Fusion Partner Genes Research Articles

Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer.

Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

Alk-rearranged lung adenocarcinoma: From molecular genetics to therapeutic targeting.

Anaplastic Lymphoma Kinase (ALK) is a potent oncogenic driver of lung adenocarcinoma (LUAD). ALK is constitutively activated by gene fusion events between the ALK and other gene fusion partners in about 2-3% of LUADs, characterized by few other gene alterations. ALK-fusions are a druggable target through potent pharmacological inhibitors of tyrosine kinase activity. Thus, several ALK-TKIs (Tyrosine Kinase Inhibitors) of first-, second- and third-generation have been developed that improved the outcomes of ALK-rearranged LUADs when used as first- or second-line agents. However, resistance mechanisms greatly limit the durability of the therapeutic effects elicited by these TKIs. The molecular mechanisms responsible for these resistance mechanisms have been in part elucidated, but overcoming acquired resistance to ALK-derived therapy remains a great challenge. Some new therapeutic strategies under investigation aim to induce long-term remission in ALK-fusion positive LUADs.

ANO7 African-ancestral genomic diversity and advanced prostate cancer

BackgroundProstate cancer (PCa) is a significant health burden for African men, with mortality rates more than double global averages. The prostate specific Anoctamin 7 (ANO7) gene linked with poor patient outcomes has recently been identified as the target for an African-specific protein-truncating PCa-risk allele.MethodsHere we determined the role of ANO7 in a study of 889 men from southern Africa, leveraging exomic genotyping array PCa case-control data (n = 780, 17 ANO7 alleles) and deep sequenced whole genome data for germline and tumour ANO7 interrogation (n = 109), while providing clinicopathologically matched European-derived sequence data comparative analyses (n = 57). Associated predicted deleterious variants (PDVs) were further assessed for impact using computational protein structure analysis.ResultsNotably rare in European patients, we found the common African PDV p.Ile740Leu (rs74804606) to be associated with PCa risk in our case-control analysis (Wilcoxon rank-sum test, false discovery rate/FDR = 0.03), while sequencing revealed co-occurrence with the recently reported African-specific deleterious risk variant p.Ser914* (rs60985508). Additional findings included a novel protein-truncating African-specific frameshift variant p.Asp789Leu, African-relevant PDVs associated with altered protein structure at Ca2+ binding sites, early-onset PCa associated with PDVs and germline structural variants in Africans (Linear regression models, −6.42 years, 95% CI = −10.68 to −2.16, P-value = 0.003) and ANO7 as an inter-chromosomal PCa-related gene fusion partner in African derived tumours.ConclusionsHere we provide not only validation for ANO7 as an African-relevant protein-altering PCa-risk locus, but additional evidence for a role of inherited and acquired ANO7 variance in the observed phenotypic heterogeneity and African-ancestral health disparity.

Updates in pathobiological aspects of anaplastic large cell lymphoma.

Anaplastic large cell lymphomas (ALCL) encompass several distinct subtypes of mature T-cell neoplasms that are unified by the expression of CD30 and anaplastic cytomorphology. Identification of the cytogenetic abnormality t(2;5)(p23;q35) led to the subclassification of ALCLs into ALK+ ALCL and ALK- ALCL. According to the most recent World Health Organization (WHO) Classification of Haematolymphoid Tumours as well as the International Consensus Classification (ICC) of Mature Lymphoid Neoplasms, ALCLs encompass ALK+ ALCL, ALK- ALCL, and breast implant-associated ALCL (BI-ALCL). Approximately 80% of systemic ALCLs harbor rearrangement of ALK, with NPM1 being the most common partner gene, although many other fusion partner genes have been identified to date. ALK- ALCLs represent a heterogeneous group of lymphomas with distinct clinical, immunophenotypic, and genetic features. A subset harbor recurrent rearrangement of genes, including TYK2, DUSP22, and TP63, with a proportion for which genetic aberrations have yet to be characterized. Although primary cutaneous ALCL (pc-ALCL) is currently classified as a subtype of primary cutaneous T-cell lymphoma, due to the large anaplastic and pleomorphic morphology together with CD30 expression in the malignant cells, this review also discusses the pathobiological features of this disease entity. Genomic and proteomic studies have contributed significant knowledge elucidating novel signaling pathways that are implicated in ALCL pathogenesis and represent candidate targets of therapeutic interventions. This review aims to offer perspectives on recent insights regarding the pathobiological and genetic features of ALCL.

