Fusion Tyrosine Kinase Research Articles

Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.

Profiling Receptor Tyrosine Kinase Fusions in Chinese Breast Cancers.

BackgroundReceptor tyrosine kinases (RTKs) are a class of tyrosine kinases that regulate cell-to-cell communication and control a variety of complex biological functions. Dysregulation of RTK signaling partly due to chromosomal rearrangements leads to novel tyrosine kinase fusion oncoproteins that are possibly driver alterations to cancers. Targeting some RTK fusions with specific tyrosine kinases inhibitors (TKIs) is an effective therapeutic strategy across a spectrum of RTK fusion-related cancers. However, there is still a paucity of extensive RTK fusion investigations in breast cancer. This study aims to characterize RTK fusions in Chinese breast cancer patients.MethodsAn in-house DNA sequencing database of 1440 Chinese breast cancer patients with a capture-based panel (520 gene or 108 gene-panel) was thoroughly reviewed. A total of 2,229 samples including 1,045 tissues and 1,184 plasmas were analyzed. RTK fusion was defined as an in-frame fusion with the tyrosine kinase domain of the RTK completely retained. Concomitant mutations were also analyzed and tumor mutational burden (TMB) was calculated. Patients’ clinical characteristics were retrieved from case records.ResultsA total of 30 RTK fusion events were identified from 27 breast cancer patients with a prevalence of 1.875%%. FGFR2 fusions were seen the most commonly (n=7), followed by RET (n=5), ROS1 (n=3), NTRK3 (n=3), BRAF (n=2), and NTRK1 (n=2). Other RTK fusions including ALK, EGFR, FGFR1, FGFR3, MET, and NTRK2 were identified in one patient each. A total of 27 unique resultant fusion proteins (22 with a novel partner) were discovered including 19 intrachromosomal rearrangements and 8 interchromosomal ones. Twenty-one fusions had the tyrosine kinase domain in-frame fused with a partner gene and six were juxtaposed with an intergenic space. Among the 27 fusions, FGFR2-WDR11 (E17: intergenic) (n=3) and ETV6-NTRK3 (E5:E15) (n=2) occurred recurrently. Of note, the normalized abundance of RTK fusion (fusion AF/max AF) correlated negatively with TMB (r=-0.48, P=0.017). Patients with TMB < 8 (Mutations/Mb) displayed a higher fusion abundance than those with TMB ≥ 8 (Mutations/Mb) (P=0.025). Moreover, CREBBP mutation only co-occurred with FGFR2 fusion (P=0.012), while NTRK3 fusion and TP53 mutation were mutually exclusive (P=0.019).ConclusionThis is the first study comprehensively delineating the prevalence and spectrum of RTK fusions in Chinese breast cancers. Further study is ongoing to identify the enriched subpopulation who may benefit from RTK fusion inhibitors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Clinical Utility of Plasma-Based Comprehensive Molecular Profiling in Advanced Non-Small-Cell Lung Cancer.

PURPOSEComprehensive molecular profiling (CMP) plays an essential role in clinical decision making in metastatic non–small-cell lung cancer (mNSCLC). Circulating tumor DNA (ctDNA) analysis provides possibilities for molecular tumor profiling. In this study, we aim to explore the additional value of centralized ctDNA profiling next to current standard-of-care protocolled tissue-based molecular profiling (SoC-TMP) in the primary diagnostic setting of mNSCLC in the Netherlands.METHODSPretreatment plasma samples from 209 patients with confirmed mNSCLC were analyzed retrospectively using the NGS AVENIO ctDNA Targeted Kit (Roche Diagnostics, Basel, Switzerland) and compared with paired prospective pretreatment tissue-based molecular profiling from patient records. The AVENIO panel is designed to detect single-nucleotide variants, copy-number variations, insertions or deletions, and tyrosine kinase fusion in 17 genes.RESULTSPotentially targetable drivers were detected with SoC-TMP alone in 34.4% of patients. Addition of clonal hematopoiesis of indeterminate potential–corrected, plasma-based CMP increased this to 39.7% (P < .001). Concordance between SoC-TMP and plasma-CMP was 86.6% for potentially targetable drivers. Clinical sensitivity of plasma-CMP was 75.2% for any oncogenic driver. Specificity and positive predictive value were more than 90% for all oncogenic drivers.CONCLUSIONPlasma-CMP is a reliable tool in the primary diagnostic setting, although it cannot fully replace SoC-TMP. Complementary profiling by combined SoC-TMP and plasma-CMP increased the proportion of patients who are eligible for targeted treatment.

