Deep sequencing of the T-cell receptor repertoire in CD8+ T-large granular lymphocyte leukemia identifies signature landscapes.

New massively parallel sequencing technology enables, through deep sequencing of rearranged T-cell receptor (TCR) Vß complementarity-determining region 3 (CDR3) regions, a previously inaccessible level of TCR repertoire analysis. The CDR3 repertoire diversity reflects clonal composition, the potential antigenic recognition spectrum, and the quantity of available T-cell responses. In this context, T-large granular lymphocyte (T-LGL) leukemia is a chronic clonal lymphoproliferation of cytotoxic T cells often associated with autoimmune diseases and various cytopenias. Using CD8(+) T-LGL leukemia as a model disease, we set out to evaluate and compare the TCR deep-sequencing spectra of both patients and healthy controls to better understand how TCR deep sequencing could be used in the diagnosis and monitoring of not only T-LGL leukemia but also reactive processes such as autoimmune disease and infection. Our data demonstrate, with high resolution, significantly decreased diversity of the T-cell repertoire in CD8(+) T-LGL leukemia and suggest that many T-LGL clonotypes may be private to the disease and may not be present in the general public, even at the basal level.

STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Whole-exome sequencing enhances prognostic classification of myeloid malignancies.

PURPOSE: To date the standard nosology and prognostic schemes for myeloid neoplasms have been based on morphologic and cytogenetic criteria. We sought to test the hypothesis that a comprehensive, unbiased analysis of somatic mutations may allow for an improved classification of these diseases to predict outcome (overall survival). EXPERIMENTAL DESIGN: We performed whole-exome sequencing (WES) of 274 myeloid neoplasms, including myelodysplastic syndrome (MDS, N=75), myelodysplastic/myeloproliferative neoplasia (MDS/MPN, N=33), and acute myeloid leukemia (AML, N=22), augmenting the resulting mutational data with public WES results from AML (N=144). We fit random survival forests (RSFs) to the patient survival and clinical/cytogenetic data, with and without gene mutation information, to build prognostic classifiers. A targeted sequencing assay was used to sequence predictor genes in an independent cohort of 507 patients, whose accompanying data were used to evaluate performance of the risk classifiers. RESULTS: We show that gene mutations modify the impact of standard clinical variables on patient outcome, and therefore their incorporation hones the accuracy of prediction. The mutation-based classification scheme robustly predicted patient outcome in the validation set (log rank P=6.77 × 10(-21); poor prognosis vs. good prognosis categories HR 10.4, 95% CI 3.21-33.6). The RSF-based approach also compares favorably with recently-published efforts to incorporate mutational information for MDS prognosis. CONCLUSION: The results presented here support the inclusion of mutational information in prognostic classification of myeloid malignancies. Our classification scheme is implemented in a publicly available web-based tool (http://myeloid-risk. CASE: edu/).

Laboratory measurement of the anticoagulant activity of edoxaban: a systematic review.

Edoxaban, an oral direct inhibitor of factor Xa, was recently approved in the United States and Japan for prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation and for treatment of venous thromboembolism (VTE). It is also licensed in Japan for prevention of VTE after major orthopedic surgery. Although routine laboratory monitoring of edoxaban is not required, laboratory measurement may be desirable in special circumstances. Our objective was to provide a systematic review of current evidence on laboratory measurement of the anticoagulant activity of edoxaban. PubMed and the Cochrane Library were searched for studies that reported a relationship between coagulation tests and plasma edoxaban levels. Study quality was assessed using Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). We identified 9 eligible studies. Anti-Xa activity is linear across a broad range of drug levels (R (2) > 0.95) and may be used for edoxaban quantification. The assay shows greater variability at above on-therapy drug concentrations. The PT is less sensitive to edoxaban. A normal prothrombin time may not exclude clinically relevant on-therapy drug levels. The activated partial thromboplastin time has insufficient sensitivity to edoxaban for measurement of its anticoagulant activity. Edoxaban exhibits variable effects on coagulation assays. Understanding these effects facilitates interpretation of test results in edoxaban-treated patients. More data on the relationship between drug levels, coagulation test results, and clinical outcomes in patients are needed.

mTOR inhibitors and their clinical application in cervical, endometrial and ovarian cancers: a critical review.

OBJECTIVES: The mechanistic (mammalian) targets of rapamycin (mTOR) inhibitors with known growth Inhibitory effect are currently in clinical trial for treatment of human cancer. The aim of this review is to present current incorporating these new drugs as single agents or in combination with other therapeutic modalities for treatment of gynecologic cancer. METHODS: A PubMed search was conducted on "mTOR inhibitors" and "human cancer". The relevant studies published between the year 2000 to present were reviewed. Those related to gynecologic cancer (cervical, endometrial and ovarian) were selected for this manuscript. The result of published data and their clinical application in gynecologic malignancies are presented. RESULTS: mTOR is directly involved in many cell signaling pathways, and mTOR inhibitors have demonstrated anti-tumor activity against a variety of human malignancies, including gynecologic cancers. Combinations of mTOR inhibitors with other treatment modalities, e.g. cytotoxic chemotherapy, hormonal therapies, and other targeted molecular agents, have shown encouraging results particularly in endometrial and ovarian cancer. CONCLUSIONS: Patients with advanced or recurrent gynecologic cancers who have failed initial treatment are need of new treatment modalities. There is strong evidence that mTOR inhibitors limit tumor proliferation and progression. The PI3k/AKT/mTOR pathway is often deregulated in gynecologic cancer. Patients with PIK3CA mutations are more responsive to PI3K/AKT/mTOR inhibitors than patients without these mutations. Routine screening for PIK3CA mutations warrants further investigation when PI3K/AKT/mTOR inhibitors are considered in treatment of patients with gynecologic cancer.