Screening Streptomyces secondary metabolites against non-small cell lung cancer using computational approaches

Secondary metabolites from bacterial sources are being seen as alternatives to identify new medicines for various disease conditions. Streptomyces is known to be a warehouse of secondary metabolites and is a promising source to be looked for the discovery and development of new anti-tumorigenic agents. Such identifications could alleviate the issue of lung cancer that takes over 2 million lives a year. In the current study, we have screened secondary metabolites from Streptomyces for their potential to act as drugs for non-small cell lung cancer. The metabolites were identified for their ability to inhibit RET protein. Gene fusion partners cause alteration to the RET protein that causes the cancer condition. The metabolites identified were based on docking, pharmacokinetics, toxicity and pass prediction. 16 metabolites from 13 different species of Streptomyces showed binding affinity to the target protein. Among the metabolites, melanin was the one that had strongest evident to act as a drug candidate for non-small cell lung cancer therapeutics.

Mutation status analysis of 58 patients with advanced ALK fusion gene positive non small cell lung cancer

PurposeTo analyze the characteristics and prognostic values of Anaplastic Lymphoma Kinase (ALK) fusion gene partner, gene subtype and abundance in tumor tissues of advanced Non Small Cell Lung Cancer (NSCLC) patients with positive ALK fusion gene and to explore the best treatment mode of ALK-Tyrosine Kinase Inhibitors(TKIs).MethodsCases of advanced NSCLC patients with ALK positive confirmed by both Next Generation Sequencing (NGS) and immunohistochemistry were retrospectively collected. The relationships of Overall Survival (OS)/Progression Free Survival (PFS) between different mutation subtypes, mutation abundance, clinicopathological features were analyzed. OS/PFS between different treatment mode of ALK inhibitors were compared.ResultsFifty-eight patients were enrolled. There were diverse fusion partners. Five subtypes of Echinoderm Microtubule-associated protein-Like 4 gene (EML4)-ALK fusion mutation were detected: V1,V2,V3,V5 and V7. The mutation abundance ranged from 0.13 to 27.77%, with a median of 5.34%. The abundance of V2 and V5 was higher than V1 and V3 respectively. There was no difference in OS between the low abundance group(≤ 5.34%) and the high abundance group(>5.34%) (P = 0.434). PFS of second-generation ALK inhibitors as first-line treatment was longer than that of Crizotinib as first-line (P<0.001). Never smokers had longer OS than current smokers(P = 0.001).ConclusionsThere are differences in abundance between different fusion partners and subtypes in advanced NSCLC with positive ALK. OS is not associated with subtypes, mutation abundance and first line treatment option of either generation of ALK inhibitors. Smoking is a poor prognostic factor.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

MET Fusions in NSCLC: Clinicopathologic Features and Response to MET Inhibition

IntroductionMET fusions have been described only rarely in NSCLC. Thus, data on patient characteristics and treatment response are limited. We here report histopathologic data, patient demographics, and treatment outcome including response to MET tyrosine kinase inhibitor (TKI) therapy in MET fusion-positive NSCLC. MethodsPatients with NSCLC and MET fusions were identified mostly by RNA sequencing within the routine molecular screening program of the national Network Genomic Medicine, Germany. ResultsWe describe a cohort of nine patients harboring MET fusions. Among these nine patients, two patients had been reported earlier. The overall frequency was 0.29% (95% confidence interval: 0.15–0.55). The tumors were exclusively adenocarcinoma. The cohort was heterogeneous in terms of age, sex, or smoking status. We saw five different fusion partner genes (KIF5B, TRIM4, ST7, PRKAR2B, and CAPZA2) and several different breakpoints. Four patients were treated with a MET TKI leading to two partial responses, one stable disease, and one progressive disease. One patient had a BRAF V600E mutation as acquired resistance mechanism. ConclusionsMET fusions are very rare oncogenic driver events in NSCLC and predominantly seem in adenocarcinomas. They are heterogeneous in terms of fusion partners and breakpoints. Patients with MET fusion can benefit from MET TKI therapy.