Abstract 1113: Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models

Abstract Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy and relapse within short time, leaving no further therapeutic option than bone marrow transplant, when feasible. This highlights the need to identify new first-line therapies for high-risk patients.We explored the potential of upfront drug combinations to prevent the rise of resistant disease, in vitro and in vivo. Crizotinib was combined with CHOP chemotherapy, an epigenetic drug (decitabine), another targeted inhibitor (trametinib- MEK inhibitor), or a second-generation ALK inhibitor. ALK+ ALCL cells were kept in culture for ~100 days in the presence of different single or combined drugs. We found that in most cases, combined treatments were more effective than single agents in the long-term control of cells expansion. Upon further functional characterization using quantitative PCR and western blot, we found that combination treatments upregulated cell cycle inhibitors and pro-apoptotic genes as well as modulated survival factors to induce deeper inhibition of oncogenic signalling and higher rates of apoptosis. Crizotinib+decitabine combination treatment promoted downregulation of DNMT1 and increase of TGFB1, both of which indirectly affect cell cycle progression. In murine model, crizotinib+decitabine significantly delayed tumour growth, compared to single treatments. Our results indicate that combining a selective drug with a less specific anticancer agent, which allows unspecific killing of resistant clones that might potentially arise, would be beneficial.In conclusion, our data suggest that a polypharmacology approach combining ALK inhibition with other agents could be a valuable therapeutic option for ALK+ ALCL patients, to prevent relapses and improve outcomes. Citation Format: Geeta G. Sharma, Giulia Arosio, Matteo Villa, Mario Mauri, Marina Zappa, Vera Magistroni, Monica Ceccon, Sara Redaelli, Ilaria Crespiatico, Diletta Fontana, Cristina Mastini, Anna Garbin, Federica Lovisa, Lara Mussolin, Rocco Piazza, Carlo Gambacorrti-Passerini, Luca Mologni. Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1113.

