Race, Genotype, and Azathioprine Discontinuation : A Cohort Study.

BACKGROUND: Thiopurines are an important class of immunosuppressants despite their risk for hematopoietic toxicity and narrow therapeutic indices. Benign neutropenia related to an ACKR1 variant (rs2814778-CC) is common among persons of African ancestries. OBJECTIVE: To test whether rs2814778-CC was associated with azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine dosing. DESIGN: Retrospective cohort study. SETTING: Two tertiary care centers. PATIENTS: Thiopurine users with White or Black race. MEASUREMENTS: Azathioprine discontinuation attributed to hematopoietic toxicity. Secondary outcomes included weight-adjusted final dose, leukocyte count, and change in leukocyte count. RESULTS: The rate of azathioprine discontinuation attributed to hematopoietic toxicity was 3.92 per 100 person-years among patients with the CC genotype (n = 101) and 1.34 per 100 person-years among those with the TT or TC genotype (n = 1365) (hazard ratio [HR] from competing-risk model, 2.92 [95% CI, 1.57 to 5.41]). The risk remained significant after adjustment for race (HR, 2.61 [CI, 1.01 to 6.71]). The risk associated with race alone (HR, 2.13 [CI, 1.21 to 3.75]) was abrogated by adjustment for genotype (HR, 1.13 [CI, 0.48 to 2.69]). Lower last leukocyte count and lower dosing were significant among patients with the CC genotype. Lower dosing was validated in an external cohort of 94 children of African ancestries prescribed the thiopurine 6-mercaptopurine (6-MP) for acute lymphoblastic leukemia. The CC genotype was independently associated with lower 6-MP dose intensity relative to the target daily dose of 75 mg/m2 (median, 0.83 [IQR, 0.70 to 0.94] for the CC genotype vs. 0.94 [IQR, 0.72 to 1.13] for the TT or TC genotype; P = 0.013). LIMITATIONS: Unmeasured confounding; data limited to tertiary centers. CONCLUSION: Patients with the CC genotype had higher risk for azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine doses. Genotype was associated with those risks, even after adjustment for race. PRIMARY FUNDING SOURCE: National Institutes of Health.

HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-like Receptor KIR3DS1.

NK cells elicit important responses against transformed and virally infected cells. Carriage of the gene encoding the activating killer Ig-like receptor KIR3DS1 is associated with slower time to AIDS and protection from HIV infection. Recently, open conformers of the nonclassical MHC class Ib Ag HLA-F were identified as KIR3DS1 ligands. In this study, we investigated whether the interaction of KIR3DS1 on primary NK cells with HLA-F on the HLA-null cell line 721.221 (221) stimulated KIR3DS1+ NK cells. We used a panel of Abs to detect KIR3DS1+CD56dim NK cells that coexpressed the inhibitory NK cell receptors KIR2DL1/L2/L3, 3DL2, NKG2A, and ILT2; the activating NK cell receptors KIR2DS1/S2/S3/S5; and CCL4, IFN-γ, and CD107a functions. We showed that both untreated and acid-pulsed 221 cells induced a similar frequency of KIR3DS1+ cells to secrete CCL4/IFN-γ and express CD107a with a similar intensity. A higher percentage of KIR3DS1+ than KIR3DS1- NK cells responded to 221 cells when either inclusive or exclusive (i.e., coexpressing none of the other inhibitory NK cell receptors and activating NK cell receptors detected by the Ab panel) gating strategies were employed to identify these NK cell populations. Blocking the interaction of HLA-F on 221 cells with KIR3DS1-Fc chimeric protein or anti-HLA-F Abs on exclusively gated KIR3DS1+ cells reduced the frequency of functional cells compared with that of unblocked conditions for stimulated KIR3DS1+ NK cells. Thus, ligation of KIR3DS1 activates primary NK cells for several antiviral functions.

HIV exposed seronegative (HESN) compared to HIV infected individuals have higher frequencies of telomeric Killer Immunoglobulin-like Receptor (KIR) B motifs; Contribution of KIR B motif encoded genes to NK cell responsiveness.

