ALK Overexpression Research Articles

Abstract 7504: Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase positive non-small cell lung cancer (ALK+NSCLC) represents a distinct subtype of NSCLC characterized by oncogenic ALK gene mutations leading to ALK overexpression and activation in tumor cells. ALK+ NSCLC will frequently develop mutations during treatment, leading to nonresponse and eventually cancer relapse. Therefore, there is a critical need for sensitive technologies to monitor ALK+ NSCLC disease status in patients over time. To facilitate longitudinal monitoring, there has been a rapid evolution in diagnostic technologies leveraging circulating tumor cells (CTCs) as a liquid biopsy tool. CTCs can be collected non-invasively at various intervals throughout patient treatment. Moreover, they shed from solid tumors and disseminate into the bloodstream, which makes isolating CTCs a valuable source of genomic, transcriptomic, and proteomic insights pertaining to the primary tumor. In this study, we present a comprehensive workflow for conducting multi-omic analysis on CTCs derived from ALK+ NSCLC patients. Methods: Blood samples were collected from a cohort of ALK+ NSCLC patients, and CTCs were isolated using a high throughput, label free microfluidic device, Labyrinth. Enriched CTCs were immunofluorescently stained for cytokeratin, CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers. Heterogeneous CTC populations with hybrid epithelial/mesenchymal (EM) phenotypes are reported. Fluorescence in situ hybridization (FISH) was performed on enriched CTC samples. DNA was extracted and sequenced using a 523 gene targeted sequencing panel. Single cell RNA-seq technology was performed in parallel on CTC samples to investigate the gene expression throughout treatment. Results: The median concentration of CTCs in the ALK+ NSCLC cohort (n=33) is 137 CTCs per ml of blood, and 64% of CTCs are in an EM hybrid state. In ALK+ NSCLC patients (n=3) with an average of 4.6 visits per sample, we observed a trend of decreasing CTC concentration for patients with partial response to the treatment. The presence of ALK fusion in ALK+ NSCLC patient’s CTC is confirmed by FISH. Oncogenic mutations (JAK1, SPEN, ERBB4, NTRK3) are observed in bulk CTC samples from two NSCLC patients. Single cell RNA sequencing was performed on three ALK+ NSCLC patients; 58, 199, and 700 potential CTCs were detected with positive cytokeratin expression and no white blood cell marker expression. Conclusions: This study examined the feasibility of using CTCs for studying and monitoring the clinical status of the ALK+ NSCLC patients as well as the genomic and transcriptomic signatures within their tumors. More patients will need to be processed in order to validate our workflow and reinforce our conclusions. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Bryce Vandenburg, Varun Kathawate, Larua Goo, Peter Ulintz, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7504.

Prognostic value of ALK overexpression and molecular abnormalities in high-grade serous ovarian carcinoma.

ALK receptor tyrosine kinase (ALK) aberrations have an established role in pathogenesis of many neoplasms, but their clinical significance in high grade serous ovarian carcinoma (HGSOC) is unclear. To analyse the frequency of ALK overexpression, molecular abnormalities of ALK, and their impact on the progression-free survival (PFS) and overall survival (OS) in HGSOC. Protein expression was examined by immunohistochemistry (IHC) using three different clones of anti-ALK antibody. The presence of translocations was analysed using fluorescent in situ hybridization. Next-generation sequencing was used for studying the copy number variation, as well as point mutation and translocations involving other commonly rearranged genes. ALK overexpression was demonstrated in up to 52% of tumours, whereas ALK copy gains in 8.2%, with no clear impact on survival. ALK point mutations were identified in 13 tumours (8.9%), with 3 belonging to the class IV showing significantly better OS. A trend suggesting better PFS was also noticed in these cases. Additionally, three gene fusions were found: ERBB2-GRB7, PRKCA-BRCA1 and SND1-BRAF, none of which has been previously described in HGSOC. HGSOC harbouring activating ALK mutations might be associated with a better survival, while ALK overexpression and ALK amplification does not impact the prognosis.