Bleeding and Thrombotic Adverse Events in Hospitalized Patients Under Empiric Treatment for Suspected Heparin-Induced Thrombocytopenia While Awaiting Confirmatory Testing.

Empiric management in suspected heparin-induced thrombocytopenia (HIT) is challenging due to imperfect prediction models, latency while awaiting test results and risks of empiric therapies. When there is high clinical suspicion for HIT, cessation of heparin and empiric non-heparin anticoagulation with FDA-approved argatroban is recommended. Alternatively off-label fondaparinux or watchful waiting have been utilized in clinical practice. Outcomes of patients empirically managed for HIT have not been compared directly in clinical trials and patients that ultimately do not have HIT are often overlooked. Clinicians need studies investigating empiric management to guide decision making in suspected HIT. In this study, adverse events (AE) were categorized and compared in patients being evaluated for HIT while undergoing empiric management by non-heparin anticoagulation with argatroban or fondaparinux, both at therapeutic or reduced doses, or watchful waiting with or without heparin. AE were defined as new thrombosis confirmed on imaging or new bleeding event after HIT was first suspected. A retrospective chart review of 312 patients tested for HIT at an academic hospital was conducted. 170 patients met inclusion criteria. Patients were excluded if the 4Ts score was < 4. The 4Ts score is a pretest probability for HIT based on thrombocytopenia degree, timing, alternative causes and presence of thrombosis. Included patients were divided according to management groups and compared with logistic regression analysis. Bleeding risk significantly differed between management groups (p = 0.002). Despite adjustment for bleeding risk, fondaparinux was associated with increased AE, (p = 0.03, OR = 5.81), while argatroban was not. There was no difference in AE based on time to initiation of empiric treatment and no advantage to reduced dosing with either anticoagulant. These findings challenge assumptions surrounding empiric HIT management.

Occult cancer detection in patients with hemostatic disorder and venous thromboembolism.

There are physiologic ties between Von Willebrand Factor (VWF) and circulating tumor cells. VWF appears to play a role in tumor biology, but it is unclear whether cancer behavior differs in Von Willebrand Disease. In patients presenting with venous thromboembolism (VTE), occult cancer is frequently considered as an underlying cause. The prevalence of occult cancer after provoked VTE is low (3%); therefore, cancer screening in these patients is not routinely recommended. In those with unprovoked VTE, occult cancer is more prevalent, estimated between 4 and 10%. Due to this elevated risk, occult cancer screening is recommended in this population. Multiple studies have investigated whether a "limited" approach (including history and physical exam, basic labs, and chest X-ray) versus "extensive" approach (addition of advanced imaging, such as computer tomography) is more effective. Current data fails to demonstrate extensive screening strategies diagnose more occult cancer, miss fewer cancers during follow up, or improve cancer-related mortality. Furthermore, many patients may be needlessly exposed to unnecessary diagnostic procedures with their associated complications and costs, as well as significant anxiety. Therefore, the decision to perform additional testing should be made on a case-by-case basis. Additional studies are needed to identify subgroups of patients with unprovoked VTE at highest risk for occult cancer.

Dynamics of clonal evolution in myelodysplastic syndromes.

To elucidate differential roles of mutations in myelodysplastic syndromes (MDS), we investigated clonal dynamics using whole-exome and/or targeted sequencing of 699 patients, of whom 122 were analyzed longitudinally. Including the results from previous reports, we assessed a total of 2,250 patients for mutational enrichment patterns. During progression, the number of mutations, their diversity and clone sizes increased, with alterations frequently present in dominant clones with or without their sweeping previous clones. Enriched in secondary acute myeloid leukemia (sAML; in comparison to high-risk MDS), FLT3, PTPN11, WT1, IDH1, NPM1, IDH2 and NRAS mutations (type 1) tended to be newly acquired, and were associated with faster sAML progression and a shorter overall survival time. Significantly enriched in high-risk MDS (in comparison to low-risk MDS), TP53, GATA2, KRAS, RUNX1, STAG2, ASXL1, ZRSR2 and TET2 mutations (type 2) had a weaker impact on sAML progression and overall survival than type-1 mutations. The distinct roles of type-1 and type-2 mutations suggest their potential utility in disease monitoring.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance.

OBJECTIVES: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). METHODS: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. RESULTS: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. CONCLUSIONS: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests.