Abstract 6734: Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor that occurs in childhood and the most common cancer in infancy. Fewer than 5% of neuroblastomas occur in adolescents and young adults (AYAs), for whom the disease has an indolent and fatal course. Chen et al. previously reported that missense mutations in the protein- tyrosine kinase domain of anaplastic lymphoma kinase (ALK) occur in approximately 10% of cases of sporadic neuroblastoma, and these missense mutations result in the activation of the ALK protein kinase domain, which plays a key role in the tumorigenic process as seen in ALK fusion proteins. Since the initial discovery of the NPM-ALK fusion protein in human anaplastic lymphoma cell lines, more than 24 different ALK fusion proteins have been discovered in various malignancies. Till date, ALK-containing chimeric proteins have not been reported in neuroblastoma. In this study, a translocation within chromosome 2p and 4q was found to bring about the formation of an in-frame fusion gene that was composed of portions of the teneurin transmembrane protein 3 (TENM3) gene and ALK gene in tumor cells from an AYA patient with neuroblastoma. The patient was 19-year-old female diagnosed with stage 4 neuroblastoma (unfavorable histology, poorly differentiated subtype, MYCN DNA not amplified). Computed tomography revealed a mass in the left adrenal gland with bone, spine, liver, and lung metastasis. ALK rearrangement was confirmed via FISH analysis. Crizotinib resulted in no disease progression for 5 months, except for metastatic spinal disease, but it did not exhibit high efficacy compared with its effects against lymphoma and lung cancer. This 5′ in-frame fusion partner gene in neuroblastoma has not yet been identified, and its role is not well-known in oncogenesis. We generated the cell line with the expression of the full length TENM3-ALK cDNA in NIH-3T3 cells led to the formation of a fusion protein. A cell-based evaluation of this predicted ALK protein variant revealed activation of downstream targets-STAT3, AKT and ERK- and ALK inhibitors, crizotinib or lorlatinib, treatment inhibited the growth of xenograft tumors with stable TENM3-ALK expression. We determined whether TENM3-ALK affects transformation and proliferation using a soft agar colony formation assay. As expected, the cells that expressed an empty vector and wild type ALK line did not produce a considerable number of colonies, whereas TENM3-ALK transfected cells showed anchorage-independent growth, resulting in the production of a higher number of colonies. The evidence for the role of ALK receptor signaling in stimulating neuroblastoma tumor cell growth and the demonstrated in vitro and in vivo efficacy of the ALK selective inhibitors in this study provide biological and clinical justification for the further exploration of this combination and testing in patients with recurrent or refractory neuroblastoma. Citation Format: Mitsuteru Hiwatari, Masafumi Seki, Ryosuke Matsuno, Kenichi Yoshida, Takeshi Nagasawa, Aiko Sato-Otsubo, Shohei Yamamoto, Motohiro Kato, Kentaro Watanabe, Masahiro Sekiguchi, Satoru Miyano, Seishi Ogawa, Junko Takita. Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6734.

