Purpose: The European Organization for Research and Treatment of Cancer (EORTC) clinical phase II trial 90101 “CREATE” showed high antitumor activity of crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK)/ROS1, in patients with advanced inflammatory myofibroblastic tumor (IMFT). However, recent findings suggested that other molecular targets in addition to ALK/ROS1 might also contribute to the sensitivity of this kinase inhibitor. We therefore performed an in-depth molecular characterization of archival IMFT tissue, collected from patients enrolled in this trial, with the aim to identify other molecular alterations that could play a role in the response to crizotinib. Experimental Design: Twenty-four archival IMFT samples were used for histopathological assessment and DNA/RNA evaluation to identify gene fusions, copy-number alterations (CNA), and mutations in the tumor tissue. Results were correlated with clinical parameters to assess a potential association between molecular findings and clinical outcomes. Results: We found 12 ALK fusions with 11 different partners in ALK-positive IMFT cases by Archer analysis whereas we did not identify any ROS1-rearranged tumor. One ALK-negative patient responding to crizotinib was found to have an ETV6–NTRK fusion in the tumor specimen. The CNA profile and mutational landscape of IMFT revealed extensive molecular heterogeneity. Loss of chromosome 19 (25% of cases) and PIK3CA mutations (9% of cases) were associated with shorter progression-free survival in patients receiving crizotinib. Conclusions: We identified multiple genetic alterations in archival IMFT material and provide further insight into the molecular profile of this ultra-rare, heterogeneous malignancy, which may potentially translate into novel treatment approaches for this orphan disease.

Purpose: European Organisation for Research and Treatment of Cancer (EORTC) 90101 (CREATE) was a prospective, multicentric, non-randomised, open-label phase II basket trial to assess the efficacy and safety of crizotinib in patients with different types of cancers, including advanced inflammatory myofibroblastic tumour (IMT) with or without anaplastic lymphoma kinase (ALK) rearrangements. Here, we report updated results with long-term follow-up. Patients/methods: After central reference pathology, eligible ALK-positive and ALK-negative patients with advanced/metastatic IMT deemed incurable with surgery, radiotherapy or systemic therapy received oral crizotinib 250 mg twice daily. The ALK status was assessed centrally using immunohistochemistry and fluorescence in situ hybridisation. The primary endpoint was the proportion of patients who achieved an objective response (i.e. complete or partial response). If 6 ALK-positive patients achieved a confirmed response, the trial would be deemed successful. Results: At data cut-off on 28th January 2021, we performed the final analysis of this trial. Of the 20 eligible and treated patients (19 of whom were evaluable for efficacy), with a median follow-up of 50 months, five were still on crizotinib treatment (4/12 ALK-positive and 1/8 ALK-negative patients). The updated objective response rate (ORR) was 66.7% (95% confidence interval [CI] 34.9e90.1%) in ALK-positive patients and 14.3% (95% CI 0.0e57.9%) in ALK-negative patients. In the ALK-positive and ALK-negative patients, the median progression-free survival was 18.0 months (95% CI 4.0eNE) and 14.3 months (95% CI 1.2 e31.1), respectively; 3-year overall survival rates were 83.3% (95% CI 48.2e95.6) and 34.3% (95% CI 4.8e68.5). Safety results were consistent with previously reported data. Conclusion: These updated results confirm previous findings that crizotinib is effective, with durable responses, in patients with locally advanced or metastatic ALK-positive IMT. With further follow-up after the original primary analysis, the ORR increased, as patients derived long-term benefit and some responses converted from stable disease to partial responses. Clinical trial number: EORTC 90101, NCT01524926.

Introduction: There has been an increased demand for mismatch repair (MMR) status testing in sarcoma patients after the success of immune checkpoint inhibition (ICI) in MMR deficient tumors. However, data on MMR deficiency in bone and soft tissue tumors is sparse, rendering it unclear if routine screening should be applied. Hence, we aimed to study the frequency of MMR deficiency in bone and soft tissue tumors after we were prompted by two (potential) Lynch syndrome patients developing sarcomas. Methods: Immunohistochemical expression of MLH1, PMS2, MSH2 and MSH6 was assessed on tissue micro arrays (TMAs), and included 353 bone and 539 soft tissue tumors. Molecular data was either retrieved from reports or microsatellite instability (MSI) analysis was performed. In MLH1 negative cases, additional MLH1 promoter hypermethylation analysis followed. Furthermore, a systematic literature review on MMR deficiency in bone and soft tissue tumors was conducted. Results: Eight MMR deficient tumors were identified (1%), which included four leiomyosarcoma, two rhabdomyosarcoma, one malignant peripheral nerve sheath tumor and one radiation-associated sarcoma. Three patients were suspected for Lynch syndrome. Literature review revealed 30 MMR deficient sarcomas, of which 33% were undifferentiated/unclassifiable sarcomas. 57% of the patients were genetically predisposed. Conclusion: MMR deficiency is rare in bone and soft tissue tumors. Screening focusing on tumors with myogenic differentiation, undifferentiated/unclassifiable sarcomas and in patients with a genetic predisposition / co-occurrence of other malignancies can be helpful in identifying patients potentially eligible for ICI.

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Clear cell sarcoma (CCSA) is characterized by a chromosomal translocation leading to EWSR1 rearrangement, resulting in aberrant transcription of multiple genes, including MET. The EORTC 90101 phase II trial evaluated the MET inhibitor crizotinib in CCSA but resulted in only sporadic responses. We performed an in-depth histopathological and molecular analysis of archival CCSA samples to identify alterations potentially relevant for the treatment outcome. Immunohistochemical characterization of MET signaling was performed using a tissue microarray constructed from 32 CCSA cases. The DNA from 24 available tumor specimens was analyzed by low-coverage whole-genome sequencing and whole-exome sequencing for the detection of recurrent copy number alterations (CNAs) and mutations. A pathway enrichment analysis was performed to identify the pathways relevant for CCSA tumorigenesis. Kaplan–Meier estimates and Fisher’s exact test were used to correlate the molecular findings with the clinical features related to crizotinib treatment, aiming to assess a potential association with the outcomes. The histopathological analysis showed the absence of a MET ligand and MET activation, with the presence of MET itself in most of cases. However, the expression/activation of MET downstream molecules was frequently observed, suggesting the role of other receptors in CCSA signal transduction. Using sequencing, we detected a number of CNAs at the chromosomal arm and region levels. The most common alteration was a gain of 8q24.21, observed in 83% of the cases. The loss of chromosomes 9q and 12q24 was associated with shorter survival. Based on exome sequencing, 40 cancer-associated genes were found to be mutated in more than one sample, with SRGAP3 and KMT2D as the most common alterations (each in four cases). The mutated genes encoded proteins were mainly involved in receptor tyrosine kinase signaling, polymerase-II transcription, DNA damage repair, SUMOylation and chromatin organization. Disruption in chromatin organization was correlated with longer progression-free survival in patients receiving crizotinib. Conclusions: The infrequent activation of MET may explain the lack of response to crizotinib observed in the majority of cases in the clinical trial. Our work describes the molecular heterogeneity in CCSA and provides further insight into the biology of this ultra-rare malignancy, which may potentially lead to better therapeutic approaches for CCSA.