Integrated Molecular Profiling of Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that occurs in infancy and early childhood, characterized by excessive myelomonocytic cell proliferation and granulocyte-macrophage colony-stimulating factor hypersensitivity. Over 80% of JMML patients have been reported to harbor mutually exclusive somatic and/or germline mutations in canonical RAS pathway genes (e.g., PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several poor prognostic factors, including the presence of two or more mutations, acute myeloid leukemia-like (AML-like) gene expression profiles, and selected gene promoter methylation profiles. However, the molecular pathogenesis in 10%-20% of patients and the relationships among biomarkers have not been well defined. This study aimed to assess the molecular pathogenesis in patients and the relationships among several biomarkers and outcomes.Patients and Methods: We performed comprehensive genetic analyses in 150 JMML patients, including 15 with Noonan syndrome-associated myeloproliferative disorder (NS/MPD), using whole-exome sequencing (n = 69, 46%), targeted deep sequencing (n = 92, 61%), RNA sequencing (n = 129, 86%), and genome-wide methylation analysis (n = 106, 71%).Results: We identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in three of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro . Therefore, we administered crizotinib to a patient with the RANBP2-ALK fusion gene. This patient achieved complete molecular remission after the administration of crizotinib for 51 days and successfully bridged to allogeneic stem cell transplantation. This patient survived without disease recurrence for 15 months after transplantation.We performed a genome-wide methylation analysis in 106 patients and combined the data with repository data from normal CD34+ samples (n = 5) and AML CD34+CD38− samples (n = 14) for an unsupervised consensus clustering of CpG methylation profiles, which yielded two distinct subgroups, hypermethylation profile (n = 45, 42%) and hypomethylation profile (n = 61, 58%). All AML samples had a hypermethylation profile, whereas all normal CD34+ and NS/MPD samples had a hypomethylation profile. A gene set enrichment analysis revealed marked enrichment of AML-associated genes in the hypermethylation cluster. The 5-year OS rate was 46.0% [95% confidence interval (CI), 31.0%-59.8%] among patients with a hypermethylation profile and 73.4% (95% CI, 57.6%-84.1%) among those with a hypomethylation profile (p = 0.0028). The 5-year TFS was significantly poorer in the hypermethylation group than in the hypomethylation group [2.2% (95% CI, 0.2%-10.1%) vs. 41.2% (95% CI, 27.1%-54.8%), p = 3.1 × 10−10]. Differential gene expression analysis for the hypermethylation and hypomethylation profiles revealed overexpression of LIN28B and its downstream target HMGA2 in the hypermethylation profile.Lastly, we integrated the molecular analyses of JMML, including genome-wide methylation profiling, RNA sequencing, and resequencing data and found that the hypermethylation profile was highly correlated with the most established risk factors for JMML, including older age, higher HbF level, lower platelet count, PTPN11/NF1 mutations, presence of two or more mutations, LIN28B overexpression, and AML-like expression profiles, as well as poor survival.Discussion: We identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations. Crizotinib represents a promising candidate drug for treating JMML in patients with ALK/ROS1 fusions. In addition, we established a risk profiling system based on genome-wide methylation analysis and showed that the hypermethylation profile is highly associated with poor survival and most established poor prognostic biomarkers for JMML. These finding will contribute to the development of precision treatments for JMML patients. DisclosuresNo relevant conflicts of interest to declare.

An Update on Molecular Genetic Aberrations in Spitz Melanocytic Proliferations: Correlation with Morphological Features and Biological Behavior.

The aim of the paper is to give an update on molecular genetic aberrations in Spitz melanocytic proliferations with special emphasis on their correlation with morphological features and biological behavior. The Spitz group of melanocytic proliferations is defined by a combination of distinctive morphological features and driver molecular genetic events. Morphologically, these neoplasms are characterized by large, oval, polygonal, or spindled melanocytes with abundant eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, often in association with epidermal hyperplasia. Molecular aberrations in Spitz melanocytic proliferations can be divided into two main groups, according to the driver genetic change: 1) 11p amplification/HRAS mutation, present in about 20% of cases, and 2) kinase fusions, present in about 50%, further subdivided into tyrosine kinase fusions (ALK, ROS1, NTRK1, NTRK3, MET, RET) or serine-threonine kinase fusions (MAP3K8, BRAF). Driver genetic aberrations can be detected along the whole biological spectrum of Spitz melanocytic proliferations, and are mutually exclusive. Although driver genetic aberrations enable proliferation of melanocytes, additional genetic events (often biallelic inactivation of CDKN2A and TERT promoter mutations) are necessary for the development of overt Spitz malignancy. CONCLUSIONS: Recent studies have demonstrated that certain driver genetic aberrations are more often associated with the benign spectrum of Spitz melanocytic proliferations and indolent biological behavior (11p amplification/HRAS mutation, tyrosine kinase fusions). In contrast, some driver aberrations are more frequent in the atypical/malignant spectrum of Spitz melanocytic proliferations with a potential for aggressive biological behavior (serine-threonine kinase fusions). In addition, certain driver aberrations are often associated with distinctive morphological features. However, none of the morphological features is entirely specific for any of these driver genetic aberrations. Immunohistochemistry for ALK, ROS1, and pan-TRK can be used for screening purposes to detect corresponding fusion proteins.