Evaluation of noncytotoxic DNMT1-depleting therapy in patients with myelodysplastic syndromes.

BACKGROUND: Mutational inactivation in cancer of key apoptotic pathway components, such as TP53/p53, undermines cytotoxic therapies that aim to increase apoptosis. Accordingly, TP53 mutations are reproducibly associated with poor treatment outcomes. Moreover, cytotoxic treatments destroy normal stem cells with intact p53 systems, a problem especially for myeloid neoplasms, as these cells reverse the low blood counts that cause morbidity and death. Preclinical studies suggest that noncytotoxic concentrations of the DNA methyltransferase 1 (DNMT1) inhibitor decitabine produce p53-independent cell-cycle exits by reversing aberrant epigenetic repression of proliferation-terminating (MYC-antagonizing) differentiation genes in cancer cells. METHODS: In this clinical trial, patients with myelodysplastic syndrome (n=25) received reduced decitabine dosages (0.1-0.2 mg/kg/day compared with the FDA-approved 20-45 mg/m2/day dosage, a 75%-90% reduction) to avoid cytotoxicity. These well-tolerated doses were frequently administered 1-3 days per week, instead of pulse cycled for 3 to 5 days over a 4- to 6-week period, to increase the probability that cancer S-phase entries would coincide with drug exposure, which is required for S-phase-dependent DNMT1 depletion. RESULTS: The median subject age was 73 years (range, 46-85 years), 9 subjects had relapsed disease or were refractory to 5-azacytidine and/or lenalidomide, and 3 had received intensive chemoradiation to treat other cancers. Adverse events were related to neutropenia present at baseline: neutropenic fever (13 of 25 subjects) and septic death (1 of 25 subjects). Blood count improvements meeting the International Working Group criteria for response occurred in 11 of 25 (44%) subjects and were highly durable. Treatment-induced freedom from transfusion lasted a median of 1,025 days (range, 186-1,152 days; 3 ongoing), and 20% of subjects were treated for more than 3 years. Mutations and/or deletions of key apoptosis genes were frequent (present in 55% of responders and in 36% of nonresponders). Noncytotoxic DNMT1 depletion was confirmed by serial BM γ-H2AX (DNA repair/damage marker) and DNMT1 analyses. MYC master oncoprotein levels were markedly decreased. CONCLUSION: Decitabine regimens can be redesigned to minimize cytotoxicity and increase exposure time for DNMT1 depletion, to safely and effectively circumvent mutational apoptotic defects. TRIAL REGISTRATION: Clinicaltrials.gov NCT01165996. FUNDING: NIH (R01CA138858, CA043703); Department of Defense (PR081404); Clinical and Translational Science Award (CTSA) (UL1RR024989); and the Leukemia and Lymphoma Society (Translational Research Program).

Preservation of Fertility in Female Cancer Patients Desiring Future Child Bearing; What is Available and What can be Offered.

The aim of this review is to present an overview of available methods for preservation of ovarian function and fertility in female cancer patients who desire to maintain their child-bearing capacity for future pregnancies. A Medline search was conducted. Published articles from American and European studies from 1976 to present were reviewed. The effect of cancer treatment on the ovary, as well as different methods of fertility preservation and their reproductive outcomes are presented. Pregnancy rates vary according to the type of primary malignancy, stage of disease, method of fertility preservation (for example, hormonal therapy, cryopreservation, fertility-sparing surgery), and other confounding factors such as the patient's age, reproductive capacity, status of partnership, and genetic disposition. The highest rates of successful pregnancy were observed with embryo cryopreservation. Today, higher cure rates and longer survival are a result of earlier cancer diagnosis and treatment. In conjunction with the advances in assisted reproduction, the preservation of ovarian function and fertility is a major part of multidisciplinary care that should be offered to any young female patient with cancer. Fertility preservation in young cancer patients raises a number of ethical issues particularly regarding standard versus experimental therapies, and long-term financial cost.

Therapeutic rationale for mTOR inhibition in advanced renal cell carcinoma.

Inhibitors of the mammalian target of rapamycin (mTOR) have entered the landscape of treatment for advanced RCC. Their development has been based on their unique biology and their potential to simultaneously inhibit both tumor cell proliferation and angiogenesis. Despite the solid biologic rationale for their development, existing clinical data is somewhat mixed. Although Temsirolimus is capable of improving overall survival it does so only in a minority of selected mRCC patients and its effects on tumor burden reduction and PFS are minimal. Similarly the activity and clinical utility of Everolimus in the refractory setting is questionable. First, because it is unknown if mTOR becomes the major driver or cancer growth after developing progressive disease on a VEGF inhibitor and secondly because existing sequential VEGF data in same setting appears to be the same if not a bit more robust to that reported with Everolimus. Combination of mTOR and VEGF inhibitors has been disappointing due to the excessive toxicities encountered in early trials without a noticeable difference in efficacy. Efforts are now placed in a series of novel compounds capable of inhibiting mTOR and the upstream signaling pathway of PI3K/AKT.

Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.

The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.