Daniel E. Sabath

Identification of a human heme exporter that is essential for erythropoiesis

FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

High-Throughput Sequencing Detects Minimal Residual Disease in Acute T Lymphoblastic Leukemia

High-throughput sequencing can detect minimal residual disease comparable to multiparametric flow cytometry in T-ALL patients. Finding a Needle in a Haystack Even in seemingly successful cancer therapy, a small number of cells can survive treatment and persist in patients in remission. This minimal residual disease (MRD) is a major cause of cancer relapse, and until recently was undetectable. New ways to track MRD can determine whether cancer has been eradicated, compare the efficacy of different treatments, monitor patient remission status, and aid in treatment selection. Wu et al. use high-throughput sequencing (HTS) of lymphoid receptor genes to track MRD in T-lineage acute lymphoblastic leukemia/lymphoma (T-ALL). The authors sequence the variable regions of two T cell antigen receptor genes (TCRB and TCRG) using multiplexed polymerase chain reaction. First, they identified clonal T cell receptor (TCR) sequences in individual T-ALL patients and then looked in the same patients after treatment. Their strategy identified clonality at diagnosis in most cases and also detected subsequent MRD. In a subset of cases, HTS detected MRD in patients where it was not detected by flow cytometry, which is currently used in the clinic. Thus, HTS may lower the threshold of detection for MRD and affect treatment decisions. High-throughput sequencing (HTS) of lymphoid receptor genes is an emerging technology that can comprehensively assess the diversity of the immune system. Here, we applied HTS to the diagnosis of T-lineage acute lymphoblastic leukemia/lymphoma. Using 43 paired patient samples, we then assessed minimal residual disease (MRD) at day 29 after treatment. The variable regions of TCRB and TCRG were sequenced using an Illumina HiSeq platform after performance of multiplexed polymerase chain reaction, which targeted all potential V-J rearrangement combinations. Pretreatment samples were used to define clonal T cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, and paired posttreatment samples were evaluated for MRD. Abnormal T lymphoblast identification by multiparametric flow cytometry was concurrently performed for comparison. We found that TCRB and TCRG HTS not only identified clonality at diagnosis in most cases (31 of 43 for TCRB and 27 of 43 for TCRG) but also detected subsequent MRD. As expected, HTS of TCRB and TCRG identified MRD that was not detected by flow cytometry in a subset of cases (25 of 35 HTS compared with 13 of 35, respectively), which highlights the potential of this technology to define lower detection thresholds for MRD that could affect clinical treatment decisions. Thus, next-generation sequencing of lymphoid receptor gene repertoire may improve clinical diagnosis and subsequent MRD monitoring of lymphoproliferative disorders.

Interferon-α treatment of posttransplant lymphoproliferative disorder in recipients of solid organ transplants

Posttransplant lymphoproliferative disorder (PTLD) has been treated with decreased immunosuppression, antiviral medications, anti-B lymphocyte agents, radiation therapy, and/or chemotherapy. However, a standardized stepwise approach to treatment has not been previously evaluated. In the present study, 19 consecutive patients presenting to a single institution with newly diagnosed PTLD were treated according to a sequential protocol that consisted of (1) a reduction in immunosuppressive medications plus, if feasible, resection or definitive radiation therapy of localized disease, (2) interferon-alpha, and (3) systemic chemotherapy. Of the 3 patients presenting exclusively with localized disease, two were treated with resection of pulmonary parenchymal nodules and one was treated with radiation therapy to a paraspinous mass, without evidence of recurrence at a mean follow-up of 31 months (range, 8 to 46 months). Sixteen patients presented with PTLD not amenable to local therapy, and they were treated daily with 3x10(6) units/m2 subcutaneous interferon-alpha. Total regression of PTLD (defined as disappearance of the tumor mass by physical examination or computed tomography scanning) was found in 8 of 14 patients who received at least 3 weeks of interferon therapy. Interferon-alpha therapy was continued for 6 to 9 months in the eight patients judged to be responders. None of these patients have relapsed to date with the same neoplastic clone. Two patients, however, developed new neoplastic clones. Seven patients received systemic chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) (n=1), EPOCH (etoposide, vincristine, and doxorubicin administered as a continuous infusion, with an intravenous bolus of cyclophosphamide and oral prednisone) (n=4), or EPOCH followed by DHAP (dexamethasone, cytarabine, and cisplatin) (n=2) after failure of interferon-alpha; five patients had a complete response. Only 1 of the 19 patients died of uncontrolled PTLD. These results suggest that the majority of solid organ transplant recipients who develop PTLD can be safely and successfully treated using a sequential approach to therapy.

Transferred melanoma-specific CD8+ T cells persist, mediate tumor regression, and acquire central memory phenotype

Adoptively transferred tumor-specific T cells offer the potential for non–cross-resistant therapy and long-term immunoprotection. Strategies to enhance in vivo persistence of transferred T cells can lead to improved antitumor efficacy. However, the extrinsic (patient conditioning) and intrinsic (effector cell) factors contributing to long-term in vivo persistence are not well-defined. As a means to enhance persistence of infused T cells in vivo and limit toxicity, 11 patients with refractory, progressive metastatic melanoma received cyclophosphamide alone as conditioning before the infusion of peripheral blood mononuclear cell-derived, antigen-specific, CD8+ cytotoxic T-lymphocyte (CTL) clones followed by low-dose or high-dose IL-2. No life-threatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 wk, and 1 of 11 had a complete remission that continued for longer than 3 y. On-target autoimmune events with the early appearance of skin rashes were observed in patients with stable disease or complete remission at 4 wk or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to nonvascular compartments, such as skin and lymph nodes. CTL clones, on infusion, were characterized by an effector memory phenotype, and CTL that persisted long term acquired phenotypic and/or functional qualities of central memory type CTLs in vivo. The use of a T-cell product composed of a clonal population of antigen-specific CTLs afforded the opportunity to demonstrate phenotypic and/or functional conversion to a central memory type with the potential for sustained clinical benefit.