Profiling receptor tyrosine kinase fusions in Chinese breast cancers.

e15092 Background: Receptor tyrosine kinases (RTKs) are a class of tyrosine kinases that regulate cell-to-cell communication and control a variety of complex biological functions. Dysregulation of RTK signaling partly due to chromosomal rearrangements leads to novel tyrosine kinase fusion oncoproteins which are possibly driver alterations to cancers. Targeting some RTK fusions with specific tyrosine kinases inhibitors (TKIs) is an effective therapeutic strategy across a spectrum of RTK fusion-related cancers. However, there is still a paucity of extensive RTK fusion investigations in breast cancer. We aimed to characterize RTK fusions in Chinese breast cancer patients. Methods: An in-house sequencing database of 1440 Chinese breast cancer patients using a 520-gene NGS sequencing panel was thoroughly reviewed. RTK fusion was defined as an in-frame fusion with the tyrosine kinase domain of the RTK completely retained with the only exception of ERBB2 fusion which was not counted due to its unclear significance. Concomitant mutations and TMB were also analyzed and calculated. Patients’ clinical characteristics were retrieved from case records. Results: 27 RTK fusion-positive breast cancers (12 tissues + 15 plasmas) were identified, patients had a median age of 52 years. Triple-negative breast cancer subtype comprised 37% with luminal and HER2 positive subtypes being 40.8% and 22.2%, respectively. 77.8% of patients were at stage IV and 22.2% at stage I-III. Ten were treatment naïve. RTK fusions occurred in 2% of breast cancers in our database, compared with the prevalence of 0.6% and 1.3% in MSKCC and TCGA, respectively. In the subset of stage IV patients, our database showed a significantly higher RTK fusion frequency than that in MSKCC (3.2% vs. 0.6%, p = 0.013). FGFR2 fusions were seen most commonly (n = 7), followed by RET (n = 4), ROS1 (n = 3), NTRK3 (n = 3), BRAF (n = 2), and NTRK1 (n = 2). Other RTK fusions including ALK, EGFR, FGFR1, FGFR3, MET, and NTRK2 were identified in one patient each. Of note, the normalized abundance of RTK fusion (fusion AF/max AF) correlated negatively with TMB (r = -0.48, p = 0.017). Patients with TMB &lt; 4 (Muts/Mb) displayed a higher fusion abundance than those with TMB ≥ 4 (Muts/Mb) (p = 0.018), suggesting a higher likelihood of subclonal nature for RTK fusions in TMB-high patients. Moreover, CREBBP mutation only co-occurred with FGFR2 fusion (p = 0.012), while NTRK3 fusion and TP53 mutation were mutually exclusive (p = 0.019). Conclusions: This is the first study comprehensively delineating the prevalence and spectrum of RTK fusions in Chinese breast cancers. Further study is ongoing to identify the enriched subpopulation which may benefit from RTK fusion inhibitors.

Oncologist management of NTRK fusion-positive NSCLC improves after virtual patient simulation participation.

e23008 Background: Although relatively rare, neurotrophic receptor tyrosine kinase (NTRK) fusions, represent a clinically relevant subgroup of NSCLC that can derive benefit from targeted therapies. Relatively quick and durable responses have been achieved with these targeted therapies in advanced NSCLC and intracranial activity has also been observed. The objective of this study was to determine if an online, virtual patient simulation (VPS)-based continuing medical education (CME) intervention improved performance of oncologists in using appropriate strategies to diagnose and manage patients with NTRK fusion-positive advanced NSCLC. Methods: The CME intervention consisted of two patient cases presented in a VPS platform that allowed learners to order lab tests, diagnoses and treatments in a manner matching the scope and depth of actual practice. Clinical decisions made by the learners using open field entries within an EHR interface were analyzed and, after each decision, tailored clinical guidance (CG) was provided based on current evidence and expert recommendation. Learner decisions were collected post-CG and compared with each user’s baseline (pre-CG) data using a McNemar’s test to determine P values. Results were then aggregated for the two cases based on clinical decisions and stratified by practice setting. The activity posted July 31, 2020; data were collected through December 1, 2020. Results: Overall significant improvements were seen after CG in clinical decisions made by oncologists in both the community and academic settings [reported as % absolute improvement (% correct pre-CG vs % correct post-CG); P value]. Conclusions: This analysis demonstrated that VPS that immerses and engages oncologists in an authentic and practical learning experience can significantly improve evidence-based clinical decisions related to the diagnosis and management of patients with NTRK fusion-positive advanced NSCLC. Despite the improvements, additional educational activities are needed to address any residual gaps and further increase oncologists’ ability in this clinical setting.[Table: see text]

NTRK, RET, BRAF, and ALK fusions in thyroid fine-needle aspirates (FNAs).