Clinical and pathological heterogeneity of four common fusion subtypes in Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinoma (Xp11.2 tRCC) is a group of rare and highly heterogeneous renal cell carcinoma (RCC). The translocation involving TFE3 and different fusion partners lead to overexpression of the chimeric protein. The purpose of this study is to explore the clinicopathological features of Xp11.2 tRCC with four common fusion subtypes. We screened out 40 Xp11.2 tRCC patients from January 2007 to August 2021 in our institution. The diagnosis was initially confirmed by TFE3 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assay and their fusion partners were verified by RNA sequencing. Then the 40 cases were divided into two groups (DBHS family and non-DBHS family group) and a clinical comparison among the four common fusion subtypes was performed. Among the 40 cases, 11 cases with SFPQ-TFE3 gene fusion and 7 cases with NONO-TFE3 gene fusion were classified in DBHS group, the remaining cases with ASPL-TFE3 (11 cases) or PRCC-TFE3 (11 cases) gene fusion were classified in non-DBHS group. Lymph node (LN) metastasis (P=0.027) and distant metastasis (P=0.009) were more common seen in non-DBHS family group than DBHS family group and cases in DBHS family group have better progressive-free survival (PFS) (P=0.02). In addition, ASPL-TFE3 fusion was associated with worse outcome (P=0.03) while NONO-TFE3 fusion (P=0.04) predicted a better prognosis. Different fusion partner genes may play a functional role in various morphology, molecular and biological features of Xp11.2 tRCCs. The impact of fusion partners on clinical characteristics of Xp11.2 tRCCs deserves further exploration.

Molecular Identification and In Silico Protein Analysis of a Novel BCOR-CLGN Gene Fusion in Intrathoracic BCOR-Rearranged Sarcoma.

BCOR (BCL6 corepressor)-rearranged sarcomas (BRSs) are a heterogeneous group of sarcomas previously classified as part of the group of "atypical Ewing" or "Ewing-like" sarcomas, without the prototypical ESWR1 gene translocation. Due to their similar morphology and histopathological features, diagnosis is challenging. The most common genetic aberrations are BCOR-CCNB3 fusion and BCOR internal tandem duplication (ITD). Recently, various new fusion partners of BCOR have been documented, such as MAML3, ZC3H7B, RGAG1, and KMT2D, further increasing the complexity of such tumor entities, although the molecular pathogenetic mechanism remains to be elucidated. Here, we present an index case of intrathoracic BRS that carried a novel BCOR-CLGN (calmegin) gene fusion, exhibited by a 52-year-old female diagnosed initially by immunohistochemistry due to the positivity of a BCOR stain; the fusion was identified by next-generation sequencing and was confirmed by Sanger sequencing. In silico protein analysis was performed to demonstrate the 3D structure of the chimera protein. The physicochemical properties of the fusion protein sequence were calculated using the ProtParam web-server tool. Our finding further broadens the fusion partner gene spectrum of BRS. Due to the heterogeneity, molecular ancillary tests serve as powerful tools to discover these unusual variants, and an in silico analysis of the fusion protein offers an appropriate approach toward understanding the exact pathogenesis of such a rare variant.

Novel transporter screening technology for chemical production by microbial fermentation.

In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.