Novel NONO::TFE3 fusion and ALK co-expression identified in a subset of cutaneous microcystic/reticular schwannoma.

Microcystic/reticular schwannoma (MRS) is a benign variant of schwannoma with a predilection for the gastrointestinal tract and skin. To date, genetic characterization of this tumor is limited. Prompted by the identification of TFE3::NONO fusion and ALK overexpression in an index case of MRS, a cohort of tumors was collected from institutional and consultation archives of two institutions. Next-generation sequencing (NGS), TFE3 fluorescence in situ hybridization (FISH), and TFE3 and ALK immunohistochemistry were performed, while clinicopathologic variables were documented. Eighteen MRS cases were identified (35 to 85 years) arising in the skin (n=8), gastrointestinal tract (n=5), adrenal gland (n=3), abdominal wall (n=1), and unknown site (n=1). Tumors showed a circumscribed to multinodular to plexiform low-power architecture with variable amounts of microcystic/reticular and solid schwannian components. Mitotic figures were scarce (0-1/10 HPFs), and atypia was absent. S100 protein and/or SOX10 immunoreactivity was noted in the microcystic/reticular and schwannian areas of all cases. NGS performed on two cutaneous tumors yielded NONO exon 12 fusion with TFE3 exon 4, and these lesions also showed HMB45 and ALK expression. Two additional cases showed ALK expression (1 weak), while a third was positive for TFE3, but these cases failed to show ALK or TFE3 rearrangement by FISH/NGS. There were no morphologic variables that correlated with the presence of NONO::TFE3. We identified a subset of microcystic/reticular schwannomas with NONO::TFE3 fusions and ALK co-expression, adding to the cohort of mesenchymal neoplasms that show ALK overexpression without rearrangement of the ALK gene.

Spectrum of kinase gene rearrangements in a large series of paediatric inflammatory myofibroblastic tumours.

Inflammatory myofibroblastic tumours (IMTs), being an exceptionally rare category of paediatric neoplasms, often contain druggable gene rearrangements involving tyrosine kinases. This study presents a large consecutive series of IMTs which were analysed for the presence of translocations by the PCR test for 5'/3'-end ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 unbalanced expression, variant-specific PCR for 47 common gene fusions and NGS TruSight RNA fusion panel. Kinase gene rearrangements were detected in 71 of 82 (87%) IMTs (ALK: n = 47; ROS1: n = 20; NTRK3: n = 3; PDGFRb: n = 1). The test for unbalanced expression had 100% reliability in identifying tumours with ALK fusions, but failed to reveal ROS1 rearrangements in eight of 20 (40%) ROS1-driven IMTs; however, ROS1 alterations were detectable by variant-specific PCR in 19 of 20 (95%) cases. ALK rearrangements were particularly common in patients below 1 year of age (10 of 11 (91%) versus 37 of 71 (52%), P = 0.039). ROS1 fusions occurred more often in lung IMTs than in tumours of other organs (14 of 35 (40%) versus six of 47 (13%), P = 0.007). Among 11 IMTs with no kinase gene rearrangement identified, one tumour demonstrated ALK activationvia gene amplification and overexpression, and another neoplasm carried COL1A1::USP6 translocation. PCR-based pipeline provides a highly efficient and non-expensive alternative for molecular testing of IMTs. IMTs with no detectable rearrangements need further studies.

A Rare Case of Anaplastic Lymphoma Kinase-Positive Large B-Cell Lymphoma Involving Epidural Tissue