6083 Background: Receptor tyrosine kinase (RTK) fusions may be targeted by small molecule inhibitors to treat various advanced tumors, including thyroid cancer. Clinical trials have studied selective inhibitors of ALK, BRAF, NTRK and RET, leading to several FDA-approved therapies. The Afirma Genomic Sequencing Classifier (GSC) classifies cytologically indeterminate thyroid nodules as molecularly benign or suspicious. The Xpression Atlas reports 905 genomic variants and 235 fusion pairs on GSC Suspicious, Suspicious for Malignancy (SFM), and Malignant FNA samples at the time of diagnosis. Here we report the prevalence of these fusion genes in real-world clinical practice. Methods: We analyzed anonymized data from 50,644 consecutive Bethesda III-VI nodule FNA samples submitted to the Veracyte CLIA laboratory for molecular testing using whole transcriptome RNA sequencing (RNA-Seq). Gene pairs are listed alphabetically. Results: 32,080 Bethesda III/IV nodules were classified as GSC Benign and 278 were Parathyroid Classifier positive. No ALK, BRAF, NTRK1/3, or RET fusions were identified among these samples. Among 16,594 Bethesda III/IV GSC Suspicious FNAs, 3% (n = 529) were positive for ALK, BRAF, NTRK1/3 or RET fusions. Among the 1,692 Bethesda V/VI FNAs, the proportion of positive nodules was 8% (n = 135). Among these combined cohorts of Bethesda III/IV GSC Suspicious and Bethesda V/VI, the most common gene fusions observed for each of the 5 studied RTK genes was: ETV6/NTRK3 (n = 164, 72% of NTRK3 fusions), CCDC6/RET (n = 104, 55% of RET), BRAF/SND1 (n = 32, 20% of BRAF), ALK/STRN (n = 20, 37% of ALK), and NTRK1/TPM3 (n = 14, 50% of NTRK1). BRAF showed the highest diversity of fusions, with 80 gene partners. Different gene partners with RET, ALK, NTRK1, and NTRK3 numbered 25, 11, 9, and 5 , respectively . Conclusions: Whole-transcriptome RNA-seq on small sample thyroid FNA specimens can identify clinically relevant ALK, BRAF, NTRK, and RET fusions across Bethesda categories. The prevalence ranges from 3% in Bethesda III/IV Afirma GSC Suspicious specimens to 8% among Bethesda V/VI specimens. Future studies need to determine if detection of precision medicine candidates by pre-operative FNA can optimize initial treatment, predict response to treatment, and prioritize selective targeted therapy should systemic treatment be needed.[Table: see text]

Detection of FGFR fusions in intrahepatic cholangiocarcinoma using targeted RNA sequencing.

e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.

Treatment of PCM1-JAK2 Fusion Tyrosine Kinase Gene-Related Acute Lymphoblastic Leukemia with Stem Cell Transplantation

PCM1-JAK2 fusion mutations are rare variants that activate a tyrosine kinase leading to a variety of neoplasms that can involve any hematologic cell line. They most often present as myelodysplasia (MPD) and can demonstrate prominent eosinophilia and/or erythrodysplasia. Transformation to acute leukemia is often seen, as is de novo leukemia. Lymphomas have also been reported. The diagnosis can often be made with routine cytogenetic analysis but specific probes and detailed next generation sequencing may be necessary. JAK2 inhibitors are active in MPD as is stem cell transplantation. Transplantation has occasionally been successful in leukemic phase as well. The current case highlights both the difficulties in diagnosis as well as the second successful treatment of MPD, transformed into acute lymphoblastic leukemia.

RTKs Can Form Membraneless Granules to Drive Cytoplasmic RAS Signaling.

