disorders in a single patient.

AbstractObjectiveHLA‐DPB1 matching may impact allogeneic hematopoietic stem cell transplantation (ASCT) outcomes; however, this locus is not in linkage disequilibrium with the remainder of the HLA genes. After classifying HLA‐DPB1 mismatches based on T‐cell epitope, avoiding non‐permissive mismatches may impact survival. We tested this hypothesis at a single academic institution.MethodsRetrospective HLA‐DPB1 genotyping was performed on 153 adult patients who underwent ASCT and unrelated donors matched for HLA‐A, B, C, DRB1, and DQB1 loci (10/10). Using the ImMunoGeneTics/HLA T‐cell epitope matching algorithm, mismatch status was classified as permissive or non‐permissive.ResultsOf 153 donor‐recipient pairs, 22 (14.4%) were HLA‐DPB1 matches, 64 (42.8%) permissive mismatches, and 67 (43.8%) non‐permissive mismatches. DPB1 mismatch increased risk of chronic graft‐versus‐host disease (cGVHD; RR 2.89 [1.19‐9.53], P=.016) compared with DPB1‐matched transplants, but there were no differences in overall mortality, risk of relapse, or acute GVHD (aGVHD). Combining matches and permissive mismatches and comparing to non‐permissive mismatches, there was no significant difference in overall survival or relapse; however, patients receiving non‐permissive mismatched transplants experienced greater risk of aGVHD overall and severe aGVHD (RR 1.66 [1.13‐2.44], P=.010 and RR 1.97 [1.10‐3.59], P=.024, respectively).ConclusionIn this single‐center study, HLA‐DPB1 matching influenced outcomes of patients undergoing ASCT for hematologic malignancy.

Abstract Objective: Onset and symptoms of menopause, and response to hormone therapy (HT) show large interindividual variability. SULT1A1 encodes for a highly expressed enzyme that metabolizes estrogens. We evaluated the relationship between genetic variation in SULT1A1, menopause age, symptoms, and response to HT. Methods: Women enrolled in the Kronos Early Estrogen Prevention Study at Mayo Clinic were randomized to 48 months of treatment with oral conjugated equine estrogen (n = 34), transdermal 17β-estradiol (E2) (n = 33), or placebo (n = 35). Linear regression models and ANOVA were used to test for association of SULT1A1 copy number, rs3760091, rs750155, and rs9282861 (SULT1A1 ∗ 2), with age at menopause and symptoms, levels of estrogens (estrone [E1], estrone sulfate [E1S], E2, and estradiol sulfate [E2S]), before and after HT. Results: SULT1A1 gene copy number affected the minor allele frequency for each single nucleotide polymorphisms tested. Before administration of exogenous hormones, increasing number of G alleles at rs9282861 was associated with earlier age at menopause (P = 0.014), lower frequency of night sweats (P = 0.009), and less severe insomnia (P = 0.046). After 48 months of treatment, SULT1A1 genotype was not associated with the presence of menopausal symptoms. In women randomized to oral conjugated equine estrogen, increasing number of the A allele at rs750155 was associated with lower E1S and E2S (P = 0.004 and 0.017), whereas increasing number of the C allele at rs3760091 was associated with lower E2S/E2 (P = 0.044). Conclusions: Interindividual variability in onset of menopause and symptoms before initiation of HT is explained in part by genetic variation in SULT1A1 and may represent a step toward individualizing HT treatment decisions.