Abstract Introduction/Objective Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK-positive LBCL) is a rare, aggressive type of non-Hodgkin’s B-cell lymphoma with characteristic rearrangements and overexpression of ALK. Diagnosis of ALK-positive LBCL is challenging because of its rarity and uncharacteristically low expression of B- lineage antigens usually needed for diagnosis of B-cell lymphomas. In this report, we describe a case of ALK-positive large B cell lymphoma involving epidural soft tissue in a young adult. Methods/Case Report 29 year old presented to emergency department with a two week history of back pain and a three day history of worsening numbness and weakness in the lower extremities. MRI of the thoracic spine showed an epidural soft tissue mass posterior to T5, T6 and T7 vertebral bodies with abnormal signal in the T6 spinous process. Histological examination from the epidural mass revealed sheets of atypical lymphoid cells with abundant pale cytoplasm and round vesicular nuclei with prominent single to occasional multiple nucleoli. Immunohistochemical stains demonstrated neoplastic cell positivity for ALK, VS38c, CD138, EMA, MUM1, CD45, PAX5 and MCK. They were negative for CD3, CD20, CD19, CD79a, CD30, CD2, CD117, SOX10, HMB45, cytokeratin, synaptophysin and chromogranin. These overall findings were consistent with ALK-positive large B-cell lymphoma. Results (if a Case Study enter NA) NA. Conclusion ALK-positive large B-cell lymphoma is a non-Hodgkin lymphoma associated with high morbidity and mortality. Absence of reliable B-cell marker expression in this B-cell lymphoma requires recognition of morphological findings to order confirmatory testing such as immunohistochemical stains for CD138 and ALK. This particular case also showed an unusual anatomic presentation in epidural tissue.

Novel neoplasms associated with syndromic pediatric medulloblastoma: integrated pathway delineation for personalized therapy

Medulloblastoma is the most common pediatric embryonal brain tumor, and may occur in cancer predisposition syndromes. We describe novel associations of medulloblastoma with atypical prolactinoma and dural high-grade sarcoma in Li-Fraumeni syndrome (LFS), and epidural desmoid fibromatosis in familial adenomatous polyposis (FAP)/Turcot syndrome. Genomic analysis showing XRCC3 alterations suggested radiotherapy as contributing factor to the progression of LFS-associated medulloblastoma, and demonstrated different mechanisms of APC inactivation in the FAP-associated tumors. The integrated genomic-transcriptomic analysis uncovered the growth pathways driving tumorigenesis, including the prolactin-prolactin receptor (PRLR) autocrine loop and Shh pathway in the LFS-associated prolactinoma and medulloblastoma, respectively, the Wnt pathway in both FAP-associated neoplasms, and the TGFβ and Hippo pathways in the soft tissue tumors, regardless of germline predisposition. In addition, the comparative analysis of paired syndromic neoplasms revealed several growth pathways susceptible to therapeutic intervention by PARP, PRLR, and selective receptor tyrosine kinase (RTK) inhibitors. These could target the defective DNA damage repair in the LFS-associated medulloblastoma, the prolactin autocrine loop in the atypical prolactinoma, the EPHA3/7 and ALK overexpression in the FAP-associated medulloblastoma, and the multi-RTK upregulation in the soft tissue neoplasms. This study presents the spatiotemporal evolution of novel neoplastic associations in syndromic medulloblastoma, and discusses the post-radiotherapy risk for secondary malignancies in syndromic pediatric patients, with important implications for the biology, diagnosis, and therapy of these tumors.-9uTLpjCeHFTYhThd81HRBVideo

821 ALK expression in metastatic cutaneous squamous cell carcinoma: A pilot study

Background: Cutaneous squamous cell carcinoma (SCC) is a common skin cancer, with over 1,000,000 cases and up to 9,000 deaths annually in the United States. Treatment options for metastatic SCC are limited, although inhibitors of PD-1 and EGFR have shown some success clinically. Our group identified ALK as a potential driver mutation in metastatic SCC through targeted sequencing. Here, we examine the role of ALK overexpression in SCC. Methods: All metastatic SCCs at our institution from 2010-2018 with tissue available were included, which yielded fifteen primary SCCs and fifteen patient-matched lymph node metastases.