Oncogenic receptor tyrosine kinase fusions assembled into functional cytoplasmic protein granules.

Calcified chondroid mesenchymal neoplasms with FN1-receptor tyrosine kinase gene fusions including FGFR2, FGFR1, MERTK, NTRK1, and TEK: a molecular and clinicopathologic analysis.

Translocations involving FN1 have been described in a variety of neoplasms that share the presence of a cartilage matrix and may also contain a variable extent of calcification. Fusions of FN1 to FGFR1 or FGFR2 have been reported in nine soft tissue chondromas, mostly demonstrated indirectly by FISH analysis. Delineation of FN1 fusions with various partner genes will facilitate our understanding of the pathogenesis and diagnostic classification of these neoplasms. In this study, we present molecular, clinical, and pathologic features of 12 cartilaginous soft tissue neoplasms showing a predilection for the TMJ region and the distal extremities. We analyzed for gene fusions with precise breakpoints using targeted RNA-seq with a 115-gene panel. We detected gene fusions in ten cases, including three novel fusions, FN1-MERTK, FN1-NTRK1, and FN1-TEK, each in one case, recurrent FN1-FGFR2 fusion in five cases, FN1-FGFR1 in one case, and FGFR1-PLAG1 in one case. The breakpoints in the 5' partner gene FN1 ranged from exons 11-48, retaining the domains of a signal peptide, FN1, FN2, and/or FN3, while the 3' partner genes retained the transmembrane domain, tyrosine kinase (TK) domains, and/or Ig domain. The tumors are generally characterized by nodular/lobular growth of polygonal to stellate cells within a chondroid matrix, often accompanied by various patterns of calcification, resembling those described for the chondroblastoma-like variant of soft tissue chondroma. Additional histologic findings include extensive calcium pyrophosphate dihydrate deposition in two cases and features resembling tenosynovial giant cell tumor (TGCT). Overall, while the tumors from our series show significant morphologic overlap with chondroblastoma-like soft tissue chondroma, we describe findings that expand the morphologic spectrum of these neoplasms and therefore refer to them as "calcified chondroid mesenchymal neoplasms." These neoplasms represent a spectrum of chondroid/cartilage matrix-forming tumors harboring FN1-receptor TK fusions that include those classified as soft tissue chondroma as well as chondroid TGCT.

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in ROS1 Fusion-Positive Lung Cancer.

Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib. Biopsies from patients with ROS1 + NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing. From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%). ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

NTRK fusion‐positive colorectal cancer in Japanese population

ALK, ROS1 and NTRK fusions are involved in the tumorigenesis of various organs, including colorectal cancer. This study aims to clarify the prevalence of these fusions in colorectal cancer in the Japanese population. Immunohistochemical analysis of 1012 specimens of colorectal cancer revealed two NTRK-positive cases (0.2%) whereas no ALK- or ROS1-positive cases were identified. Reverse transcription polymerase chain reaction (RT-PCR) detected an LMNA-NTRK1 fusion in a case of adenosquamous carcinoma and a TPM3-NTRK1 fusion in a case of tubular adenocarcinoma. Both NTRK1 fusion-positive cases lacked activating mutations in KRAS and BRAF and were mismatch repair-deficient with loss of MLH1 and PMS2 expression and MLH1 promoter methylation. Our results show that receptor tyrosine kinase fusions are rare but present in colorectal cancers in Japanese patients, with a prevalence similar to that reported in other countries.

Molecular Characterization and Therapeutic Targeting of Colorectal Cancers Harboring Receptor Tyrosine Kinase Fusions.

Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer. We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Myeloid/lymphoid neoplasms with eosinophilia: clinical picture and therapeutic approaches

Myeloid/lymphoid neoplasms with eosinophilia (M/Leo) and tyrosine kinase (TK) fusion genes constitute a separate category within the 2016 World Health Organization (WHO) classification. All these are characterized by blood or tissue eosinophilia and the presence of a unique genetic abnormality. M/Leo may have diverse clinical manifestations with variable response to TK inhibitors (TKI). PDGFRA -rearranged neoplasms (usually with detectable FIP1L1-PDGFRA ) are found to be extremely sensitive to low dose of imatinib (IM at 100 mg daily) with nearly 100% hematological complete response rate. Moreover, >90% of IM treated patients may achieve long-term molecular response. IM discontinuation may result in sustained remission in c.50–60% of patients. An excellent response to IM (but at 400 mg/day) was also demonstrated for patients with PDGFRB rearrangements, but trials on IM cessation were not attempted. The FGFR1 -rearranged neoplasms are associated with an aggressive disease course and allogeneic stem cell transplantation (allo-SCT) is the only potentially curative approach. Participation in clinical trials should be recommended. Recently, pemigatinib was found to be effective in a proportion of FGFR1 -rearranged individuals. An aggressive outcome with rapid blast transformation is also characteristic for the JAK2 -rearranged neoplasms. These patients should be included in clinical trials or attempted with ruxolitinib or fedratinib as a ‘bridge’ to allo-SCT. A new category of neoplasms with eosinophilia and FLT3 and ABL1 rearrangements has not yet been incorporated into the WHO 2016 classification. The prognosis is poor with a tendency to evolve into resistant acute leukemia. The treatment includes TKI with known activity against FLT3/ABL1 followed by allo-SCT.

Assessment of the leukemogenic mechanism of Ph-like acute lymphocytic leukemia for the identification of a novel therapeutic target

Ph-like acute lymphocytic leukemia (ALL) is a subtype of Ph-negative B precursor ALL, and its gene expression profile is similar to that of Ph+ALL. In recent decades, comprehensive genomic analyses have revealed that Ph-like ALL has two types. The first type is associated with the ABL-class tyrosine kinase fusion gene, and the second type with fusion genes involving cytokine receptors or molecules, including CRLF2, which are correlated with the activation of the JAK/STAT pathway. Based on these findings, tyrosine kinase or JAK inhibitors were found to be effective for Ph-like ALL. Genetic abnormalities identified in Ph-like ALL, except for CRLF2 rearrangement, are quite rare. Thus, functional studies regarding each genomic abnormality are relevant for establishing targeted therapies for Ph-like ALL. To develop a targeted molecular therapy, a functional study of NCOR1-LYN, which is a novel ABL-class fusion gene, was conducted on pediatric patients with Ph-like ALL.

HGG-43. CONGENITAL GLIOBLASTOMA MULTIFORME: A CASE REPORT OF A RARE PEDIATRIC BRAIN TUMOR, MOLECULAR ANALYSIS, AND REVIEW OF THE LITERATURE

Congenital brain tumors are rare, accounting for less than 4% of all pediatric brain tumors. Congenital glioblastoma multiforme (GBM) is rarer still, accounting for 3–15% of congenital brain tumors. There is literature to suggest that these tumors differ from pediatric and adult GBM clinically and molecularly, and as such should be treated as their own distinct entity. Our case is a 4 week old male who initially presented to his pediatrician for enlarging head circumference and upward gaze palsy. An MRI was obtained revealing a right parietal mass. He underwent gross total resection the following day with pathology revealing glioblastoma, WHO grade IV. Further analysis revealed ATRX retained, p53 immunoreactivity in 15–20% of nuclei, IDH1 and IDH2 wildtype, MGMT promoter not methylated, H3K27M wildtype, no 1p and/or 19q deletion/codeletion. Interestingly, RNA analysis of his tumor detected the PPP1CB-ALK fusion transcript as well as amplification of the ALK gene. Co-occurrence of these mutations has been reported in a small number of pediatric glioblastoma patients and PPP1CB-ALK fusions are one of the most common receptor tyrosine kinase fusions in infantile gliomas. ALK rearrangements and amplifications suggest a potential therapeutic target with tyrosine kinase inhibitors in glioblastoma. This patient serves as an example of a rare congenital glioblastoma with unique molecular features that may suggest novel treatment opportunities. We present his clinical course along with a pertinent review of the literature.