Probes for HER2

As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.

Background: Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically evince immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR confers on the macrophages the ability to selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in many cancers, including but not limited to breast and gastroesophageal cancers. CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden and prolonged survival in relevant models. CT-0508 cells were safe in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Methods: This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in approximately 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2 who have failed available therapies including anti-HER2 therapies when indicated. Filgrastim will be used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product will be manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. Group 1 subjects will receive CT-0508 infusion split over D1, 3 and 5. Subjects will be continually assessed for acute and cumulative toxicity. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), persistence, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others.

Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically evince immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR on the macrophages confers the ability to selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis and is seen in many cancers, including but not limited to breast and gastroesophageal cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreasing tumor burden and prolonging survival in relevant models. CT-0508 cells were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in approximately 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2, who have failed available therapies including anti-HER2 therapies where indicated.Filgrastim is being used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product is manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. The study is enrolling Group 1 subjects, who receive CT-0508 infusion split over D1, 3 and 5. Subjects will be continually assessed for acute and cumulative toxicity. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will follow, and will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial registry number: NCT04660929 Table 1.HER2 Positivity Frequencies Across Tumor TypesTumor typeHER2 positivity (%)ReferenceBladder cancer8-70Gandour-Edwards et al, 2002;Caner et al, 2008;Laé et al, 2010; Fleischmann et al, 2011;Charfi et al, 2013;Yan et al, 2015Breast cancer11.0-25.0Varga et al, 2013;Stenehjem et al, 2014Cervical cancer2.8-3.9Chavez-Blanco et al, 2004;Yan et al, 2015Colorectal cancer1.6-5.0Schuell et al, 2006;Ingold Heppner et al, 2014;Seo et al, 2014Esophageal cancer12.0-14.0König et al, 2013;Yoon et al, 2013;Wang et al, 2014Extrahepatic Cholangiocarcinoma6.3-9.0Yoshikawa et al, 2008;Yan et al, 2015Gallbladder cancer9.8-12.8Roa et al, 2014;Yan et al, 2015Gastric adenocarcinoma7.0-34.0Rüschoff et al, 2012;Hofmann et al, 2008Ovarian cancer26Slamon et al, 1989Salivary mucoepidermoid carcinomas17.6Glisson et al, 2004Salivary duct carcinoma30-40Skálová et al, 2003; Cornolti et al, 2007; Nardi et al, 2013Testicular cancer2.4Yan et al, 2015Uterine cancer3.0Yan et al, 2015 Citation Format: Yara George Abdou, Debora Barton, Amy Ronczka, Daniel Cushing, Michael Klichinsky, Kim Reiss Binder. A phase 1, first in human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors [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 OT1-03-01.

Background: Adoptive T cell therapies have led to remarkable advances in hematologic cancers but with less effect in ST. Actively recruited tumor associated macrophages (TAM) are abundant in the ST microenvironment (TME) and typically display immunosuppressive behavior. Macrophages engineered to be proinflammatory may be an ideal vector for adoptive ST cellular therapy. Engineered CAR-M selectively recognize and phagocytose antigen overexpressing cancer cells, reprogram TME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies show that CT-0508 induces targeted cancer cell phagocytosis while sparing normal cells, decreases tumor burden and prolongs survival, and was safe and effective in a semi-immunocompetent mouse model of human HER2-overexpressing ovarian cancer. Methods: This FIH Phase 1 study is evaluating safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary efficacy in 18 subjects with locally advanced/unresectable or metastatic ST overexpressing HER2, with progression on available therapies, including anti-HER2 therapies. Filgrastim is used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis prior to CT-0508 CAR macrophage infusion. Group 1 subjects receive CT-0508 on D1, 3, & 5. Group 2 subjects will receive full dose on D1. A Safety Review Committee will review dose limiting toxicities. Pre/post-treatment biopsies and blood samples will be collected for correlative analysis of immunogenicity, trafficking (PCR, RNA scope), CT-0508 persistence in blood and tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Table 1. Her2 Overexpression Across Tumor Types Tumor HER2 Overexpression (%) Bladder 8-70 Salivary duct 30-40 Gastric 7-34 Ovarian 26 Breast 11-25 Salivary mucoepidermoid 17.6 Esophageal 12-14 Gallbladder 9.8-12.8 Cholangiocarcinoma 6.3-9 Colorectal 1.6-5 Cervical 2.8-3.9 Uterine 3 Testicular 2.4 Citation Format: Kim A. Reiss, Yuan Yuan, Naoto T. Ueno, Yara Abdou, Debora Barton, Ramona F. Swaby, Amy Ronczka, Daniel J. Cushing, Sascha Abramson, Thomas Condamine, Michael Klichinsky, E. Claire Dees. A phase 1, first in human (FIH) study of autologous anti-HER2 chimeric antigen receptor macrophages (CAR-M) in HER2-overexpressing solid tumors (ST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT524.

The development and subsequent adaptation of mass cytometry for the histological analysis of tissue sections has allowed the simultaneous spatial characterization of multiple components. This is useful to find the correlation between the genotypic and phenotypic profile of tumor cells and their environment in clinical-translational studies. In this revision, we provide an overview of the most relevant hallmarks in the development, implementation and application of multiplexed imaging in the study of cancer and other conditions. A special focus is placed on studies based on imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI). The purpose of this review is to help our readers become familiar with the verification techniques employed on this tool and outline the multiple applications reported in the literature. This review will also provide guidance on the use of IMC or MIBI in any field of biomedical research.

Although RNA plays a vital role in the process of gene expression, it is less used as an in situ biomarker for clinical diagnostics compared to DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules themselves. To tackle this issue, methods that are sensitive and specific are needed. Here we present an RNA single molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity on the RNA molecules, they form V shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We not only successfully applied our method to assess HER2 RNA mRNA expression status in invasive breast cancer tissue, but also to investigate the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicates the great potential of our method to be applied in the diagnosis of disease using RNA biomarkers.

Pachyonychia congenita (PC) is a rare keratinizing disorder characterized by painful palmoplantar keratoderma (PPK) for which there is no standard current treatment. PC is caused by dominant mutations in keratin 6A, 6B, 6C, 16, and 17 genes involved in stress, wound healing, and epidermal barrier formation. Mechanisms leading to pain and PPK in PC remain elusive. Here, we show overexpression of EGFR ligands epiregulin and TGF-α as well as HER1-EGFR and HER2 in the upper spinous layers of PC lesions. EGFR activation was confirmed by upregulated MAPK/ERK and mTOR signaling. Abnormal late terminal keratinization was associated with elevated transglutaminase-1 (TG1) activity. Additionally, the Ca2+ permeable channel TRPV3 was significantly increased in PC-lesional skin suggesting a predominant role of the TRPV3/EGFR signaling complex in PC. We hypothesized that this complex contributes to promoting TG1 activity and induces the expression and shedding of EGFR ligands. To counteract this biological cascade, we treated 3 PC patients with oral erlotinib for 6 to 8 months. The treatment was well tolerated and led to an early, drastic, and sustained reduction of neuropathic pain with a major improvement of quality-of-life. Our study provides evidence that targeted pharmacological inhibition of EGFR is an effective strategy in PC.