Primary Intraosseous Rhabdomyosarcoma: Rare Subtype Involving Mandible with Unique Translocation

<h3>Introduction</h3> A rare subtype of spindled and epithelioid rhabdomyosarcoma (RMS) with unique gene rearrange- ments (FUS-TFCP2 or EWSR1-TFCP2) was first described in 2018 (J Pathol 245:29–40). These aggressive tumors occur in a wide age range with a female predilection and develop almost exclusively within a variety of bone sites, in par- ticular, the craniofacial bones. Majority of affected individuals died of disease within 15 months. FUS-TFCP2 is the predominant translocation associated with this RMS subtype. This case report describes a rare mandibular sarcoma in a child with a complex, oncologic, medical history. <h3>Clinical Presentation and Pathology Findings</h3> 15-year-old female experienced acute lymphoblastic leukemia at 11 months of age, with oncologic resolution. At age 7, anthracycline-induced cardiomyopathy was diagnosed, ne- cessitating heart transplantation at age 9. Subsequently, EBV-associated diffuse large cell lymphoma involving the retropharynx was diagnosed at age 10, with successful oncologic management. At age 15, a left mandibular en- largement was noted. Fine needle core biopsy identified a spindled and epithelioid sarcoma, diffusely positive for nuclear MyoD1 with rare cytoplasmic desmin and rare nuclear myogenin. ALK and S100 immunostaining were positive in a certain tumor cell population. The diagnosis of spindled and epithelioid RMS was made. Conventional cytogenetics demonstrated a complex hyperdiploid tumor (78XXX with numerous derivative aberrations). Next- generation sequencing identified a FUS-TFCP2 translocation, which appears to be tumor-defining with this primary intrabony RMS subtype. Oncologic sarcoma management was performed with a marginal response to chemother- apy and low-dose radiation therapy. Left mandible resection was performed with negative bone and soft tissue margins (3.5cm tumor size). <h3>Conclusion</h3> Primary intraosseous RMS is a rare and aggressive subtype, which has tumor-defining translocations and expresses MyoD1 to a greater extent than Desmin or Myogenin. Of interest, is the overexpression of ALK, which may provide a target for ALK inhibitors.

A novel fusion between CDC42BPB and ALK in a patient with quadruple wild‐type gastrointestinal stromal tumor

BackgroundGastrointestinal stromal tumors (GISTs) are the most common type of mesenchymal tumor in gastrointestinal tract, with striking features of morphology and immunohistochemistry. But GISTs in pregnancy could seldom be found. Pathogenic activating mutations of the proto‐oncogene KIT and PDGFRA are detected in majority GISTs, and adjuvant imatinib therapy targeting KIT and PDGFRA mutations is recommended for patients with high‐risk GIST. However, some rare subgroups with distinct molecular features remain uncovered and more therapeutic targets need to be revealed.MethodsThe DNA/RNA samples were detected using the NGS‐based YuanSu450 gene panel. After identifying the CDC42BPB‐ALK fusion by NGS, this novel fusion was further confirmed by Sanger sequencing. Subsequently, FISH analysis was performed using the Vysis ALK Break Apart FISH Probe kit to testify the ALK status. ALK protein expression was confirmed by IHC (D5F3 and 5A4).ResultsHerein, we reported the first case of quadruple wild‐type (WT) GIST with ALK‐CDC42BPB fusion and ALK (D5F3) overexpression. In this study, we described a 33‐year‐old pregnant patient in lactation who had a massive space occupying lesion (with the maximum diameter of 22 cm) in the stomach and was eventually diagnosed as quadruple WT GIST (KIT WT/PDGFRA WT/SDH WT/RAS‐P WT).ConclusionWe unexpectedly found that this GIST patient showed ALK (D5F3) overexpression and harbored a novel fusion CDC42BPB exon 24‐ALK in exon 20.