A Novel NGS Assay to Detect Any KMT2A fusion Transcript at Low Levels

Introduction: Measurable residual disease (MRD) detection is associated with relapse. Leukemias with KMT2A rearrangement (KMT2Ar) have an adverse prognosis with more than 80 possible fusion partner genes (FPG) identified. With the advent of effective targeted therapies for KMT2Ar leukemia such as menin inhibitors, there is an unmet need for sensitive assays to detect these various fusions, not only to guide and monitor therapy, but also to predict and intercept relapse. Multicolor flow cytometry (MFC) is the only readily available test for MRD in KMT2Ar acute myeloid leukemia (AML), not requiring prior knowledge of exact fusions and breakpoints for design of patient-specific assays. Standard next-generation sequencing (NGS) methods cannot accurately detect genetic alterations at low frequencies because of inherent technical errors and the need for high sequencing depth. Blocker Displacement Amplification (BDA) technology enables selective detection of such rare alterations with NGS (Wu LR, Nat Biomed Eng 2017). Here, we demonstrate the effective detection of KMT2Ar and its various FPG with a novel NGS assay leveraging BDA. Methods: The NuProbe RNA fusion proprietary technology uses BDA (FusionBDA) where primers flank any known exon junction of cDNA corresponding to transcripts of the gene of interest, while non-extensible oligonucleotides (blockers) span the wildtype (WT) exon junction, therefore suppressing amplification of WT while differentially amplifying fusion junctions. As a result, final libraries are enriched with reads mapping to fusion genes. This allows de novo calls and minimizes read requirements. We designed a custom panel targeting 35 exons of KMT2A (forward and reverse) and developed a pipeline using 3 previously established fusion detection methods (Arriba, STAR-Fusion, FusionCatcher). We validated this assay in a KMT2Ar cell line. Limit-of-detection (LoD) was assessed by blending RS4;11 (leukemia with KMT2A::AFF1) with HT-1197 cells (bladder cancer, no KMT2Ar), to attain contrived samples with 0.001% to 100% tumor fractions (400ng input). Limit-of-input (LoI) was assessed using cDNA with 1- 400ng RNA from 100% and 0.1% tumor fraction samples. In a blinded analysis, we tested 50 bone marrow samples from 35 patients with KMT2Ar AML, chosen deliberately with various 11q23 translocations including rare ones. Results: Following LoD experiment, KMT2Ar was detected accurately down to 0.005% tumor fraction (Figure 1). Subsequently, LoI assay revealed the ability to detect KMT2Ar in 100% VAF tumor fraction samples using as low as 1ng of input RNA and using 400ng in 1% VAF samples. Among samples with morphologic evidence of leukemia, FusionBDA detected various KMT2Ar in 44/45 samples (97.8%) except 1 sample with a t(11;19)(q23;p13.1) (Table 1). This sample had a suboptimal amount and quality of cDNA loaded. We evaluated concordance of predicted FPG based on bands by cytogenetics (CG) and the published literature on KMT2A recombinome. We found that FusionBDA identified the same predicted FPG (same band) in 41/45 samples (91%). In samples with various t(11;19) translocations, different FPG were identified all located on 11p13 within a few bands from ones by CG, likely representing the accurate FPG given the low resolution of CG and the associated error in determining arm band accurately. Finally, we explored the potential of this assay for MRD detection by examining 5 samples at morphologic remission following treatment. All samples where fluorescence in situ hybridization (FISH) detected a KMT2A fusion had FusionBDA+ (31/31 samples including 1 in morphologic remission). 1 sample with no detectable KMT2A fusion by FISH and MRDneg by MFC, had detectable fusion by FusionBDA, indicating possibly an improved sensitivity. 3 samples with MRD+ by MFC also had detectable KMT2Ar by FusionBDA (lowest MRD+ level by flow was 0.5%). 1 sample was MRD+ by MFC and had a detectable KMT2Ar by FusionBDA in 2/3 callers (detectable by FusionCatcher and StarFusion not Arriba), therefore not meeting our stringent criteria though likely concordant with MRD by MFC. Further analysis in the MRD setting is planned. Conclusion: FusionBDA is a novel, sensitive, and specific assay for KMT2Ar acute leukemia. This assay is able to agnostically detect KMT2Ar with various fusions at a predicted sensitivity of at least 0.005%, therefore allowing both MRD detection and target identification. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LILRB4 Is a Novel Target for KMT2A Rearranged Acute Leukemia

