Novel gene fusion discovery in Spitz tumours and its relevance in diagnostics.

In addition to morphologic analysis, molecular diagnostic work up of Spitz tumours is often of great value for their accurate diagnosis/classification. Nowadays, next-generation sequencing (NGS) is the predominant screening method in molecular diagnostics. Up to 80% of these melanocytic neoplasms comprise gene fusions as genetic anomalies for which the driver codes for a protein harbouring a kinase domain. However, because of the variety of fusion partners the use of PCR-based targeted enrichment NGS methods is not recommended. We describe a series of four Spitz tumour samples in which distinct gene fusions were detected by hybridisation-based capture NGS (TPM3::ALK, LIMA1::ROS1, LRRFIP2::ROS1 and MYO5A::RET). Two of these fusions are not previously described. All 4 fusions were confirmed by reverse transcription-PCR. These findings demonstrate the need for molecular analysis that can detect unknown fusions in Spitz neoplasms for optimal diagnosis.

HLA-I diversity and tumor mutational burden by comprehensive next-generation sequencing as predictive biomarkers for the treatment of non-small cell lung cancer with PD-(L)1 inhibitors.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) improved outcomes in non-small cell lung cancer (NSCLC) patients. We report the predictive utility of human leukocyte antigen class I (HLA-I) diversity and tumor mutational burden (TMB) by comprehensive next-generation sequencing. METHODS: 126 patients were included. TMB high was defined as ≥ 10 nonsynonymous mutations/Mb. Patients exhibit high HLA-I diversity if at least one locus was in the upper 15th percentile for DNA alignment scores. RESULTS: No difference in response rate (RR; 44.4% versus 30.9%; p = 0.1741) or 6-month survival rate (SR; 75.6% versus 77.8%; p = 0.7765) was noted between HLA-I high diversity and low diversity patients. HLA-I high diversity patients did significantly more often exhibit durable clinical benefit (DCB), defined as response or stable disease lasting minimally 6 months (64.4% [29/45] versus 43.2% [35/81]; p = 0.0223). TMB high patients exhibited higher RR (49.1% versus 25.4%; p = 0.0084) and SR 6 months after start ICI (85.5% versus 70.4%; p = 0.0468) than TMB low patients. The proportion of patients with DCB, did not differ significantly between TMB high and low subgroups (60.0% [33/55] versus 42.3% [30/71]; p = 0.0755). Patients with combined dual high TMB and HLA-I diversity had higher RR (63.2% versus 22.2%; p = 0.0033), but SR at 6 months did not differ significantly (84.2% versus 64,4%; p = 0.1536). A significantly higher rate of patients experienced DCB in dual high compared to the dual low group (73.7% [14/19] versus 35.6% [16/45]; p = 0.0052). Triple positive patients (high TMB and HLA-I diversity and PD-L1 positive) had higher RR (63.6% versus 0.0%; p = 0.0047) and SR at 6 months (100% versus 66.7%; p = 0.0378) compared to triple-negative patients. CONCLUSION: HLA-I diversity was able to predict durable clinical benefit in ICI treated NSCLC patients, but failed to confirm as a predictor of response or survival. TMB confirmed as a predictive biomarker.

Diagnostic Validation of a Comprehensive Targeted Panel for Broad Mutational and Biomarker Analysis in Solid Tumors.

The use of targeted Next Generation Sequencing (NGS) for the diagnostic screening of somatic variants in solid tumor samples has proven its high clinical value. Because of the large number of ongoing clinical trials for a multitude of variants in a growing number of genes, as well as the detection of proven and emerging pan-cancer biomarkers including microsatellite instability (MSI) and tumor mutation burden (TMB), the currently employed diagnostic gene panels will become vastly insufficient in the near future. Here, we describe the validation and implementation of the hybrid capture-based comprehensive TruSight Oncology (TSO500) assay that is able to detect single-nucleotide variants (SNVs) and subtle deletions and insertions (indels) in 523 tumor-associated genes, copy-number variants (CNVs) of 69 genes, fusions with 55 cancer driver genes, and MSI and TMB. Extensive validation of the TSO500 assay was performed on DNA or RNA from 170 clinical samples with neoplastic content down to 10%, using multiple tissue and specimen types. Starting with 80 ng DNA and 40 ng RNA extracted from formalin-fixed and paraffine-embedded (FFPE) samples revealed a precision and accuracy >99% for all variant types. The analytical sensitivity and specificity were at least 99% for SNVs, indels, CNVs, MSI, and gene rearrangements. For TMB, only values around the threshold could yield a deviating outcome. The limit-of-detection for SNVs and indels was well below the set threshold of 5% variant allele frequency (VAF). This validated comprehensive genomic profiling assay was then used to screen 624 diagnostic samples, and its success rate for adoption in a clinical diagnostic setting of broad solid tumor screening was assessed on this cohort.

