Mark Klinger

Improved Survival with T Cell Clonotype Stability After Anti-CTLA-4 Treatment in Cancer Patients

CTLA-4 blockade induces T cell repertoire turnover, but high-frequency clonotypes are maintained in patients with favorable outcomes. Cancer’s Glass Jaw In the body’s bout against cancer, antibodies that block CTLA-4 (cytotoxic T lymphocyte–associated antigen-4) induce the immune system to enter the ring. But precisely how these antibodies affect the broad tumor-restricted T cell response remains unclear. Now, Cha et al. use next-generation sequencing to show that blocking CTLA-4 in cancer sufferers drives turnover of the T cell repertoire. However, increasing T cell diversity is not the whole story—patients with more favorable clinical outcomes maintained certain high-frequency T cells with treatment, whereas patients who lost T cells prevalent before therapy had poorer prognosis. These data suggest that although CTLA-4 blockade induces T cell repertoire diversification, maintenance of high-frequency—and presumably high-avidity—clones may be relevant for the antitumor immune response. Cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) blockade can promote antitumor T cell immunity and clinical responses. The mechanism by which anti–CTLA-4 antibodies induces antitumor responses is controversial. To determine the effects of CTLA-4 blockade on the T cell repertoire, we used next-generation deep sequencing to measure the frequency of individual rearranged T cell receptor β (TCRβ) genes, thereby characterizing the diversity of rearrangements, known as T cell clonotypes. CTLA-4 blockade in patients with metastatic castration-resistant prostate cancer and metastatic melanoma resulted in both expansion and loss of T cell clonotypes, consistent with a global turnover of the T cell repertoire. Overall, this treatment increased TCR diversity as reflected in the number of unique TCR clonotypes. The repertoire of clonotypes continued to evolve over subsequent months of treatment. Whereas the number of clonotypes that increased with treatment was not associated with clinical outcome, improved overall survival was associated with maintenance of high-frequency clones at baseline. In contrast, the highest-frequency clonotypes fell with treatment in patients with short overall survival. Stably maintained clonotypes included T cells having high-avidity TCR such as virus-reactive T cells. Together, these results suggest that CTLA-4 blockade induces T cell repertoire evolution and diversification. Moreover, improved clinical outcomes are associated with less clonotype loss, consistent with the maintenance of high-frequency TCR clonotypes during treatment. These clones may represent the presence of preexisting high-avidity T cells that may be relevant in the antitumor response.

Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia

The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

Activation of an oncogenic microRNA cistron by provirus integration

Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.

Mll5 contributes to hematopoietic stem cell fitness and homeostasis

MLL5 is a novel trithorax group gene and a candidate tumor suppressor gene located within a 2.5-Mb interval of chromosome band 7q22 that frequently is deleted in human myeloid malignancy. Here we show that inactivation of the Mll5 gene in mice results in a 30% reduction in the average representation of hematopoietic stem cells and in functional impairment of long-term hematopoietic repopulation potential under competitive conditions. Bone marrow cells from Mll5-deficient mice were defective in spleen colony-forming assays, and the mutant mice showed enhanced susceptibility to 5-fluorouracil-induced myelosuppression. Heterozygous and homozygous Mll5 mutant mice did not spontaneously develop hematologic cancers, and loss of Mll5 did not alter the phenotype of a fatal myeloproliferative disorder induced by oncogenic Kras in vivo. Collectively, the data reveal an important role for Mll5 in HSC homeostasis and provide a basis for further studies to explore its role in leukemogenesis.

Detection of circulating tumour DNA in patients with aggressive B-cell non-Hodgkin lymphoma