Background: KMT2a gene rearrangement (KMT2Ar) is a recurrent mutation in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), occurring both in children and adults. Patients with KMT2Ar acute leukemia have poor outcomes related to increased rates of relapsed and refractory disease. Novel therapies including BITE and CAR-T cells has proven efficacious for subgroups of patients with acute leukemia, however those with KMT2Ar are at risk for lineage switch relapse following immunotherapy. In a recent analysis, there have been no reported survivors following lineage switch relapse. Therefore, it is critical to identify novel targets and treatment strategies for KMT2Ar acute leukemia. We have previously demonstrated leukocyte immunoglobulin like receptor B4 (LILRB4) is a novel tumor associated antigen with complete expression on AML with monocytic differentiation (FAB M5), which can be effectively targeted by a novel anti-LILRB4 CAR-T cell we developed and validated in preclinical models. A majority of acute monocytic leukemia cell lines and patient samples contain the KTMT2Ar, which led us to hypothesize LILRB4 is expressed on other KMT2Ar acute leukemias including B-ALL, and may retain expression on lineage switch relapsed disease. In the current study, we evaluate the anti-leukemia function of anti-LILRB4 CAR-T cells against KMT2Ar ALL and lineage switch disease. Methods and results: We first analyzed the expression of LILRB4 on a panel of B-ALL cell lines by flow cytometry. The KMT2Ar cell lines, RS411 [t(4;11)] and KOPN8 [t(11;19)] demonstrated complete expression of LILRB4 regardless of fusion gene partner, while cell lines without KMT2Ar including 697 and NALM6, were negative for LILRB4. We next analyzed expression of LILRB4 on primary patient samples of de novo, relapsed or refractory acute leukemia identified as KMT2Ar from our institution biospecimen repository. In five cases of B-ALL with KMT2Ar, LILRB4 was co-expressed with CD19 on leukemia blasts as demonstrated by flow cytometry. In one patient with multiply relapsed AML with KMT2Ar, expression of LILRB4 was retained on each relapse following stem cell transplant. Lastly, in an infant with refractory KMTAr B-ALL who developed lineage switch AML following treatment with Blinatumomab, LILRB4 was strongly expressed on the complete population of leukemia blasts. We next evaluated the effector function of anti-LILRB4 CAR-T cells against LILRB4-positive, KMT2Ar B-ALL cell lines. Human T-cells were activated with CD3/CD28 beads, then transduced with lentiviral vector encoding the second generation anti-LILRB4 CAR transgene, incorporating a 4-1BB co-stimulatory and CD3-zeta activation domain, and were expanded for 7-10 days with IL7 and IL15. In vitro, anti-LILRB4 CAR-T cells demonstrated potent cytotoxicity in mixed culture against KOPN8, and similarly displayed significantly increased cytokine release of IL2, TNF-alpha and Interferon-gamma compared to control-T cells. To evaluate the function of anti-LILRB4 CAR-T cell in vivo, we engrafted KOPN8 cells in NSG immunocompromised mice, followed by treatment with control or CAR-T cells. Anti-LILRB4 CAR-T cell treated mice demonstrated significantly decreased leukemia burden and prolonged survival compared to control treated mice. Patient derived mouse xenografts (PDX) of KMT2Ar relapsed AML (PDX1) and lineage switch leukemia (PDX2) were also established. Significantly, when utilizing autologous anti-LILRB4 CAR-T cells, leukemia was eliminated in the PDX1 model. And strikingly in PDX2, we demonstrate elimination of KMT2Ar lineage switch leukemia in mice treated with anti-LILRB4 CAR-T cells. Conclusion: In this preclinical work, we demonstrate LILRB4 is expressed on KMT2Ar acute leukemias including B-ALL, and importantly displays a retained pattern of expression on fatal lineage switch relapsed AML. Furthermore, we demonstrate potent anti-leukemia activity of anti-LILRB4 CAR-T cells against LILRB4 expressing KMT2Ar acute leukemia, broadening the application of this cell therapy. This work offers a new treatment strategy for patients with dismal outcomes and will be tested in a Phase I clinical trial for children and adults with LILRB4 expressing acute leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pralsetinib in RET fusion-positive non-small-cell lung cancer: A real-world data (RWD) analysis from the Italian expanded access program (EAP)