Spitz nevus with EHBP1‐ALK fusion and distinctive membranous localization of ALK

ALK rearrangements define a histopathologically distinctive yet diverse subset of Spitz tumors characterized by fusiform to epithelioid melanocytes with frequent fascicular growth and ALK overexpression. Molecularly, these tumors are characterized by fusions between ALK and a variety of gene partners, most commonly TPM3 and DCTN1. We describe an unusual case of a Spitz nevus occurring in a 13-year-old female that manifested ALK immunopositivity with cell membrane localization. The proliferation was polypoid and composed of elongated nests of epithelioid melanocytes with enlarged nuclei, prominent nucleoli, and abundant cytoplasm without significant atypia and lacking mitotic figures. The nevus exhibited strong and diffuse expression of p16. Targeted next-generation RNA sequencing revealed an in-frame EHBP1-ALK fusion, which has been reported only once in the literature. EHBP1 encodes an adaptor protein with plasma membrane targeting potential. Together, these findings suggest that the 5' ALK fusion partner in Spitz tumors may dictate the subcellular localization of the ALK chimeric oncoprotein. In summary, this case highlights a rare ALK fusion associated with a distinct immunohistochemical staining pattern and further expands the spectrum of ALK-rearranged melanocytic tumors.

Morphologic features in a series of 352 Spitz melanocytic proliferations help predict their oncogenic drivers.

Spitz nevi are indolent melanocytic tumors arising preferentially during and after childhood. Over the last decades, recurrent oncogenic drivers, sparsely detected in melanoma, were identified in Spitz melanocytic proliferations. Therefore, the detection of such drivers appears as a relevant diagnostic tool to distinguish both entities. Interestingly, morphologic features might correlate with the oncogenic drivers. Thus, the goal of this study was to assess the performances of previously identified morphological criteria to predict the presence of specific drivers. In total, 352 Spitz melanocytic proliferations either with a genetically identified oncogenic driver or investigated for ALK, ROS1, and NTRK1 overexpression by immunohistochemistry were enrolled in the present study. The microscopic features of the cases were assessed blindly with regards to the molecular status and, performances of previously described morphological criteria to predict the molecular status were assessed applying the likelihood-ratio test (LHR). Overall, an oncogenic driver was identified in 76% of the cases (n = 268/352). No microscopic features allowed the reliable prediction of ROS1- and NTRK1-overexpressing cases. By contrast, a plexiform pattern can contribute to the recognition of ALK-overexpressing cases (LHR(+) = 6.14). Importantly, the pseudo-schwannoma variant was highly suggestive of NTRK3-rearranged cases (LHR(+) = 43). Moreover, atypical/malignant tumor (LHR(+) = 5.18), severe cellular atypia (LHR(+) = 5.07), and p16 loss (LHR(+) = 14) contribute to the recognition of MAP3K8-rearranged cases, while the presence of a sheet-like architecture (LHR(+) = 5.39) and a marked fibrosis of the stroma (LHR(+)=5.06) were predictive of BRAF-fused tumors. To conclude, our study confirms ALK-overexpressing, NTRK3-, MAP3K8-, and BRAF-rearranged cases harbored distinct morphologic features allowing their microscopic recognition.

A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma.

Simple SummaryNeuroblastoma is a tumor of the sympathetic nervous system that substantially contributes to childhood cancer mortality. Neuroblastoma originates from the neural crest cells that are subjected to genetic alterations during embryonic development. These impairments hit key genes, whose expression is activated/repressed by transcription factors and inhibited by negative regulators, named microRNAs, thereby promoting tumorigenesis. Here, we have focused on the interactions between the transcription factors MYCN and PHOX2B with their target genes ALK and LIN28B and the miRNAs let-7, miR-34 and miR-204, which should act as negative regulators of their expression. In neuroblastoma, the physiologic regulatory networks among these genes and microRNAs are disrupted, leading to a complex and aberrant oncogene expression that underlies the development of the tumor. We also looked into the role of these genetic determinants of neuroblastoma starting from their physiological role in neural crest development and ending with their pathogenic dysregulation that leads to neuroblastoma oncogenesis.Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

Anaplastic Large-Cell Lymphomas in Adults: The Cytogenetic and Molecular Genetic Characteristics, Treatment Results (a single institutional experience)