Previously, we showed that Killer Immunoglobulin-like Receptor (KIR)3DS1 homozygotes (hmz) are more frequent in HIV exposed seronegative (HESN) than in recently HIV infected (HIV+) individuals. KIR3DS1 encodes an activating Natural Killer (NK) cell receptor (NKR). The link between KIR genotype and HIV outcomes likely arises from the function that NK cells acquire through expression of particular NKRs. An initial screen of 97 HESN and 123 HIV+ subjects for the frequency of KIR region gene carriage observed between-group differences for several telomeric KIR region loci. In a larger set of up to 106 HESN and 439 HIV+ individuals, more HESN than HIV+ subjects were KIR3DS1 homozygotes, lacked a full length KIR2DS4 gene and carried the telomeric group B KIR haplotype motif, TB01. TB01 is characterized by the presence of KIR3DS1, KIR2DL5A, KIR2DS3/5 and KIR2DS1, in linkage disequilibrium with each other. We assessed which of the TB01 encoded KIR gene products contributed to NK cell responsiveness by stimulating NK cells from 8 HIV seronegative KIR3DS1 and TB01 motif homozygotes with 721.221 HLA null cells and evaluating the frequency of KIR3DS1+/-KIR2DL5+/-, KIR3DS1+/-KIR2DS1+/-, KIR3DS1+/-KIR2DS5+/- NK cells secreting IFN-γ and/or expressing CD107a. A higher frequency of NK cells expressing, versus not, KIR3DS1 responded to 721.221 stimulation. KIR2DL5A+, KIR2DS1+ and KIR2DS5+ NK cells did not contribute to 721.221 responses or modulate those by KIR3DS1+ NK cells. Thus, of the TB01 KIR gene products, only KIR3DS1 conferred responsiveness to HLA-null stimulation, demonstrating its ligation can activate ex vivo NK cells.

Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

Relationships between epidermal growth factor receptor expression and human papillomavirus status as markers of prognosis in oropharyngeal cancer.

PURPOSE: This study examines the prognostic significance of epidermal growth factor receptor (EGFR) expression in relation to human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (SCC). MATERIALS AND METHODS: Pathological diagnosis of 270 oropharyngeal SCCs was verified by the study pathologist; clinical details were extracted from institutional databases. Recurrence in any form or death from any cause was recorded for a median of 2.5 (range: 0-19.3) years after diagnosis. HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry; EGFR expression was evaluated by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox regression with censoring at dates of last follow-up. RESULTS: Thirty-seven percent of cancers were HPV-positive (91% type 16). HPV was a predictor of loco-regional recurrence, event-free and overall survival after adjustment for clinicopathological variables and EGFR. Patients with EGFR-positive cancers were 5-fold more likely to have loco-regional failure relative to those with EGFR-negative cancers. Patients with HPV-negative/EGFR-positive cancers had an adjusted 13-fold increased risk of having a loco-regional failure, an almost 4-fold increased risk of having an event and more than a 4-fold increased risk of dying of any cause relative to those with HPV-positive/EGFR-negative cancers. There was weak evidence that the effects of EGFR on outcome were limited to patients with HPV-negative cancers. CONCLUSIONS: HPV and EGFR are independent prognostic markers in oropharyngeal SCC. Combining testing for HPV and EGFR appears to provide additional prognostic information.

Mohs micrographic surgery of a papillary eccrine adenoma.

BACKGROUND: Papillary eccrine adenoma (PEA) is a rare benign sweat gland neoplasm first described by Rulon and Helwig in 1977. Although these lesions typically behave in a benign fashion, PEA's on the volar surfaces may demonstrate more aggressive biologic behavior. Additionally, aggressive digital papillary adenomas (ADPA) may histologically simulate PEAs and behave in a more malignant fashion. OBJECTIVE: To present a case report of a patient with an incompletely excised PEA that was successfully extirpated using Mohs micrographic surgery (MMS). METHODS: A 51-year-old black woman was evaluated for the treatment of an incompletely excised PEA located on the dorsum of her left hand at the base of the thumb. Mohs micrographic surgery was felt to be the ideal treatment choice because of incomplete prior resections, ill-defined clinical borders, the need for conservative surgical excision to preserve sensory and motor function of the left hand, and the previously reported more aggressive nature of this tumor when located on volar surfaces. The patient underwent a two-stage, six section micrographically controlled excision using the fresh tissue technique. RESULTS: Complete resection of the PEA without significant damage to neurovascular structures. CONCLUSION: This case demonstrates the increasingly important role MMS is playing in the surgical management of a wide variety of cutaneous tumors. To our knowledge, this is the first time MMS has been used in the resection of a PEA.

Diffuse cutaneous mucinoses.

The cutaneous mucinoses are a complex group of dermatologic diseases with local, follicular, or diffuse disease. The diffuse cutaneous mucinoses are remarkable not only for their dermal disease, but also for the numerous systemic manifestations. It is important that the clinical dermatologist be able accurately to diagnose and differentiate scleredema, scleromyxedema, REM, generalized myxedema of hypothyroidism, and pretibial myxedema of hyperthyroidism. Because of the variability of associated systemic manifestations, some with substantial morbidity and mortality, accurate diagnosis is vital for awareness and appropriate management.