Current methods for detecting the presence of disease in patients with diffuse large B cell lymphoma (DLBCL) or mediastinal large B-cell lymphoma (MLBCL) rely primarily on imaging methods, which are associated with significant cost and radiation exposure. Very few patients with DLBCL have evidence of circulating disease using flow cytometric assays (Mancuso et al, 2010). This has so far precluded the development of minimal residual disease (MRD) assessment tools in those diseases, in contrast to tumours with a circulating component, where MRD assays are emerging as important methods (Ferrero et al, 2011). The availability of high-throughput sequencing techniques now provides an opportunity to probe for the presence of very small amounts of circulating tumour genetic material in peripheral blood (PB). If sequencing-based methods can reliably detect circulating disease, they could eventually find a role in the treatment and monitoring of patients with those tumours. We present here a pilot study of a sequencing method designed to examine whether tumour DNA is detectable in patients with newly diagnosed DLBCL/MLBCL, and whether it becomes undetectable after therapy. Tumour samples obtained from PB samples and formalin-fixed paraffin-embedded (FFPE) or frozen tissue were analysed using the Sequenta LymphoSIGHT method (Sequenta Inc., South San Francisco, CA, USA), as previously described (Faham et al, 2012a, 2012b). Briefly, genomic DNA was extracted from tumour cells, peripheral blood mononuclear cells (PBMCs) or plasma, amplified using locus-specific primer sets for IGH and IGK rearrangements, and sequenced. A frequency >5% in the diagnostic tumour sample was considered to represent one of the tumour clonotypes; the frequency of the tumour clonotype(s) in PB was calculated relative to the total number of reads in the sample. We approached consecutive patients with newly diagnosed DLBCL or MLBCL who presented for care at Dana-Farber Cancer Institute (DFCI). Independently, five patients with DLBCL who had banked tumour and blood samples at the M.D. Anderson Cancer Center (MDACC) were enrolled on a similar study. Written informed consent was obtained from all patients. All patients had a sample of tumour from their diagnostic biopsy sent to Sequenta Inc. for analysis. We collected a sample of whole blood before the first cycle of therapy; when possible, we also collected a sample after the conclusion of therapy (only in the DFCI cohort). Seventeen patients were enrolled on this study (Table I). None of 13 patients biopsied had bone marrow involvement. At least one dominant tumour clonotype could be established from the diagnostic biopsy for all 16 patients with sufficient amounts of amplifiable DNA. The diagnostic biopsy sample for one patient had <0.3 ng of DNA, and a dominant tumour clonotype was not identified. Among the 16 patients with an established tumour clonotype, circulating tumour DNA could be detected pre-treatment in plasma in 11 of the 16 (69%, 90% confidence interval [CI] 45–87%), in PBMCs in eight of the 16 (50%, 90%CI 28–72%), and in either in 13 of the 16 (81%, 90%CI 58–95%) (Table I, Fig 1A, B). The three patients in whom no DNA could be detected at diagnosis all had stage I disease and had no evidence of disease by positron emission tomography-computerized tomography (PET-CT) after their diagnostic procedure at the time that the study sample was drawn. Therefore, tumour DNA was detected in all 13 (100%) of patients with evidence of fluorodeoxyglucose-avid disease at the time of sample collection. There was no apparent correlation between the level of circulating tumour DNA and clinical characteristics including histology, stage, burden of disease, or International Prognostic Index (The International Non-Hodgkin’s Lymphoma Prognostic Factors Project, 1993). There was also no association between the presence or level of circulating DNA and lactate dehydrogenase elevation pre-treatment. All patients were treated with chemo-immunotherapy; one patient with MLBCL also received radiotherapy. Seven patients from the DFCI cohort returned for collection of post-therapy samples; the remaining received their care outside of DFCI and did not return for follow-up. At a median follow-up of 269 (range, 220–281) days from the beginning of therapy, none of the patients have relapsed. Among the seven evaluable patients, six (86%, 90%CI 48–99%) had no detectable circulating tumour DNA at the conclusion of chemoimmunotherapy (Table I, Fig 1C). Using a novel high-throughput sequencing technique, we could determine the dominant tumour clonotype in 94% of 17 patients with DLBCL/MLBCL from routine diagnostic tumour samples, and in 100% of those with adequate amount of tumour material for analysis. Among those patients, 81% had detectable circulating tumour DNA in plasma or PBMCs; the proportion was 100% among the patients with evidence of disease by PET-CT scan after their diagnostic surgical biopsy. Furthermore, the tumour DNA correspondence

Impact of the TCR Signal on Regulatory T Cell Homeostasis, Function, and Trafficking

Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naïve and memory CD4+ T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56Lck, we have examined the importance of TCR signaling in Treg cells. Inactivation of p56Lck resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56Lck in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR.

Thymic OX40 Expression Discriminates Cells Undergoing Strong Responses to Selection Ligands

OX40 is a member of the TNF receptor family expressed on activated and regulatory T (Treg) cells. Using an Ox40-cre allele for lineage marking, we found that a subpopulation of naive T cells had also previously expressed OX40 in the thymus. Ox40-cre was induced in a small fraction of thymocytes that were OX40+, some of which were CD25high Treg cell precursors. Thymic OX40 expression distinguished cells experiencing a strong signaling response to positive selection. Naive T cells that had previously expressed OX40 demonstrated a partially activated phenotype that was distinct from that of most naive T cells. The results are consistent with the selection of Treg cells and a minor subpopulation of naive T cells being dependent on strong signaling responses to thymic self ligands.

Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

RasGRP1 is a Ras-specific exchange factor, which is activated by T-cell receptor (TCR) and promotes TCR-dependent positive selection of thymocytes. RasGRP1 is highly expressed on most T lymphocytic leukemias and is a common site of proviral insertion in retrovirus-induced murine T-cell lymphomas. We used RasGRP1 transgenic mice to determine if deregulated expression of RasGRP1 has a causative role in the development of T-cell malignancies. Thymic lymphomas occurred in three different RasGRP1 transgenic mouse lines. Thymocyte transformation correlated with high transgene expression in early stage lymphomas, indicating that deregulated RasGRP1 expression contributed to the initiation of lymphomagenesis. Expression of the positively selectable H-Y TCR accelerated lymphomagenesis in RasGRP1 transgenic mice. However, the transformed thymocytes lacked markers of positive selection and lymphomas occurred when positive selection was precluded by negative selection of the H-Y TCR. Therefore, initiation of lymphomagenesis via RasGRP1 was not associated with TCR-dependent positive selection of thymocytes. Thymic lymphomas occurred in RasGRP1 transgenic/Rag2−/− mice, demonstrating that neither TCR nor pre-TCR were required for RasGRP1-driven lymphomagenesis. The RasGRP1 transgene conferred pre-TCR-independent survival and proliferation of immature thymocytes, suggesting that deregulated expression of RasGRP1 promotes lymphomagenesis by expanding the pool of thymocytes which are susceptible to transformation.

Deep Sequencing Reveals Myeloma Cells in Peripheral Blood in Majority of Multiple Myeloma Patients

INTRODUCTION The evaluation of myeloma cells in multiple myeloma (MM) patients has generally been limited to the assessment of bone marrow involvement because of the sensitivity limitations of traditional minimal-residual-disease-detection methods. MATERIALS AND METHODS We developed a sequencing-based method to identify myeloma cells in bone marrow (BM) and peripheral blood (PB) samples, based on their unique immunoglobulin gene rearrangements, that can detect cancer clones at levels well below 1 in 1 million leukocytes (0.0001%). In this multisite study, we used this sequencing method to determine the fraction of patients with myeloma cells in their PB at diagnosis and posttreatment time points. RESULTS Using this sequencing approach, we detected myeloma cells in the PB in the vast majority of MM patients (44/46, 96%). We demonstrated a clear correlation (R(2) = 0.57) between myeloma clone levels in paired BM and PB samples, and noted that PB clone levels were approximately 100-fold lower than levels in BM samples. The sequencing assay demonstrated a clear sensitivity advantage in the BM compartment and at least equivalent sensitivity in the PB compared with that of monoclonal-protein results. CONCLUSION This study highlights the promise of a blood-based, sequencing minimal-residual-disease assay that can be used to measure MM disease burden at different time points and various disease stages.

Reporter alleles that inform on differences in Cre recombinase expression.

Alleles that express reporters after Cre recombination allow for fate-mapping studies when used in combination with appropriate cre alleles. In this study, we describe two fluorescent reporter alleles that differentially mark populations of cells as a function of their level of expression of Cre recombinase. Mice carrying these alleles were generated and used to demonstrate the usefulness of the reporter alleles for informing on prior Cre recombinase expression in lymphocytes. The alleles expand the range of genetic tools available for understanding how differences in gene expression result in divergent developmental fates during the development and differentiation of lymphocytes and other cells.