Harvey A. Greisman

Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.

The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

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.

IgH partner breakpoint sequences provide evidence that AID initiates t(11;14) and t(8;14) chromosomal breaks in mantle cell and Burkitt lymphomas

Previous studies have implicated activation-induced cytidine deaminase (AID) in B-cell translocations but have failed to identify any association between their chromosomal breakpoints and known AID target sequences. Analysis of 56 unclustered IgH-CCND1 translocations in mantle cell lymphoma across the ~ 344-kb bcl-1 breakpoint locus demonstrates that half of the CCND1 breaks are near CpG dinucleotides. Most of these CpG breaks are at CGC motifs, and half of the remaining breaks are near WGCW, both known AID targets. These findings provide the strongest evidence to date that AID initiates chromosomal breaks in translocations that occur in human bone marrow B-cell progenitors. We also identify WGCW breaks at the MYC locus in Burkitt lymphoma translocations and murine IgH-MYC translocations, both of which arise in mature germinal center B cells. Finally, we propose a developmental model to explain the transition from CpG breaks in early human B-cell progenitors to WGCW breaks in later stage B cells.

Rapid High-Resolution Mapping of Balanced Chromosomal Rearrangements on Tiling CGH Arrays

The diagnosis and classification of many cancers depends in part on the identification of large-scale genomic aberrations such as chromosomal deletions, duplications, and balanced translocations. Array-based comparative genomic hybridization (array CGH) can detect chromosomal imbalances on a genome-wide scale but cannot reliably identify balanced chromosomal rearrangements. We describe a simple modification of array CGH that enables simultaneous identification of recurrent balanced rearrangements and genomic imbalances on the same microarray. Using custom tiling oligonucleotide arrays and gene-specific linear amplification primers, translocation CGH (tCGH) maps balanced rearrangements to ∼100-base resolution and facilitates the rapid cloning and sequencing of novel rearrangement breakpoints. As proof of principle, we used tCGH to characterize nine of the most common gene fusions in mature B-cell neoplasms and myeloid leukemias. Because tCGH can be performed in any CGH-capable laboratory and can screen for multiple recurrent translocations and genome-wide imbalances, it should be of broad utility in the diagnosis and classification of various types of lymphomas, leukemias, and solid tumors.

BCL6 breaks occur at different AID sequence motifs in Ig–BCL6 and non-Ig–BCL6 rearrangements

BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig-BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig-BCL6 and non-Ig-BCL6 rearrangements.

Convergent BCL6 and lncRNA promoters demarcate the major breakpoint region for BCL6 translocations

To the editor: We recently published a study showing that BCL6 translocations occur at distinct sequence motifs within intron 1 of the BCL6 gene that are recognized and deaminated by activation-induced cytidine deaminase (AID).[1][1] It has been unclear, however, why these breakpoints cluster in an

Flow cytometric evaluation of skin biopsies for mycosis fungoides

Multicolor flow cytometry (FC) is indispensable for lymphoma diagnosis and classification, but its utility in evaluating skin biopsies for mycosis fungoides (MF) is not well established. We describe the largest series to date of skin biopsies evaluated by FC for MF (n = 33), and we compare the flow cytometric results with the histologic, molecular, and clinical findings. Abnormal T-cell populations were identified by FC in 14 of 18 patients (78%) having histologically confirmed MF and in no patient whose histology was negative or indeterminate for MF (n = 14). One patient had histologic, flow cytometric, and molecular findings compatible with MF, but this patients clinical course was more suggestive of a drug eruption. Fourteen of 15 abnormal T-cell populations showed definitive aberrant expression of at least 2 surface antigens, including CD2 (47%), CD3 (67%), CD4, CD5 (87%), CD7, and CD45 (67%); most cases (67%) had light scatter properties suggesting increased cell size and/or cytoplasmic complexity. The high specificity of FC suggests that it will be a useful adjunct to routine histology in the evaluation of diagnostic skin biopsies for MF.

Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization

Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --(SEA) and --(FIL). In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods.

A Multiplex Approach to the Molecular Diagnosis of β-Thalassemia

This Commentary discusses a new method for detecting B-thalassemia mutation as described by Xiong et al (J Mol Diagn 13:427-435).