ObjectivesThe selective RET-inhibitor pralsetinib has shown therapeutic activity in early clinical trials in patients with non-small cell lung cancer (NSCLC) harboring rearranged during transfection (RET) gene fusions. To date, the real-world efficacy of pralsetinib in this population is unknown. Materials and methodsA retrospective efficacy and safety analysis was performed on data from patients with RET-fusion positive NSCLC enrolled in the pralsetinib Italian expanded access program between July 2019 and October 2021. ResultsOverall, 62 patients with RET-fusion positive NSCLC received pralsetinib at 20 Italian centers. Next-generation sequencing was used to detect RET alterations in 44 patients (73 %). The most frequent gene fusion partner was KIF5B (75 % of 45 evaluable). Median age was 62 years (range, 36–90), most patients were female (57 %) and never smokers (53 %). Brain metastases were known in 18 patients (29.5 %) at the time of pralsetinib treatment. 13 patients were treatment naïve (unfit for chemotherapy), 48 were pretreated (median number of previous lines: 1, range, 1–4). The objective response rate (ORR) was 66 % [95 % confidence interval (CI), 53–81] in the evaluable population (n = 59). The disease control rate (DCR) was 79 %. After a median follow-up of 10.1 months, the median progression free survival was 8.9 months (95 %CI, 4.7–NA). In patients with measurable brain metastases (n = 6) intracranial ORR was 83 %, intracranial DCR was 100 %. Overall, 83.6 % of patients experienced any-grade treatment-related adverse events (TRAEs), 39 % grade 3 or greater (G ≥ 3). The most common G ≥ 3 TRAEs were neutropenia (9.8 %), dry mouth/oral mucositis (8.2 %), and thrombocytopenia (6.6 %). Seven patients (12 %) discontinued pralsetinib due to TRAEs, twenty-six had at least one dose level modification due to TRAEs. Two treatment-related deaths were observed (1 sepsis, 1 typhlitis). ConclusionsIn the real-world setting, pralsetinib confirmed durable systemic activity and intracranial response in RET-fusion positive NSCLC. Toxicity profile was consistent with previous reports.

MRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

EP16.03-003 Systematic Population-based Identification of NTRK Fusion Genes Among Hispanic Patients with Non-Small Cell Lung Cancer (NSCLC)

Neurotrophic tropomyosin-related kinases (NTRKs) A, B, and C constitute a receptor tyrosine kinase family involved in neuronal development. They are encoded by NTRK1-3 genes, respectively. NTRK fusions create chimeric genes in which the 3' region of the NTRK gene is joined with a 5' sequence of a fusion partner gene. As a result of the fusion, a chimeric oncoprotein originates a ligand-independent constitutive activation of the kinase activity. This chimeric kinase varies among tumor histology and population, being 0.3-3% in non-squamous NSCLC; however, little is known about these mutations among the Hispanic population.

112P Beyond BRAF V600: Characterizing the genomic landscape of non-BRAF V600 mutations and BRAF fusions in 121,221 in adult patients with cancer

112P Beyond BRAF V600: Characterizing the genomic landscape of non-BRAF V600 mutations and BRAF fusions in 121,221 in adult patients with cancer

074 ALK-positive desmoplastic Spitz naevus in a patient with corresponding ALK-positive anaplastic large cell lymphoma

Aim:Spitz naevus is a benign melanocytic tumour with distinct histopathological features of spindle-shaped melanocytes. An understanding of genetic aberrations can help to characterise the different subsets of Spitzoid neoplasms. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that was first described in the pathogenesis of anaplastic large cell lymphoma (ALCL). ALK mutations have been reported in up to 10% of Spitz tumours.This study describes the rare occurrence of an ALK mutation in two different diseases in the same patient – ALCL and Spitz naevus. Method:Presentation, management, and clinical outcomes were retrospectively reviewed for a patient with ALK-positive ALCL and ALK-positive Spitz naevus at a tertiary paediatric hospital. Result:The patient was a 14-year-old female with a 12-month history of a red-brown plaque on the right shoulder.This was on a background stage III ALCL treated with chemotherapy and stem cell transplantation. She underwent surgical excision of the skin lesion. Histopathological examination showed an ALK-positive desmoplastic Spitz naevus. A bone marrow aspirate and trephine (BMAT) of the right axillary lymph node at the time of ALCL diagnosis also revealed an ALK mutation. She is currently in remission 2 years post stem cell transplantation. Conclusion:Our case reports on a rare coincidence of a patient having the same mutation for two distinct disease processes. There has been one similarly reported case in the literature in a patient with ALK-positive cutaneous ALCL and Spitz naevi with the same fusion partner gene (TPM3). Whilst this occurrence is not fully understood, further studies may identify whether this is an independent coincidence in two different cell lineages, or a fragile site similarity which may predispose to the same translocation event.

