Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells.

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T Cells.

T cell modification with genes that encode chimeric antigen receptors (CAR-T cells) has shown tremendous promise for the treatment of B cell malignancies. The successful translation of CAR-T cell therapy to other tumor types, including solid tumors, is the next big challenge. As the field advances from second- to next-generation CAR-T cells comprising multiple genetic modifications, more sophisticated methods and tools to engineer T cells are being developed. Viral vectors, especially γ-retroviruses and lentiviruses, are traditionally used for CAR-T cell engineering due to their high transduction efficiency. However, limited genetic cargo, high costs of production under good manufacturing practice (GMP) conditions, and the high regulatory demands are obstacles for widespread clinical translation. To overcome these limitations, different nonviral approaches are being explored at a preclinical or clinical level, including transposon/transposase systems and mRNA electroporation and nonintegrating DNA nanovectors. Genome editing tools that allow efficient knockout of particular genes and/or site-directed integration of the CAR and/or other transgenes into the genome are also being evaluated for CAR-T cell engineering. In this review, we discuss the development of viral and nonviral vectors used to generate CAR-T cells, focusing on their advantages and limitations. We also discuss the lessons learned from clinical trials using the different genetic engineering tools, with special focus on safety and efficacy.

VEGFR-2 redirected CAR-T cells are functionally impaired by soluble VEGF-A competition for receptor binding.

BACKGROUND: The adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101. METHODS: Primary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups. RESULTS: In line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME. CONCLUSIONS: This study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

miR-155 Overexpression in OT-1 CD8+ T Cells Improves Anti-Tumor Activity against Low-Affinity Tumor Antigen.

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones.

All systems go: converging synthetic biology and combinatorial treatment for CAR-T cell therapy.

Synthetic biology has been transformative to the treatment of advanced hematological malignancies by chimeric antigen receptor (CAR)-engineered T cells. A range of obstacles are now understood to limit the responses of solid epithelial-derived tumors to CAR therapy. For example, inefficient tumor homing and a fortified stroma can restrain the number of CAR-T cells reaching the tumor bed. Upon transendothelial migration across the tumor vasculature, CAR-T cells face a highly suppressive microenvironment that can quickly render them hypofunctional. Safety also remains a critical issue for advancing CAR therapy of solid tumors. Innovative CAR design as well as coengineering and combinatorial treatment strategies with oncolytic adenovirus, radiotherapy, vaccines, chemotherapy, small molecules and monoclonal antibodies hold tremendous potential to support CAR-T cell control of solid tumors, either by directly promoting CAR-T cell function, or/and by re-programming the TME and harnessing the endogenous immune system against the tumor.

Cooperation between Constitutive and Inducible Chemokines Enables T Cell Engraftment and Immune Attack in Solid Tumors.

We investigated the role of chemokines in regulating T cell accumulation in solid tumors. CCL5 and CXCL9 overexpression was associated with CD8+ T cell infiltration in solid tumors. T cell infiltration required tumor cell-derived CCL5 and was amplified by IFN-γ-inducible, myeloid cell-secreted CXCL9. CCL5 and CXCL9 coexpression revealed immunoreactive tumors with prolonged survival and response to checkpoint blockade. Loss of CCL5 expression in human tumors was associated with epigenetic silencing through DNA methylation. Reduction of CCL5 expression caused tumor-infiltrating lymphocyte (TIL) desertification, whereas forced CCL5 expression prevented Cxcl9 expression and TILs loss, and attenuated tumor growth in mice through IFN-γ. The cooperation between tumor-derived CCL5 and IFN-γ-inducible CXCR3 ligands secreted by myeloid cells is key for orchestrating T cell infiltration in immunoreactive and immunoresponsive tumors.

Correction to: CART cells are prone to Fas- and DR5-mediated cell death.

After publication of this article [1], it was noticed that 3 authors were missed from the author list.

CART cells are prone to Fas- and DR5-mediated cell death.

Adoptive transfer of T cells transduced with Chimeric Antigen Receptors (CAR) are now FDA-approved for the treatment of B-cell malignancies. Yet, the functionality of the endogenous TCR in CART cells has not been fully assessed. Here, we demonstrate that CART cells progressively upregulate Fas, FasL, DR5 and TRAIL, which result in their programmed cell death, independently of antigen-mediated TCR or CAR activation. CART cell apoptosis occurs even when the CAR contains a single (co-)activatory domain such as CD3ζ, CD28 or 4-1BB. Importantly, the dominant role of the Fas and DR5 pathways in CART cell apoptosis is demonstrated by the significant rescue of CART cells upon in vivo blockade by combined Fas-Fc and DR5-Fc recombinant proteins. These observations are of crucial importance for the long-term persistence of CART cells and for the development of new applications including the combined TCR and CAR activation against solid tumors.

Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy.