The Dynamics of Nucleotide Variants in the Progression from Low-Intermediate Myeloma Precursor Conditions to Multiple Myeloma: Studying Serial Samples with a Targeted Sequencing Approach.

Multiple myeloma (MM), or Kahler's disease, is an incurable plasma cell (PC) cancer in the bone marrow (BM). This malignancy is preceded by one or more asymptomatic precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). The molecular mechanisms and exact cause of this progression are still not completely understood. In this study, the mutational profile underlying the progression from low-intermediate risk myeloma precursor conditions to MM was studied in serial BM smears. A custom capture-based sequencing platform was developed, including 81 myeloma-related genes. The clonal evolution of single nucleotide variants and short insertions and deletions was studied in serial BM smears from 21 progressed precursor patients with a median time of progression of six years. From the 21 patients, four patients had no variation in one of the 81 studied genes. Interestingly, in 16 of the 17 other patients, at least one variant present in MM was also detected in its precursor BM, even years before progression. Here, the variants were present in the pre-stage at a median of 62 months before progression to MM. Studying these paired BM samples contributes to the knowledge of the evolutionary genetic landscape and provides additional insight into the mutational behavior of mutant clones over time throughout progression.

Oncogenic cooperation between TCF7-SPI1 and NRAS(G12D) requires ß-catenin activity to drive T-cell acute lymphoblastic leukemia.

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.

Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities.

Multiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient's life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

Suz12 inactivation cooperates with JAK3 mutant signaling in the development of T-cell acute lymphoblastic leukemia.

The polycomb repressive complex 2, with core components EZH2, SUZ12, and EED, is responsible for writing histone 3 lysine 27 trimethylation histone marks associated with gene repression. Analysis of sequence data from 419 T-cell acute lymphoblastic leukemia (T-ALL) cases demonstrated a significant association between SUZ12 and JAK3 mutations. Here we show that CRISPR/Cas9-mediated inactivation of Suz12 cooperates with mutant JAK3 to drive T-cell transformation and T-ALL development. Gene expression profiling integrated with ChIP-seq and ATAC-seq data established that inactivation of Suz12 led to increased PI3K/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and WNT signaling. Moreover, a drug screen revealed that JAK3/Suz12 mutant leukemia cells were more sensitive to histone deacetylase (HDAC)6 inhibition than JAK3 mutant leukemia cells. Among the broad genome and gene expression changes observed on Suz12 inactivation, our integrated analysis identified the PI3K/mTOR, VEGF/VEGF receptor, and HDAC6/HSP90 pathways as specific vulnerabilities in T-ALL cells with combined JAK3 and SUZ12 mutations.

Cooperative Enhancer Activation by TLX1 and STAT5 Drives Development of NUP214-ABL1/TLX1-Positive T Cell Acute Lymphoblastic Leukemia.

The NUP214-ABL1 fusion is a constitutively activated tyrosine kinase that is significantly associated with overexpression of the TLX1 and TLX3 transcription factors in T cell acute lymphoblastic leukemia (T-ALL). Here we show that NUP214-ABL1 cooperates with TLX1 in driving T-ALL development using a transgenic mouse model and human T-ALL cells. Using integrated ChIP-sequencing, ATAC-sequencing, and RNA-sequencing data, we demonstrate that TLX1 and STAT5, the downstream effector of NUP214-ABL1, co-bind poised enhancer regions, and cooperatively activate the expression of key proto-oncogenes such as MYC and BCL2. Inhibition of STAT5, downregulation of TLX1 or MYC, or interference with enhancer function through BET-inhibitor treatment leads to reduction of target gene expression and induction of leukemia cell death.

Single-cell sequencing reveals the origin and the order of mutation acquisition in T-cell acute lymphoblastic leukemia.

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10-20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38- multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) among various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38- cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.

HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Mutant JAK3 signaling is increased by loss of wild-type JAK3 or by acquisition of secondary JAK3 mutations in T-ALL.

