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Introduction Endobronchial ultrasound (EBUS)-guided transbronchial needle aspirate (TBNA) is a widely used method of minimally invasive lymph node sampling. The benefit of processing samples by cytologic methods versus “core biopsy” is unclear. It is unknown if safety or diagnostic yield varies by needle gauge. Materials and Methods Between June 2018 and July 2019, 40 patients (56 lesions) undergoing EBUS TBNA lymph node evaluation were enrolled in this single center prospective trial. Patients were chosen by permuted block randomization to undergo EBUS TBNA starting with 22-gauge or 19-gauge needles. Separate samples were sent for processing by cytologic methods and histopathology. Surgical pathologists and cytopathologists were blinded to needle size. The primary endpoint was diagnostic yield. Secondary endpoints compared specimen adequacy by rapid on-site evaluation (ROSE), sample adequacy for molecular testing, sample quality, and safety. Results Diagnostic yield for histopathologic examination was 87.5% and 83.9% for 19g and 22g respectively (P-value 0.625). There was no significant difference in diagnostic yield by cytologic examination based on needle size. There was no significant difference in slide quality. Molecular adequacy for core-biopsy was 77% and 80% for 22-gauge and 19-gauge needles, respectively. Molecular adequacy for cytology cell block was 77% and 80% for 22-gauge and 19-gauge needles, respectively. There were no significant procedural complications. Conclusion Both the 22-gauge and 19-gauge EBUS TBNA needles provided a similar diagnostic yield and clinical utility for ancillary testing. Processing techniques by cytologic methods or “core biopsy” showed no significant impact in diagnostic yield or utility of molecular testing.

Background: Inflammatory breast cancer (IBC) is characterized by a young age at diagnosis, high metastatic potential, and poor overall survival. To better understand the etiology of this aggressive disease, we performed a retrospective study to evaluate genomic alterations and tumor microenvironment in IBC patients. Methods: Targeted next generation sequencing (NGS) was performed using blood cell-free DNA (cfDNA) (n=32), paired DNA from tumor tissues (n=29) and DNA from whole blood or normal tissue (n=20). We also characterized the tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1 and PD-L1) through immunohistochemistry (IHC) staining. Results: Our results shown a high incidence of germline variants in patients with IBC that could be associated with an increased risk of developing the disease. Germline variants were found more frequently in patients with TN (38.9%) than non-TN IBC (28.6%). IHC staining revealed that in 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs. Patients with PD-L1&PD-1 positive biopsies had a lower OS (31 months) than patients with PD-L1&PD-1 negative (38 months) tumors, though the difference was not statistically significant. PD-L1&PD-1 positive tumors had significantly more CD8+ (cytotoxic T-cells), CD20+ (B-cells) and FoxP3+ (Tregs) cells than PD-L1&PD-1 negative ones. Furthermore, most IBC patients showed to have deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. All tumor biopsies showed positive staining for p-FAK1(Tyr 397). Conclusion: A subset of IBC patients (29%) expressed both PD-L1 and PD-1 on tumor immune cells infiltrates, providing rationale for testing PD-1/PD-L1 blocking therapies. IBC patients could have benefits from PD-1/PD-L1 blocking therapies alone or in combination with PARP or FAK (Tyr397) kinase inhibitors, both having the capacity of altering the tumor microenvironment. Citation Format: Maria F. Arisi, Yulan Gong, Rajeswari Nagarathinam, Lorenzo Gerratana, Jianming Pei, Kathy Q. Cai, Jennifer Winn, Zachary Hasse, Elias Obeid, Julio Noriega, Christopher Sebastiano, Eric Ross, R. Katherine Alpaugh, Massimo Cristofanilli, Sandra V Fernandez. Genetic variants and tumor microenvironment in inflammatory breast cancer: Clues for targeted therapies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-02.

Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test.Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS.A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material.Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.

While Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase active site inhibitors have not typically led to durable, clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DBP. We found that disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Importantly, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis (MF), suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence (OIS) in vitro. The ERK2-DBP mutation attenuated JAK2-mediated OIS by preventing the physical interaction of ERK2 with the transcription factor, Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D-domain, these data suggested that the D-domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression, and that pharmacologic targeting of the ERK2-D domain might attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D-domain has the potential to be potent therapeutic target in Ras/MAPK dependent cancers.

Background and Objective: Robotic bronchoscopy has demonstrated high navigational success in small peripheral lung nodules but the diagnostic yield is discrepantly lower. Needle based confocal laser endomicroscopy (nCLE) enables real-time microscopic imaging at the needle tip. We aim to assess feasibility, safety and needle repositioning based on real-time nCLE-guidance during robotic bronchoscopy in small peripheral lung nodules. Methods: Patients with suspected peripheral lung cancer underwent fluoroscopy and radial EBUS assisted robotic bronchoscopy. After radial EBUS nodule identification, nCLE-imaging of the target area was performed. nCLE-malignancy and airway/lung parenchyma criteria were used to identify the optimal sampling location. In case airway was visualized, repositioning of the biopsy needle was performed. After nCLE tool-in-nodule confirmation, needle passes and biopsies were performed at the same location. Measurements and Main Results: Twenty patients were included (final diagnosis n = 17 (lung) cancer) with a median lung nodule size of 14.5 mm (range 8–28 mm). No complications occurred. In 19/20 patients, good quality nCLE-videos were obtained. In 9 patients (45%), real-time nCLE-imaging revealed inadequate positioning of the needle and repositioning was performed. After repositioning, nCLE-imaging provided tool-in-nodule-confirmation in 19/20 patients. Subsequent ROSE demonstrated representative material in 9/20 patients (45%) and overall diagnostic yield was 80% (16/20). Of the three patients with malignant nCLE-imaging but inadequate pathology, two were diagnosed with malignancy during follow-up. Conclusion: Robotic bronchoscopic nCLE-imaging is feasible and safe. nCLE-imaging in small, difficult-to-access lung nodules provided additional real-time feedback on the correct needle positioning with the potential to optimize the sampling location and diagnostic yield.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death. Advancements in navigational bronchoscopy have shown encouraging results but the diagnostic yield of small lung nodules by bronchoscopic techniques is still below that of transthoracic needle aspiration. The development of robotic bronchoscopy has demonstrated a significantly improved navigational success but the diagnostic yield is regularly limited by near-miss of the target nodule. Needle-based confocal laser endomicroscopy is a novel imaging technique that allows for the real-time visualization of individual cells and structures with microscopic resolution at the tip of the needle. We present the first reported case of confocal laser endomicroscopy guided robotic bronchoscopy for the real-time diagnosis of a small, partially cystic lung nodule.

Paragangliomas are rare neuroendocrine tumors that can vary in size and metabolic activity. We report a case of giant bilateral malignant retroperitoneal paragangliomas (PGL) in a patient with germline succinate dehydrogenase B (SDHB) mutation. This patient, who presented in an emaciated and debilitated state, was managed with adrenergic blockade followed by radical primary surgery. After being metabolically and radiographically disease free for 4 years, he underwent salvage resection for recurrent retroperitoneal disease and palliative radiation to a site of solidary vertebral metastasis. We review incidence and prognosis of metastatic PGL.

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines. To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients. To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction. Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. Disclosures Levine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.