Anaplastic large cell lymphomas in adults is a group of rare diseases that are characterized by aggressive clinical course, generalization of the process, high frequency of extranodal lesions, typical morpho-immunophenotyping peculiarities (diffuse infiltration with specific anaplastic giant cells and high expression of the activating CD30 antigen). The prognosis varies greatly depending on the genetic profile of the disease (the presence of ALK, DUSP22, TP-63 genetic rearrangements). In case of presence of ALK and DUSP22 rearrangements, the prognosis is favorable (overall survival [OS] is 70-90%); triple-negative profile is characterized by intermediate prognosis (OS 40-50%), while TP63 gene rearrangements are associated with extremely poor prognosis (OS 8-17%).The goal of the study was to evaluate cytogenetic characteristics and the results of high-dose chemotherapy in patients with anaplastic large cell lymphoma.The results: 91 patients who were followed up at one center were enrolled in the study over the period 2000 to 2017. The majority of patients had advanced stages of the disease: 72 patients (79%; including ALK+ ALGL in 71% of cases and ALK- ALGL in 90% of cases) had stage III-IV disease and were in the intermediate-high and high-risk groups according to the International Prognostic Index (IPI) (60% of patients).The testing results were as follows: ALK gene rearrangements and, correspondingly, ALK overexpression were revealed in 52 patients (57%; median age 33 years). 39 patients (43%; median age 51 years) did not demonstrate any ALK-related cytogenetic aberrations. The results of FISH assay of ALK-negative ALGL were as follows: DUSP22, TP63 rearrangements and triple-negative profile were observed in 20%, 13% and 67% of cases, respectively.The majority of patients in the general group received high-dose chemotherapy - 42 patients with ALK+ ALGL, 27 patients with ALK- ALGL; the rest of the patients received CHOP/CHOEP therapy courses.ALK, DUSP22, TP63 rearrangements and triple-negative profile were associated with 83% and 79%, 100% and 100%, 8% and 10%, 60% and 39% overall survival (OS) and event-free survival (EFS) rates, respectively. The analysis of treatment failures (relapse/progression) in ALK+ ALGL group showed that detection of minimal residual disease in the bone marrow and peripheral blood specimens after 2 courses of chemotherapy was an important factor (real-time PCR demonstrated different ALK-based chimeric translocation products, including NPM-ALK, ATIC-ALK, TPM3-ALK).Thus, the results of high-dose chemotherapy in patients with anaplastic large-cell lymphoma in adults (namely overall survival and event-free survival rates) demonstrate statistically significant differences depending on the cytogenetic profile of the disease. The least favorable profiles are TP63 rearrangements in patients with ALK- ALGL, as well as minimal residual disease in ALK+ ALGL, which allows to optimize induction chemotherapy regimens DisclosuresNo relevant conflicts of interest to declare.

Molecular profiling and targeted agents in recurrent, metastatic salivary gland tumor (R/M SGT) patients (pts) treated at two academic centers.

6081 Background: Treatment selection based on actionable alterations (AAs) is an appealing strategy for pts with R/M SGT. The GEMS-001 study (NCT02069730) at Princess Margaret Cancer Centre (PM) and the Vall D´Hebron Institute of Oncology (VHIO) pre-screening program facilitate the identification of AAs for R/M SGT pts and treatment selection. Methods: We analyzed R/M SGT treated at PM and VHIO from 2015 to 2020. Clinicopathological features, molecular alterations and treatment modalities were correlated with outcomes. The primary endpoint was overall response rate (ORR) by RECIST 1.1. Clinical benefit rate (CBR) was defined by pts with partial response or stable disease ≥4 months. Clinical actionability of multigene panel testing (NGS) and immunohistochemistry (IHC) were assessed as per institutional molecular tumor boards or investigators. Pts were opportunistically matched to available therapies from each center. Results: In total 206 pts were enrolled. On IHC, HER2 overexpression was present in 9%, Androgen Receptor (AR) 33%, Estrogen/Progesterone Receptor (ER/PR) 11% and ALK overexpression 0%. On NGS, PIK3CA mutation (mut) was in 9%, NTRK fusion 6%, NOTCH1-3 mut 5%, HRAS mut 6%, ERBB2/3 alterations (alt) 4% and FGFR1-4 alt 3%. Up to 92 pts (45%) displayed at least 1 AA and 36 pts (18%) had ≥2 AAs. A total of 60 pts (29%) were matched to AAs. Of those matched, median age was 60 years (range 33-84), M:F 21:39, 95% ECOG≤1 with a median number of prior treatment lines 0 (range 0-3), and their AAs included 26 AR, 9 HER2 or ERBB2 overexpression, 9 PIK3CA mut, 3 NTRK fusion, 3 FGFR1-3 alt and 10 other AAs (2 ER/PR overexpression, 2 EGFR mut, 1 c-kit mut, 1 BAP1 mut, 1 Non-V600 BRAF mut, 1 CDKN2A mut, 1 CHEK2 mut and 1 PTCH1 mut). Overall, ORR was 27% for the matched population. See table for outcomes. Conclusions: In our cohort, almost one third of the population received therapies matched to AAs. Our results suggest that targeted therapies have promising activity in pts with R/M SGT supporting comprehensive molecular and IHC profiling in treatment determination.[Table: see text]

