Kimberly P. Dunsmore

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.

The genomic landscape of pediatric and young adult T-lineage acute lymphoblastic leukemia

Genetic alterations that activate NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors, are hallmarks of T-lineage acute lymphoblastic leukemia (T-ALL), but detailed genome-wide sequencing of large T-ALL cohorts has not been carried out. Using integrated genomic analysis of 264 T-ALL cases, we identified 106 putative driver genes, half of which had not previously been described in childhood T-ALL (for example, CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We describe new mechanisms of coding and noncoding alteration and identify ten recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOXA1 deregulated ALL, PTPN2 mutations in TLX1 deregulated T-ALL, and PIK3R1/PTEN mutations in TAL1 deregulated ALL, which suggests that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches.

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis

Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.

Pilot Study of Nelarabine in Combination With Intensive Chemotherapy in High-Risk T-Cell Acute Lymphoblastic Leukemia: A Report From the Children's Oncology Group

PURPOSE Childrens Oncology Group study AALL00P2 was designed to assess the feasibility and safety of adding nelarabine to a BFM 86-based chemotherapy regimen in children with newly diagnosed T-cell acute lymphoblastic leukemia (T-ALL). PATIENTS AND METHODS In stage one of the study, eight patients with a slow early response (SER) by prednisone poor response (PPR; ≥ 1,000 peripheral blood blasts on day 8 of prednisone prephase) received chemotherapy plus six courses of nelarabine 400 mg/m(2) once per day; four patients with SER by high minimal residual disease (MRD; ≥ 1% at day 36 of induction) received chemotherapy plus five courses of nelarabine; 16 patients with a rapid early response (RER) received chemotherapy without nelarabine. In stage two, all patients received six 5-day courses of nelarabine at 650 mg/m(2) once per day (10 SER patients [one by MRD, nine by PPR]) or 400 mg/m(2) once per day (38 RER patients; 12 SER patients [three by MRD, nine by PPR]). RESULTS The only significant difference in toxicities was decreased neutropenic infections in patients treated with nelarabine (42% with v 81% without nelarabine). Five-year event-free survival (EFS) rates were 73% for 11 stage one SER patients and 67% for 22 stage two SER patients treated with nelarabine versus 69% for 16 stage one RER patients treated without nelarabine and 74% for 38 stage two RER patients treated with nelarabine. Five-year EFS for all patients receiving nelarabine (n = 70) was 73% versus 69% for those treated without nelarabine (n = 16). CONCLUSION Addition of nelarabine to a BFM 86-based chemotherapy regimen was well tolerated and produced encouraging results in pediatric patients with T-ALL, particularly those with a SER, who have historically fared poorly.

Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: implications for minimal residual disease detection.

Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B‐ALL) have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T‐ALL) though it is presumed that CD99 and terminal deoxynucleotidyl transferase (TdT) are highly informative based on limited studies.

Safe Integration of Nelarabine into Intensive Chemotherapy in Newly Diagnosed T-cell Acute Lymphoblastic Leukemia: Children’s Oncology Group Study AALL0434

Nelarabine has shown impressive single agent clinical activity in T‐cell acute lymphoblastic leukemia (T‐ALL), but has been associated with significant neurotoxicities in heavily pre‐treated patients. We showed previously that it was safe to add nelarabine to a BFM‐86 chemotherapy backbone (AALL00P2). Childrens Oncology Group (COG) AALL0434 is a Phase III study designed to test the safety and efficacy of nelarabine when incorporated into a COG augmented BFM–based regimen, which increases exposure to agents with potential neurotoxicity compared to the historical AALL00P2 regimen.

