Stuart S. Winter

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

High frequency of PTEN, PI3K, and AKT abnormalities in T-cell acute lymphoblastic leukemia.

To more comprehensively assess the pathogenic contribution of the PTEN-PI3K-AKT pathway to T-cell acute lymphoblastic leukemia (T-ALL), we examined diagnostic DNA samples from children with T-ALL using array comparative genomic hybridization and sequence analysis. Alterations of PTEN, PI3K, or AKT were identified in 47.7% of 44 cases. There was a striking clustering of PTEN mutations in exon 7 in 12 cases, all of which were predicted to truncate the C2 domain without disrupting the phosphatase domain of PTEN. Induction chemotherapy failed to induce remission in 3 of the 4 patients whose lymphoblasts harbored PTEN deletions at the time of diagnosis, compared with none of the 12 patients with mutations of PTEN exon 7 (P = .007), suggesting that PTEN deletion has more adverse therapeutic consequences than mutational disruptions that preserve the phosphatase domain. These findings add significant support to the rationale for the development of therapies targeting the PTEN-PI3K-AKT pathway in T-ALL.

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 BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

Benign Hematogone-Rich Lymphoid Proliferations Can Be Distinguished From B-Lineage Acute Lymphoblastic Leukemia by Integration of Morphology, Immunophenotype, Adhesion Molecule Expression, and Architectural Features

Distinction of normal B-lymphoid proliferations including precursors known as hematogones from acute lymphoblastic leukemia (ALL) is critical for disease management. We present a multiparameter assessment of 27 bone marrow samples containing at least 25% hematogones (range, 25%-72%) by morphologic review. We used flow cytometry to evaluate B-cell differentiation antigen and adhesion molecule expression and immunohistochemistry on clot sections to evaluate architectural distribution. Flow cytometry revealed that intermediately differentiated cells (CD19+, CD10+) predominated, followed in frequency by CD20+, surface immunoglobulin-positive cells, with CD34+, terminal deoxynucleotidyl transferase (TdT)-positive cells as the smallest subset. Adhesion molecules (CD44, CD54) were expressed more heterogeneously compared with expression in acute lymphoblastic leukemia. Immunohistochemistry revealed that CD34+, TdT-positive cells were dispersed without significant clustering, while CD20+ cells exceeded CD34/TdT-positive cells in 24 of 25 cases. This multidisciplinary study demonstrates that hematogone-rich lymphoid proliferations exhibit a spectrum of B-lymphoid differentiation antigen expression with predominance of intermediate and mature B-lineage cells, heterogeneity of adhesion molecule expression, and nonclustered bone marrow architectural distribution.

Inactivation of LEF1 in T-cell acute lymphoblastic leukemia

To further unravel the molecular pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), we performed high-resolution array comparative genomic hybridization on diagnostic specimens from 47 children with T-ALL and identified monoallelic or biallelic LEF1 microdeletions in 11% (5 of 47) of these primary samples. An additional 7% (3 of 44) of the cases harbored nonsynonymous sequence alterations of LEF1, 2 of which produced premature stop codons. Gene expression microarrays showed increased expression of MYC and MYC targets in cases with LEF1 inactivation, as well as differentiation arrest at an early cortical stage of thymocyte development characterized by expression of CD1B, CD1E, and CD8, with absent CD34 expression. LEF1 inactivation was associated with a younger age at the time of T-ALL diagnosis, as well as activating NOTCH1 mutations, biallelic INK4a/ARF deletions, and PTEN loss-of-function mutations or activating mutations of PI3K or AKT genes. These cases generally lacked overexpression of the TAL1, HOX11, HOX11L2, or the HOXA cluster genes, which have been used to define separate molecular pathways leading to T-ALL. Our findings suggest that LEF1 inactivation is an important step in the molecular pathogenesis of T-ALL in a subset of young children.

Risk determinants for catheter‐associated blood stream infections in children and young adults with cancer

Catheter‐associated blood stream infections (CABSI) are frequent complications encountered with cancer treatment. In order to understand which factors might predispose to CABSIs in children and young adults, we evaluated risk for infection in association with tumor type, catheter type, and setting of occurrence.

Absence of Biallelic TCRγ Deletion Predicts Early Treatment Failure in Pediatric T-Cell Acute Lymphoblastic Leukemia

PURPOSE To identify children with T-cell acute lymphoblastic leukemia (T-ALL) at high risk of induction chemotherapy failure by using DNA copy number analysis of leukemic cells collected at diagnosis. PATIENTS AND METHODS Array comparative genomic hybridization (CGH) was performed on genomic DNA extracted from diagnostic lymphoblasts from 47 children with T-ALL treated on Childrens Oncology Group Study P9404 or Dana-Farber Cancer Institute Protocol 00-01. These samples represented nine patients who did not achieve an initial complete remission, 13 who relapsed, and 25 who became long-term, event-free survivors. The findings were confirmed in an independent cohort of patients by quantitative DNA polymerase chain reaction (DNA-PCR), an assay that is well suited for clinical application. RESULTS Analysis of the CGH findings in patients in whom induction chemotherapy failed compared with those in whom induction chemotherapy was successful identified the absence of biallelic TCRgamma locus deletion (ABD), a characteristic of early thymocyte precursors before V(D)J recombination, as the most robust predictor of induction failure (P < .001). This feature was also associated with markedly inferior event-free (P = .002) and overall survival (P < .001) rates: 25% versus 58% and 25% versus 72%, respectively. Using a rapid and inexpensive quantitative DNA-PCR assay, we validated ABD as a predictor of a poor response to induction chemotherapy in an independent series of patients. CONCLUSION Lymphoblasts from children with T-ALL should be evaluated at diagnosis for deletion within the TCRgamma locus. Patients lacking biallelic deletion, which confers a high probability of induction failure with contemporary therapy, should be assigned to alternative therapy in the context of a prospective clinical trial.

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.

Prognostic Value of an Activation State Marker for Epidermal Growth Factor Receptor in Tissue Microarrays of Head and Neck Cancer

Overexpression and mutation of epidermal growth factor receptors (EGFR) have been shown to be important in the prognosis of several cancers, including head and neck cancers. However, our inability to define the activation status of these and other receptors limits our ability to assess the importance of these pathways and to exploit effectively new molecularly targeted treatments directed at their catalytic activities. Here we describe the use of automated, high-throughput fluorescence lifetime imaging microscopy to measure EGFR autophosphorylation status by fluorescence resonance energy transfer (FRET) in head and neck tumors. We have correlated FRET efficiency with the clinical and survival data. The results from head and neck arrays show that high FRET efficiency is correlated with worsening disease-free survival but not with overall survival. This powerful tool could be exploited as a new independent quantitative prognostic factor in clinical decisions and cancer management.