Head and neck rhabdomyosarcoma with TFCP2 fusions and ALK overexpression: a clinicopathological and molecular analysis of 11 cases.

Primary intraosseous rhabdomyosarcoma (RMS) is a rare entity defined by EWSR1/FUS-TFCP2 or, less commonly, MEIS1-NCOA2 fusions. The lesions often show a hybrid spindle and epithelioid phenotype, frequently coexpress myogenic markers, ALK, and cytokeratin, and show a striking propensity for the pelvic and craniofacial bones. The aim of this study was to investigate the clinicopathological and molecular features of 11 head and neck RMSs (HNRMSs) characterised by the genetic alterations described in intraosseous RMS. The molecular abnormalities were analysed with fluorescence in-situ hybridisation and/or targeted RNA/DNA sequencing. Seven cases had FUS-TFCP2 fusions, four had EWSR1-TFCP2 fusions, and none had MEIS1-NCOA2 fusions. All except one case were intraosseous, affecting the mandible (n=4), maxilla (n=3), and skull (n=3). One case occurred in the superficial soft tissue of the neck. The median age was 29years (range, 16-74years), with an equal sex distribution. All tumours showed mixed epithelioid and spindle morphology. Immunohistochemical coexpression of desmin, myogenin, MyoD1, ALK, and cytokeratin was seen in most cases. An intragenic ALK deletion was seen in 43% of cases. Regional and distant spread were seen in three and four patients, respectively. Two patients died of their disease. We herein present the largest series of HNRMSs with TFCP2 fusions to date. The findings show a strong predilection for the skeleton in young adults, although we also report an extraosseous case. The tumours are characterised by a distinctive spindle and epithelioid phenotype and a peculiar immunoprofile, with coexpression of myogenic markers, epithelial markers, and ALK. They are associated with a poor prognosis, including regional or distant spread and disease-related death.

The clinical and prognostic role of ALK in glioblastoma

Backgroundanaplastic lymphoma kinase (ALK) overexpression and gene alterations have been detected in several malignancies, with prognostic and therapeutic implications. However, few studies investigated the correlation between ALK altered expression and prognosis in patients with glioblastoma (GBM). MethodsWe performed an evaluation of ALK overexpression and structural/quantitative chromosome alterations through immune-histochemical assay (IHC with D5F3 antibody) and fluorescent in situ hybridization (FISH) in patients with isocitrate dehydrogenase (IDH) wild type (wt) GBM. Assuming an ALK overexpression in 20 % of patients we planned a sample of 44 patients to achieve a probability of 90 % to include from 10 % to 30 % of patients with ALK alterations. ResultsWe evaluated 44 patients with IDH wt GBM, treated in our institution and dead due to GBM progression in 2017. ALK overexpression obtained by a composed score (the product of IHC intensity staining and rate of positive cells) was observed in 19 (43 %) patients. FISH analysis showed that 11 patients (25 %) had gene deletion, 2 patients (4.5 %) had monosomy and one patient (2.3 %) presented polysomy. Only one patient (2.3 %) demonstrated ALK rearrangement. There was no statistical difference in median OS between patients with ALK-positive (mOS = 18.9 months) and ALK-negative IHC (mOS = 18.0 months). ConclusionWe identified some rare previously unreported alterations of ALK gene in patients with IDH wt GBM. In these patients, the ALK overexpression does not influences survival.