P343: GENOMIC CHARACTERISATION OF B-OTHER ALL IN UKALL2003 PATIENTS BY NEXT GENERATION SEQUENCING

Background: Incorporating genetics into risk stratification for the treatment of childhood acute lymphoblastic leukaemia (ALL) has contributed to increased survival rates. About 30% of patients (B-other-ALL) harbour none of the known major chromosomal changes. Recently, we estimated that up to two thirds of B-other-ALL can be classified into novel subtypes using FISH and MLPA alone. We showed that ABL-class fusions and ERG deletions were linked to prognosis (Schwab et al, BJHaem, 2022). Aims: To characterise B-other-ALL in the UKALL2003 trial using next generation sequencing (NGS), to evaluate the added benefits of these techniques and validate the prognostic significance of B-other-ALL subtypes. Methods: B-other-ALL patients were tested using whole genome sequencing (WGS) (n=158) and bespoke targeted NGS (t-NGS) for the detection of abnormalities in 64 genes commonly mutated or rearranged in B-ALL (n=180), 42 patients had both WGS and t-NGS. Data were integrated with results from FISH, MLPA and cytogenetics. Results: A representative cohort of 347 B-other-ALL was classified into subgroups, including six with ≥20 cases (Figure). Among patients tested by WGS, 92% (n=146/158) were classified compared to 75% (n=104/138) tested by t-NGS. NGS-based approaches allowed for the detection of PAX5 P80R, IKZF1 N169Y and ZEB2 H1038R and fusion partner genes. Identification of DUX4-r was not possible using t-NGS. PAX5alt was the most frequently observed (n=90), including dic(9;20) (n=27), dic(9;12) (n=11), PAX5 fusions (n=21), PAX5 mutation (n=11) and PAX5-ITD (n=12). Additionally, 11 patients were observed with PAX5 P80R mutations, classified as a separate subgroup due to the associated gene expression signature. PAX5alt had an outcome similar to B-other-ALL overall (overall survival (OS) at 10yrs 82% vs 86%). There was no significant difference in outcome between PAX5 abnormalities, although an association with age was observed: dic(9;20) was more commonly observed in children aged 1-4 (p< 0.001), while both dic(9;12) and PAX5 P80R were seen in older children aged 10-15 years (p=0.005). One subgroup with DUX4 rearrangements and/or ERG deletions (DUX4-r/ERG-d) (n=78) had DUX4 involvement confirmed by WGS in 57 patients. In the remaining 21 patients ERG-d were identified by t-NGS and/or MLPA. ERG-d is exclusive to DUX4-r patients, thus can be used as a surrogate marker. In line with other studies, patients with DUX4-r had a lower relapse rate (RR) (5%) and improved OS (96%) compared to other subgroups. Patients classified as ETV6-RUNX1-like (n=21) were characterised by multiples abnormalities of ETV6, including rearrangements (n=17) and/or deletion (n=12) as well as rearrangements (n=7) and/or deletions (n=6) of IKZF1. An ETV6-RUNX1-like gene expression signature was confirmed by RNA-Seq (n=6). No relapses or deaths were reported within this subgroup. As previously reported, ABL-class fusions (n=25) were associated with an inferior outcome compared to other subgroups (RR=63% and OS=50%). Patients with rearrangements of CRLF2 (n=52) and ZNF384 (n=37) had similar outcomes to the cohort overall. Image:Summary/Conclusion: A combination of techniques has classified a representative cohort of B-other-ALL patients from UKALL2003 into clinically relevant subgroups. The identification of DUX4-r, subgroup defining mutations and fusion partner genes demonstrates the value of NGS-based approaches. We have confirmed the good and poor prognostic associations of DUX4-r and ABL-class fusions, respectively, and identified ETV6-RUNX1-like subgroup to have a good prognosis.