Abstract Background: Tumor-infiltrating lymphocytes (TIL) have prognostic and potentially predictive significance in the (neo)adjuvant treatment of high-risk breast cancer. However, quantitative TIL measurement is not routinely performed. It is unclear why some tumors attract large quantities of TIL while others do not. We sought to confirm the association between TIL and pathologic complete response rate (pCR) and to further use next generation sequencing (NGS) to identify genes and gene pathways associated with the presence/absence of TIL. Methods: We studied 140 women with high risk stage I-III breast cancer, enrolled in the Breast Cancer Genome Guided Therapy Study (BEAUTY), obtaining serial biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant chemotherapy (NAC) with taxane (+/- trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Diagnostic pre-NAC core needle biopsies and surgical resection specimens post-NAC were available from 110 patients. Stromal TIL were semi-quantitated on a scale of 1-4 (with 1: ≤10/hpf, 2: subtle infiltrate &gt;10/hpf, 3: moderate infiltrate readily visible at low power magnification, 4: dense infiltrate with innumerable lymphocytes). For this analysis, low TIL was defined as scores of 1-2 vs. high defined as 3-4. Using pre-NAC biopsies, RNAseq was performed using the Illumina HiSeq2000 and the Mayo Analysis Pipeline for RNAseq (MAP-Rseq) for quality control, sequence alignment, and gene counts. The quantity of TIL was associated with transcripts across the transcriptome after conditional quantile normalization. Differentially expressed genes were obtained using EdgeR analysis, using a false discovery rate of 0.05, and pathways were evaluated using GAGE methods. Results: The pCR and residual cancer burden (RCB)-0/I rates by stromal TIL status within each molecular subtype are presented in the table. A diverse spectrum of 1344 genes with differential expression between tumors with high vs. low stromal TIL was identified. The genes with &gt;2.0-fold change (FC) and p&lt;1e-09 included S100A7 (4.49 FC), LCN2 (2.48 FC), and ART3 (2.82 FC) (genes known to be involved in immune regulation), as well as TDRD1 (2.71 FC) (a gene related to ERG [ETS-related gene] expression). In addition, the "regulation of actin cytoskeleton" pathway was upregulated in tumors with high TIL, while the "Hedgehog signaling" and "Wnt signaling" pathways were downregulated. Molecular SubtypeStromal TILspCR rate n (%)RCB-0/I rateLuminal AHigh------Luminal ALow0/9 (0%)0/9 (0%)Luminal BHigh1/9 (11.1%)1/8 (12.5%)Luminal BLow3/24 (12.5%)6/23 (26.1%)ER+/HER2+High3/9 (33.3%)4/9 (44.4%)ER+/HER2+Low1/6 (16.7%)1/6 (16.7%)ER-/HER2+High8/9 (88.9%)7/7 (100%)ER-/HER2+Low4/8 (50.0%)6/8 (75.0%)Triple NegativeHigh10/19 (52.6%)13/19 (68.4%)Triple NegativeLow7/14 (50.0%)9/13 (69.2%) Conclusions: We identified genes and gene pathways associated with high TIL expression in breast tumors prior to NAC that provide insight into the interactions between TIL and tumors. TIL can be easily semi-quantitated on H&E and along with these novel biomarkers, may contribute to the personalization of breast cancer therapy. Citation Format: Moyer AM, Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno-Aspitia A, Northfelt DW, Gray RJ, Sinnwell JP, Carlson EE, Dockter TJ, Jones KN, Felten SJ, Conners AL, Wieben ED, Ingle JN, Wang L, Weinshilboum RM, Visscher DW, Goetz MP. Differential mRNA expression patterns in breast tumors with high vs. low quantity of stromal tumor–Infiltrating lymphocytes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-05.

Abstract Background: Tumorigenesis involves both genetic and epigenetic changes. Epigenetic alterations are reversible and are promising cancer therapeutic targets. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferase inhibitor, is FDA approved for hematological malignancies. However, the effect of decitabine in breast cancer is not completely understood. Previous reports indicated that one decitabine mechanism involves regulation of protein levels for DNMT1, the major DNA methyltransferase that methylates hemimethylated CpG di-nucleotides in DNA. However, the E3 ligase involved in this process has not been identified. Whether decitabine also regulates DNMT3A and 3B in a similar fashion remains unclear. Therefore, our goals were to 1) understand mechanisms underlying decitabine action, 2) test the antitumor activity of decitabine in breast cancer models and 3) identify biomarkers associated with response to decitabine. Methods and Results: Western blots of breast cancer cell lines showed that DNMT1, DNMT3A, and DNMT3B protein levels decreased following decitabine treatment without a reduction in mRNA levels. Bioinformatic analysis of DNA methyltransferase sequences revealed a potential TRAF6 binding motif, and the interaction with TRAF6 (TNF receptor-associated factor 6) was confirmed by IP. TRAF6 functions as an E3 ligase. To determine whether TRAF6 might be the E3 ligase responsible for the degradation of DNMTs after decitabine treatment, we knocked down TRAF6 by RNA interference or knocked out the TRAF6 gene by CRISPR/Cas9. Down regulation of TRAF6 attenuated DNMT ubiquitination and increased DNMT protein levels, suggesting that TRAF6 might mediate proteasome-dependent degradation of all three DNMTs. This was further confirmed by reconstituting the knockout cells with WT and a TRAF6-C70A mutant, followed by assessing DNMT protein levels. Global DNA methylation was also increased after TRAF6 depletion and was confirmed in TRAF6 knock out cells in which DNMT levels were unaffected by decitabine. Cell cytotoxicity and colony forming assays showed that TRAF6 knockout cells were resistant to decitabine, suggesting that a major decitabine mechanism of action is through the regulation of TRAF6 which, in turn, degrades DNMTs, leading to decreased global methylation. Finally, decitabine significantly induced TRAF6 at both mRNA and protein levels, a process that might create positive feedback leading to increased degradation of DNMT proteins upon decitabine treatment. Based on these results, we further hypothesized that levels of the three DNMTs might influence decitabine response. Using 18 breast cancer patient derived xenograft (PDX) models, we found a wide range of DNMT protein levels regardless of ER/HER2 status. DNMT levels in the PDX models were directly associated with sensitivity to decitabine treatment, confirming our hypothesis. Conclusion: Our data showed that decitabine might be an effective agent for treating breast cancer and revealed a novel mechanism underlying decitabine treatment. Baseline DNMT protein levels may serve as a biomarker for predicting decitabine drug response. Citation Format: Yu J, Qin B, Boughey JC, Moyer AM, Visscher DW, Sinnwell JP, Yin P, Thompson KJ, Docter TJ, Kalari KR, Suman VJ, Wieben ED, Felten SJ, Conners AL, Jones KN, McLaughlin SA, Copland JA III, Moreno Aspitia A, Northfelt DW, Gray RJ, Ingle JN, Lou Z, Weinshilboum R, Goetz MP, Wang L. Regulation of DNA methyltransferases via TRAF6 determines breast cancer response to decitabine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-51.