Immunocytochemical Detection of ALK and ROS1 Rearrangements in Lung Cancer Cytological Samples.

The detection of molecular alterations such as ROS1 and ALK rearrangements is performed as part of the diagnosis of advanced-stage lung adenocarcinoma. These alterations allow the treatments with tyrosine kinase inhibitors. Cytological samples are very useful as up to 40% patients are diagnosed with this type of sample. Here we describe the immunocytochemistry technique usable to reveal the overexpression of ALK or ROS1 tyrosine kinase receptors secondary to ALK and ROS1 rearrangements, respectively.

Bone morphogenetic protein 9 (BMP9) directly induces Notch effector molecule Hes1 through the SMAD signaling pathway in osteoblasts.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad-binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.

A subset of epithelioid and spindle cell rhabdomyosarcomas is associated with TFCP2 fusions and common ALK upregulation.

Rhabdomyosarcomas with TFCP2 fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2 break-apart probe (n = 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient's age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone (n = 12/14) and soft tissue (n = 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented (n = 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALK fusion on break-apart FISH (n = 5) nor next-generation sequencing (n = 14). ALK upregulation was frequently associated with an internal deletion at genomic level. TFCP2 was fused in 5' either to EWSR1 (n = 6) or FUS (n = 8). EWSR1 was involved in both soft tissue cases. FISH with TFCP2 break-apart probe was positive in all tested cases (n = 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from13 to 107.55and recurrent CDKN2A deletions. FET-TFCP2 rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2 rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.

Immunotherapies targeting the PD-1/PD-L1 checkpoint axis are of growing interest for the treatment of mesenchymal neoplasms. However, PD-L1 expression and tumor-associated lymphocytes have not been well-investigated in uterine smooth muscle tumors. Forty-nine uterine smooth muscle tumors (23 leiomyosarcomas, 8 smooth muscle tumors of uncertain malignant potential [STUMP], 7 atypical leiomyomas, and 11 benign leiomyomas) were evaluated for tumoral and tumor-associated immune PD-L1 expression and tumor-associated T-cell infiltration. ALK immunohistochemistry was performed to exclude inflammatory myofibroblastic tumors. Tumor PD-L1 expression was seen in 70% of leiomyosarcomas and 14% of atypical leiomyomas; no cases of STUMP or benign leiomyoma demonstrated tumoral PD-L1. PD-L1 positivity was seen in tumor-associated immune cells in 78% of leiomyosarcomas, 25% of STUMP, no cases of atypical leiomyomas, and 9% of benign leiomyomas. Of the 23 leiomyosarcomas, 15 (65%) had a combined positive score ≥1, while of the 26 other uterine smooth muscle tumors, only 2 (8%) had a combined positive score ≥1. Tumor-associated CD8+ cells were highest among leiomyosarcomas (mean: 87/high-power fields vs. 17/high-power fields for nonleiomyosarcomas), and were significantly associated with PD-L1 expression. One PD-L1, CD8-enriched leiomyosarcoma showed an ALK overexpression suggesting possible classification as inflammatory myofibroblastic tumor, but otherwise lacked morphologic features of this entity. Leiomyosarcomas demonstrate significantly higher PD-L1 expression and cytotoxic T-cell infiltration when compared with other uterine smooth muscle tumors. These data suggest the possibility that treatment with targeted immunotherapy may be appropriate in a selected population of patients with leiomyosarcoma and, potentially, in related tumors bearing ALK rearrangements.