Cancer immunotherapy relies upon the ability of T cells to infiltrate tumors. The endothelium constitutes a barrier between the tumor and effector T cells, and the ability to manipulate local vascular permeability could be translated into effective immunotherapy. Here, we show that in the context of adoptive T cell therapy, antitumor T cells, delivered at high enough doses, can overcome the endothelial barrier and infiltrate tumors, a process that requires local production of C3, complement activation on tumor endothelium and release of C5a. C5a, in turn, acts on endothelial cells promoting the upregulation of adhesion molecules and T-cell homing. Genetic deletion of C3 or the C5a receptor 1 (C5aR1), and pharmacological blockade of C5aR1, impaired the ability of T cells to overcome the endothelial barrier, infiltrate tumors, and control tumor progression in vivo, while genetic chimera mice demonstrated that C3 and C5aR1 expression by tumor stroma, and not leukocytes, governs T cell homing, acting on the local endothelium. In vitro, endothelial C3 and C5a expressions were required for endothelial activation by type 1 cytokines. Our data indicate that effective immunotherapy is a consequence of successful homing of T cells in response to local complement activation, which disrupts the tumor endothelial barrier.

Type II enteropathy-associated T-cell lymphoma features a unique genomic profile with highly recurrent SETD2 alterations.

Enteropathy-associated T-cell lymphoma (EATL), a rare and aggressive intestinal malignancy of intraepithelial T lymphocytes, comprises two disease variants (EATL-I and EATL-II) differing in clinical characteristics and pathological features. Here we report findings derived from whole-exome sequencing of 15 EATL-II tumour-normal tissue pairs. The tumour suppressor gene SETD2 encoding a non-redundant H3K36-specific trimethyltransferase is altered in 14/15 cases (93%), mainly by loss-of-function mutations and/or loss of the corresponding locus (3p21.31). These alterations consistently correlate with defective H3K36 trimethylation. The JAK/STAT pathway comprises recurrent STAT5B (60%), JAK3 (46%) and SH2B3 (20%) mutations, including a STAT5B V712E activating variant. In addition, frequent mutations in TP53, BRAF and KRAS are observed. Conversely, in EATL-I, no SETD2, STAT5B or JAK3 mutations are found, and H3K36 trimethylation is preserved. This study describes SETD2 inactivation as EATL-II molecular hallmark, supports EATL-I and -II being two distinct entities, and defines potential new targets for therapeutic intervention.

Tumor Regression and Delayed Onset Toxicity Following B7-H4 CAR T Cell Therapy.

B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6-8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Targeting the tumor vasculature to enhance T cell activity.

T cells play a critical role in tumor immune surveillance as evidenced by extensive mouse-tumor model studies as well as encouraging patient responses to adoptive T cell therapies and dendritic cell vaccines. It is well established that the interplay of tumor cells with their local cellular environment can trigger events that are immunoinhibitory to T cells. More recently it is emerging that the tumor vasculature itself constitutes an important barrier to T cells. Endothelial cells lining the vessels can suppress T cell activity, target them for destruction, and block them from gaining entry into the tumor in the first place through the deregulation of adhesion molecules. Here we review approaches to break this tumor endothelial barrier and enhance T cell activity.

A human ErbB2-specific T-cell receptor confers potent antitumor effector functions in genetically engineered primary cytotoxic lymphocytes.

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.

Novel recombinant human b7-h4 antibodies overcome tumoral immune escape to potentiate T-cell antitumor responses.

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo.

Adoptive immunotherapy using T lymphocytes genetically modified to express a chimeric antigen receptor (CAR-T) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy, whereby T-cell activation signal 1 (CD3z) is physically dissociated from costimulatory signal 2 (CD28) in two CARs of differing antigen specificity: mesothelin and a-folate receptor (FRa). Human T cells were genetically modified to coexpress signal 1 (anti-Meso scFv-CD3z) and signal 2 (anti-FRa scFv-CD28) CARs in trans. Trans-signaling CAR-T cells showed weak cytokine secretion against target cells expressing only one TAA in vitro, similar to first-generation CAR-T cells bearing CD3z only, but showed enhanced cytokine secretion upon encountering natural or engineered tumor cells coexpressing both antigens, equivalent to that of second-generation CAR-T cells with dual signaling in cis. CAR-T cells with dual specificity also showed potent anticancer activity and persistence in vivo, which was superior to first-generation CAR-T cells and equivalent to second-generation CARs. Importantly, second-generation CAR-T cells exhibited potent activity against cells expressing mesothelin alone, recapitulating normal tissue, whereas trans-signaling CAR-T cells did not. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CAR-T cells against cancer while minimizing parallel reactivity against normal tissues bearing single antigen.

Primary human ovarian epithelial cancer cells broadly express HER2 at immunologically-detectable levels.

The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies.