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We report on a case of EWSR1‐PATZ1 rearranged brain tumor occurring in a 17 month‐old child, originally interpreted as an infantile glioblastoma. Our case shows important analogies with the 2 previously reported cases, including the intraventricular location, the histologic appearance (pushing borders, oligodendrocyte‐like morphology, rich vascular network) and the glioneural immunophenotype, supporting the role of these features as relevant clues to the diagnosis. On the other hand, our case displays unique characteristics, i.e. the onset in an infant, the presence of a focal high‐grade component and the leptomeningeal dissemination, pointing to the importance of considering this entity in the differential diagnosis of an infantile glial/glioneural tumor.

Neurotrophic tyrosine receptor kinase (NTRK) fusions are infrequent genetic events that can occur in various tumor types. Specifically, NTRK-rearranged sarcoma has been observed in pediatric mesenchymal tumors and, to a lesser extent, in adult mesenchymal tumors like fibrosarcoma. Recently, NTRK-rearranged uterine sarcoma (US) has been identified as a rare entity characterized by constitutive activation or overexpression of the TRK receptor, which plays a role in cell proliferation and differentiation. Since its initial description in 2018, only 46 cases of NTRK-rearranged US have been reported. In this context, herein we describe an exceptional case of an STRN3::NTRK3 fused US with histologically confirmed splenic metastasis. Notably, such localization has not been previously associated with pure uterine sarcomas in the literature. The fusion involved STRN3 (exon-3) and NTRK3 (exon-14) genes and was identified through next-generation sequencing analysis. Recognizing this specific molecular rearrangement is crucial, as it not only enables targeted therapy but also holds diagnostic significance in specific clinical scenarios.

Background: Molecular confirmation of histologic diagnosis has become mandatory for the diagnosis of Ewing sarcoma family of tumors (ESFT). Aim: To validate the diagnosis made by morphology and immunohistochemistry (IHC) by fluorescence in-situ hybridization (FISH) for EWSR1 rearrangement on formalin fixed paraffin embedded (FFPE) tissues. Settings and design: A retrospective and prospective observational study. Material and methods: All patients who had FISH studies for EWSR1 rearrangement for small round cell tumors during 10 years period were included. Demographic, clinical and radiological details were obtained from medical records. Morphology was reviewed with IHC by CD99, FLI1 and others. FISH studies were performed using the break apart probe. Additional molecular studies and IHC were done to resolve the diagnosis in EWSR1 rearranged tumors. Final diagnosis was made by integrating clinical, morphology, IHC and molecular features. Results: There were 81 patients (M: F 45:36, median age 21 years) with 32 skeletal and 49 extra skeletal tumors. CD 99 was positive in 94.52%. FISH for EWSR1 were positive in 59, negative in 13 and failed in 9. The final diagnosis was made as ESFT in 67, angiomatoid fibrous histiocytoma in 3, desmoplastic small round cell tumor in 3, myxoid chondrosarcoma in 2, unclassified in one, synovial sarcoma in 3, and one each of lymphoma and small cell neuroendocrine carcinoma. FISH was positive for ESFT in 89.83% of EWSR1 rearranged tumors. FISH validated the diagnosis made on IHC in 79.10%. FISH resolved the diagnosis in 1.49% CD99 negative tumors. Conclusion: FISH is a reliable ancillary technique for the diagnosis of ESFT on FFPE tissues.

The section on mesenchymal tumors in the 5th edition of WHO classification of skin tumors has undergone several changes, the most important of which is the inclusion of newly identified tumor entities, which will be the main focus of this review article. These specifically include three novel cutaneous mesenchymal tumors with melanocytic differentiation, and rearrangements of the CRTC1::TRIM11, ACTIN::MITF, and MITF::CREM genes as well as EWSR1::SMAD3-rearranged fibroblastic tumors, superficial CD34-positive fibroblastic tumors, and NTRK-rearranged spindle cell neoplasms. Some of the other most important changes will be briefly mentioned as well.

Abstract BCOR is an epigenetic regulator and is genetically altered by mutation, deletion, or gene fusion in a range of cancers. “Central nervous system high-grade neuroepithelial tumor with BCOR alteration” is a recently described entity with characteristic internal tandem duplications within exon 15 of the BCOR gene (hereafter: CNS HGNET-BCOR ex15 ITD). In this case series of 3 patients, we report the clinicopathologic, molecular, and methylome features of gliomas with novel EP300-BCOR in-frame gene fusions, thus expanding the spectrum of BCOR alterations seen in CNS tumors. The gliomas in this series arise in children (ages 10–18), involve the supratentorial compartment, and have an infiltrative pattern of growth and a myxoid/microcystic background with frequent psammomatous calcifications and prominent chicken-wire vessels. All 3 cases had areas with low-grade morphology and 2 of them demonstrated histologic high-grade transformation. In contrast to CNS HGNET-BCOR ex15 ITD, they lack perivascular pseudorosettes. On a t-Distributed Stochastic Neighbor Embedding plot they cluster perfectly together, away from CNS HGNET-BCOR ex15ITD, consistent with a different entity. Gliomas with EP300-BCOR fusions and high-grade histology can demonstrate relatively rapid regrowth after debulking or subtotal resection.

Background Patients with ALK‐rearranged non‐small cell lung cancer (ALK+ NSCLC) inevitably acquire resistance to ALK inhibitors. Longitudinal monitoring of cell‐free plasma DNA (cfDNA) next‐generation sequencing (NGS) could predict the response and resistance to tyrosine kinase inhibitor (TKI) therapy in ALK+ NSCLC. Methods Patients with ALK+ NSCLC determined by standard tissue testing and planned to undergo TKI therapy were prospectively recruited. Plasma was collected at pretreatment, 2 months‐post therapy, and at progression for cfDNA‐NGS analysis, Guardant 360. Results Among 92 patients enrolled, circulating tumor DNA (ctDNA) was detected in 69 baseline samples (75%): 43 ALK fusions (62.3%) and two ALK mutations without fusion (2.8%). Two patients showed ALK‐resistance mutations after ceritinib; G1202R, and co‐occurring G1202R and T1151R. Eight patients developed ALK resistance mutations after crizotinib therapy; L1196M (n = 5), G1269A (n = 1), G1202R (n = 1), and co‐occurring F1174L, G1202R, and G1269A (n = 1). Absence of ctDNA at baseline was significantly associated with longer progression‐free survival (PFS; median 36.1 vs. 11.4 months, p = 0.0049) and overall survival (OS; not reached vs. 29.3 months, p = 0.0200). ctDNA clearance at 2 months (n = 29) was associated with significantly longer PFS (25.4 vs. 11.6 months, p = 0.0012) and OS (not reached vs. 26.1 months, p = 0.0307) than those without clearance (n = 22). Patients with co‐occurring TP53 alterations and ALK fusions at baseline (n = 16) showed significantly shorter PFS (7.28 vs. 13.0 months, p = 0.0307) than those without TP53 alterations (n = 25). Conclusions cfDNA‐NGS facilitates detection of ALK fusions and resistance mutations, assessment of prognosis, and monitoring dynamic changes of genomic alterations in ALK+ NSCLC treated with ALK‐TKI. To investigate whether longitudinal monitoring of cfDNA‐NGS could predict the response and resistance of TKI therapy in ALK+ NSCLC, we recruited prospectively 92 patients with ALK+ advanced NSCLC determined by standard tissue testing and planned for TKI therapy. NGS of cfDNA is useful not only for the detection of ALK fusions and resistance mutations, but also for assessing prognosis and monitoring the dynamic changes of genomic alterations in ALK+ NSCLC treated with ALK‐TKI.

CIC-rearranged sarcoma is an aggressive round cell sarcoma, and an alternative ATXN1/ATXN1L fusion has been reported. Diagnosis may be difficult, and molecular assays may suffer from imperfect sensitivity. Characteristic histology and ETV4 immunohistochemical positivity are diagnostically helpful. However, ETV4 staining is unavailable in most laboratories. Here, we explored the diagnostic utility of MUC5AC immunohistochemistry in CIC-rearranged sarcomas. All 30 cases, except one, of CIC-rearranged sarcomas and 2 ATXN1-rearranged sarcomas were positive for MUC5AC, although the number of immunopositive cells was generally low (< 5%) in most samples, representing a characteristic scattered pattern. The only MUC5AC-negative case had the lowest tumor volume. Among the 110 mimicking round cell malignancies, 12 tumors showed MUC5AC positivity, including occasional cases of synovial sarcoma and small cell carcinoma, whereas the remaining 98 samples were negative. Despite its lower specificity than that of ETV4 and sparse reactivity that requires careful interpretation, MUC5AC may serve as a useful marker for CIC/ATXN1-rearranged sarcoma because of its wider accessibility.

Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non‐small‐cell lung cancer (NSCLC) patients with ALK‐rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK‐independent. We generated tumor cell cultures from multiple regions of an ALK‐rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK‐inhibitor response. This identified a role for PI3Kβ and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K‐AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kβ. In ALK‐rearranged primary cultures, the combined inhibition of ALK and PI3Kβ prevented the EGFR‐mediated ALK‐inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial‐to‐mesenchymal transformed cells. In conclusion, combinatorial ALK‐ and PI3Kβ‐inhibitor treatment carries promise as a treatment for ALK‐rearranged NSCLC. PI3Kβ inhibitors enhance the response to ALK inhibitors in primary ALK‐rearranged lung cancer cells. PI3Kβ inhibition blocks ALK‐inhibition‐induced cytoprotective reactivation of EGFR signaling, enhancing the cytotoxic effect on both epithelial‐ and mesenchymal‐phenotype cancer cells without affecting normal lung epithelial cells. Combined inhibition of both ALK and PI3Kβ therefore represents a promising approach to improve clinical responses in ALK‐rearranged lung cancers.

Leptomeningeal metastases (LM) from solid tumors represent an unmet need of increasing importance due to an early use of MRI for diagnosis and improvement of outcome of some molecular subgroups following targeted agents and immunotherapy. In this review, we first discussed factors limiting the efficacy of targeted agents in LM, such as the molecular divergence between primary tumors and CNS lesions and CNS barriers at the level of the normal brain, brain tumors and CSF. Further, we reviewed pathogenesis and experimental models and modalities, such as MRI (with RANO and ESO/ESMO criteria), CSF cytology and liquid biopsy, to improve diagnosis and monitoring following therapy. Efficacy and limitations of targeted therapies for LM from EGFR-mutant and ALK-rearranged NSCLC, HER2-positive breast cancer and BRAF-mutated melanomas are reported, including the use of intrathecal administration or modification of traditional cytotoxic compounds. The efficacy of checkpoint inhibitors in LM from non-druggable tumors, in particular triple-negative breast cancer, is discussed. Last, we focused on some recent techniques to improve drug delivery.

Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c‐Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL‐AF9‐induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1‐knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony‐forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL‐AF9‐induced AML via maintaining the pool of LSC. In this study, we found that Six1 is a key transcription factor that is differentially expressed in normal hematopoiesis and leukemogenesis and regulates LSC pools by modulating the expression of glycolytic genes.