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

ABSTRACT New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratorys absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.

Sensitive and High-Throughput Isolation of Rare Cells from Peripheral Blood with Ensemble-Decision Aliquot Ranking†

This paper describes an approach called ensemble decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood. eDAR has a recovery of over 93% (n=9) with a zero false positive rate (n=8), and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis. We anticipate eDAR will enable new studies of various types of rare cells that circulate in blood.

V(D)J rearrangement in Nijmegen breakage syndrome.

Repair of DNA double-strand breaks is essential for maintenance of genomic stability, and is specifically required for rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) loci during development of the immune system. Abnormalities in these repair processes also contribute to oncogenic chromosomal rearrangements that underlie many lymphoid malignancies. Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition characterized by immunodeficiency, radiation sensitivity, and increased predisposition to lymphoid cancers bearing oncogenic Ig and TCR locus translocations. NBS patients fail to produce nibrin, a protein required for the nuclear localization and function of a DNA repair complex that includes Mre11 and Rad50. Mre11 has biochemical properties that suggest a potential role in V(D)J recombination. We studied V(D)J recombination in NBS cells in vitro and in vivo, using cell lines and peripheral blood leukocyte DNA from NBS patients. We found that NBS cells were competent to rejoin signal substrates with normal efficiency and high fidelity. Coding substrates were similarly rejoined efficiently, and coding end structures appeared normal. In B cells from NBS patients, the spectrums of IgH CDR3 regions were diverse and normally distributed. Moreover, the lengths and composition of Igkappa VJ joins and IgH VDJ joins derived from NBS and normal subjects were indistinguishable. Our data indicate that nibrin plays no essential role in V(D)J recombination and is not required for the generation of an apparently diverse B cell repertoire.

T-Cell Clonality Determination Using Polymerase Chain Reaction (PCR) Amplification of the T-Cell Receptor gamma-Chain Gene and Capillary Electrophoresis of Fluorescently Labeled PCR Products

We compared the effectiveness of polymerase chain reaction (PCR) and DNA blot analysis (DBA) for detecting clonal T-cell populations and investigated whether a nonradioactive PCR method could be used in routine clinical diagnosis. We analyzed DNA from 117 cases for T-cell clonality by PCR amplification. DBA was performed on 77 of these cases. Denaturing polyacrylamide gel electrophoresis (PCR-PAGE) of radiolabeled PCR products and capillary electrophoresis (PCR-CE) of fluorescently labeled PCR products were used for PCR product separation and quantitation. Complete agreement was obtained between PCR-PAGE and DBA in 67 of 77 cases. One case was positive by DBA and negative by PCR-PAGE, and 3 cases were positive by PAGE and negative by DBA. Five cases indeterminate by DBA were positive by PCR-PAGE, and 1 indeterminate case was negative by PCR-PAGE. In the comparison of PCR-PAGE and PCR-CE, of 63 cases with height ratios less than 2.0, all were negative by PCR-PAGE. Of 52 cases with height ratios of 2.0 or more, 50 were positive by PCR-PAGE. We conclude that PCR-CE is analytically equivalent to DBA and PCR-PAGE for detecting clonal T-cell populations. The PCR-CE method is semiquantitative and, therefore, may be more objective than gel-based methods.

Mutations and molecular variants of the NBS1 gene in non-Hodgkin lymphoma

Non‐Hodgkin lymphomas (NHLs) are characterized by chromosomal translocations that juxtapose loci encoding lymphoid antigen receptors with cellular proto‐oncogenes. These translocations are thought to arise from inaccurate processing of DNA breaks created during physiologic recombination of the antigen receptor genes in lymphocytes. The inherited disorders ataxia‐telangiectasia and Nijmegen breakage syndrome are caused by mutations in the ATM and NBS1 genes, respectively, and are characterized by generalized genomic instability and a high incidence of lymphoid cancers. Lymphoid cells from patients with either disorder frequently have chromosomal translocations involving T‐cell‐receptor or immunoglobulin loci. To investigate the potential role of the NBS1 gene in the pathogenesis of NHL, we screened tumor DNA samples from 91 sporadic cases of NHL and genomic DNA from 154 control individuals for mutations in all 16 exons of the NBS1 gene and in flanking intronic sequences. One NHL case with a truncating mutation in NBS1 and a second NHL case with a putative missense mutation were detected. Neither mutation was observed among controls. Three additional putative missense mutations were observed only in the normal control samples. A panel of six common polymorphisms spanning the NBS1 gene was genotyped and provided no evidence for loss of heterozygosity in the NHL cases with mutations or in the NHL population overall. These results suggest that mutations in NBS1 do not play a major role in the development of NHL in the United States.

An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.