Preclinical efficacy of daratumumab in T-cell acute lymphoblastic leukemia (T-ALL)

As a consequence of acquired or intrinsic disease resistance, the prognosis for patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Novel, less toxic drugs are clearly needed. One of the most promising emerging therapeutic strategies for cancer treatment is targeted immunotherapy. Immune therapies have improved outcomes for patients with other hematologic malignancies including B-cell ALL; however no immune therapy has been successfully developed for T-ALL. We hypothesize targeting CD38 will be effective against T-ALL. We demonstrate that blasts from patients with T-ALL have robust surface CD38 surface expression and that this expression remains stable after exposure to multiagent chemotherapy. CD38 is expressed at very low levels on normal lymphoid and myeloid cells and on a few tissues of nonhematopoietic origin, suggesting that CD38 may be an ideal target. Daratumumab is a human immunoglobulin G1κ monoclonal antibody that binds CD38, and has been demonstrated to be safe and effective in patients with refractory multiple myeloma. We tested daratumumab in a large panel of T-ALL patient-derived xenografts (PDX) and found striking efficacy in 14 of 15 different PDX. These data suggest that daratumumab is a promising novel therapy for pediatric T-ALL patients.

MLL rearrangements impact outcome in HOXA-deregulated T-lineage acute lymphoblastic leukemia: a Children's Oncology Group Study.

MLL rearrangements impact outcome in HOXA -deregulated T-lineage acute lymphoblastic leukemia: a Children’s Oncology Group Study

Intravenous Immunoglobulin G Therapy in Fetal and Neonatal Alloimmune Thrombocytopenia

SummaryNeonatal alloimmune thrombocytopenia (NAIT) is a rare disorder in the neonatal period, occurring in 1 in 2000 to 5000 births. The clinical syndrome of NAIT can be varied, but the resulting thrombocytopenia can be very severe and can result in serious haemorrhage in the fetus and neonate. At least 5 human platelet antigen systems have been described, and all have been implicated in cases of NAIT. Approximately 50% of platelet antigen-incompatible firstborn infants will be affected. The risk of recurrence in subsequent pregnancies is high, estimated at >75%, with the disease being either equally or more severe than in the first infant. Mortality in NAIT is estimated to be 10%, and is usually associated with intracranial haemorrhage. The incidence of intracranial haemorrhage is estimated to be approximately 14%, with approximately 25% occurring antenatally. In an effort to prevent these severe complications, therapy is usually instituted in infants with severe thrombocytopenia from NAIT. Treatment strategies have included appropriate, compatible platelet transfusion, corticosteroid use and exchange transfusion. However, success in treating immune-mediated thrombocytopenia with intravenous immunoglobulins (IVIg) has prompted the use of this therapy in fetuses and infants with NAIT. IVIg has demonstrated efficacy in both of these clinical situations. The literature concerning the use of IVIg, both ante-and post-natally, is reviewed, and treatment strategies for infants and fetuses affected by NAIT are discussed.

Hedgehog pathway mutations drive oncogenic transformation in high-risk T-cell acute lymphoblastic leukemia

The role of Hedgehog signaling in normal and malignant T-cell development is controversial. Recently, Hedgehog pathway mutations have been described in T-ALL, but whether mutational activation of Hedgehog signaling drives T-cell transformation is unknown, hindering the rationale for therapeutic intervention. Here, we show that Hedgehog pathway mutations predict chemotherapy resistance in human T-ALL, and drive oncogenic transformation in a zebrafish model of the disease. We found Hedgehog pathway mutations in 16% of 109 childhood T-ALL cases, most commonly affecting its negative regulator PTCH1. Hedgehog mutations were associated with resistance to induction chemotherapy (P = 0.009). Transduction of wild-type PTCH1 into PTCH1-mutant T-ALL cells induced apoptosis (P = 0.005), a phenotype that was reversed by downstream Hedgehog pathway activation (P = 0.007). Transduction of most mutant PTCH1, SUFU, and GLI alleles into mammalian cells induced aberrant regulation of Hedgehog signaling, indicating that these mutations are pathogenic. Using a CRISPR/Cas9 system for lineage-restricted gene disruption in transgenic zebrafish, we found that ptch1 mutations accelerated the onset of notch1-induced T-ALL (P = 0.0001), and pharmacologic Hedgehog pathway inhibition had therapeutic activity. Thus, Hedgehog-activating mutations are driver oncogenic alterations in high-risk T-ALL, providing a molecular rationale for targeted therapy in this disease.