The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common γ chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma with the variant RNF213-, ATIC- and TPM3-ALK fusions is characterized by copy number gain of the rearranged ALK gene.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by 2p23/ALK aberrations, including the classic t(2;5)(p23;q35)/NPM1-ALK rearrangement present in ~80% of cases and several variant t(2p23/ALK) occurring in the remaining cases. The ALK fusion partners play a key role in the constitutive activation of the chimeric protein and its subcellular localization. Using various molecular technologies, we have characterized ALK fusions in eight recently diagnosed anaplastic large cell lymphoma cases with cytoplasmic-only ALK expression. The identified partner genes included EEF1G (one case), RNF213/ALO17 (one case), ATIC (four cases) and TPM3 (two cases). Notably, all cases showed copy number gain of the rearranged ALK gene, which is never observed in NPM1-ALK-positive lymphomas. We hypothesized that this could be due to lower expression levels and/or lower oncogenic potential of the variant ALK fusions. Indeed, all partner genes, except EEF1G, showed lower expression in normal and malignant T cells, in comparison with NPM1 In addition, we investigated the transformation potential of endogenous Npm1-Alk and Atic-Alk fusions generated by clustered regularly interspaced short palindromic repeats/Cas9 genome editing in Ba/F3 cells. We found that Npm1-Alk has a stronger transformation potential than Atic-Alk, and we observed a subclonal gain of Atic-Alk after a longer culture period, which was not observed for Npm1-Alk Taken together, our data illustrate that lymphomas driven by the variant ATIC-ALK fusion (and likely by RNF213-ALK and TPM3-ALK), but not the classic NPM1-ALK, require an increased dosage of the ALK hybrid gene to compensate for the relatively low and insufficient expression and signaling properties of the chimeric gene.

Hedgehog pathway activation in T-cell acute lymphoblastic leukemia predicts response to SMO and GLI1 inhibitors.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive childhood leukemia that is caused by the accumulation of multiple genomic lesions resulting in transcriptional deregulation and increased cell proliferation and survival. Through analysis of gene expression data, we provide evidence that the hedgehog pathway is activated in 20% of T-ALL samples. Hedgehog pathway activation is associated with ectopic expression of the hedgehog ligands Sonic hedgehog (SHH) or Indian hedgehog (IHH), and with upregulation of the transcription factor GLI1 Ectopic expression of SHH or IHH in mouse T cells in vivo caused hedgehog pathway activation in both lymphoid and epithelial cells in the thymus and resulted in increased expression of important T-cell stimulatory ligands (Dll4, Il7, and Vegf) by thymic epithelial cells. In T-ALL cell lines, pharmacological inhibition or short interfering RNA-mediated knockdown of SMO or GLI1 led to decreased cell proliferation. Moreover, primary T-ALL cases with high GLI1 messenger RNA levels, but not those with low or undetectable GLI1 expression, were sensitive to hedgehog pathway inhibition by GANT61 or GDC-0449 (vismodegib) using ex vivo cultures and in vivo xenograft models. We identify the hedgehog pathway as a novel therapeutic target in T-ALL and demonstrate that hedgehog inhibitors approved by the US Food and Drug Administration could be used for the treatment of this rare leukemia.

RNA sequencing unravels the genetics of refractory/relapsed T-cell acute lymphoblastic leukemia. Prognostic and therapeutic implications.

Despite therapeutic improvements, a sizable number of patients with T-cell acute lymphoblastic leukemia still have a poor outcome. To unravel the genomic background associated with refractoriness, we evaluated the transcriptome of 19 cases of refractory/early relapsed T-cell acute lymphoblastic leukemia (discovery cohort) by performing RNA-sequencing on diagnostic material. The incidence and prognostic impact of the most frequently mutated pathways were validated by Sanger sequencing on genomic DNA from diagnostic samples of an independent cohort of 49 cases (validation cohort), including refractory, relapsed and responsive cases. Combined gene expression and fusion transcript analyses in the discovery cohort revealed the presence of known oncogenes and identified novel rearrangements inducing overexpression, as well as inactivation of tumor suppressor genes. Mutation analysis identified JAK/STAT and RAS/PTEN as the most commonly disrupted pathways in patients with chemorefractory disease or early relapse, frequently in association with NOTCH1/FBXW7 mutations. The analysis on the validation cohort documented a significantly higher risk of relapse, inferior overall survival, disease-free survival and event-free survival in patients with JAK/STAT or RAS/PTEN alterations. Conversely, a significantly better survival was observed in patients harboring only NOTCH1/FBXW7 mutations: this favorable prognostic effect was abrogated by the presence of concomitant mutations. Preliminary in vitro assays on primary cells demonstrated sensitivity to specific inhibitors. These data document the negative prognostic impact of JAK/STAT and RAS/PTEN mutations in T-cell acute lymphoblastic leukemia and suggest the potential clinical application of JAK and PI3K/mTOR inhibitors in patients harboring mutations in these pathways.

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia.

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34(+) thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34(+) and Notch inactive CD4(+)CD8(+) thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.

JAK3 mutants transform hematopoietic cells through JAK1 activation, causing T-cell acute lymphoblastic leukemia in a mouse model.

JAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase-independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.