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Purpose: HER3, a member of the EGFR receptor family, plays a central role in driving oncogenic cell proliferation in breast cancer. Novel HER3 therapeutics are showing promising results while recently developed HER3 PET imaging modalities aid in predicting and assessing early treatment response. However, baseline HER3 expression, as well as changes in expression while on neoadjuvant therapy, have not been well-characterized. We conducted a prospective clinical study, pre- and post-neoadjuvant/systemic therapy, in patients with newly diagnosed breast cancer to determine HER3 expression, and to identify possible resistance mechanisms maintained through the HER3 receptor., Experimental Design: The study was conducted between May 25, 2018 and October 12, 2019. Thirty-four patients with newly diagnosed breast cancer of any subtype (ER ± , PR ± , HER2 ±) were enrolled in the study. Two core biopsy specimens were obtained from each patient at the time of diagnosis. Four patients underwent a second research biopsy following initiation of neoadjuvant/systemic therapy or systemic therapy which we define as neoadjuvant therapy. Molecular characterization of HER3 and downstream signaling nodes of the PI3K/AKT and MAPK pathways pre- and post-initiation of therapy was performed. Transcriptional validation of finings was performed in an external dataset (GSE122630)., Results: Variable baseline HER3 expression was found in newly diagnosed breast cancer and correlated positively with pAKT across subtypes (r = 0.45). In patients receiving neoadjuvant/systemic therapy, changes in HER3 expression were variable. In a hormone receptor-positive (ER +/PR +/HER2-) patient, there was a statistically significant increase in HER3 expression post neoadjuvant therapy, while there was no significant change in HER3 expression in a ER +/PR +/HER2+ patient. However, both of these patients showed increased downstream signaling in the PI3K/AKT pathway. One subject with ER +/PR -/HER2- breast cancer and another subject with ER +/PR +/HER2 + breast cancer showed decreased HER3 expression. Transcriptomic findings, revealed an immune suppressive environment in patients with decreased HER3 expression post therapy., Conclusion: This study demonstrates variable HER3 expression across breast cancer subtypes. HER3 expression can be assessed early, post-neoadjuvant therapy, providing valuable insight into cancer biology and potentially serving as a prognostic biomarker. Clinical translation of neoadjuvant therapy assessment can be achieved using HER3 PET imaging, offering real-time information on tumor biology and guiding personalized treatment for breast cancer patients., (© 2024. The Author(s).)

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5 + stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1 + stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers., (© 2024. The Author(s).)

Although immune checkpoint inhibitors (ICI) have revolutionized cancer management, patient response can be heterogeneous, and the development of ICI resistance is increasingly reported. Novel treatment strategies are necessary not only to expand the use of ICI to previously unresponsive tumor types but also to overcome resistance. Targeted radionuclide therapy may synergize well with ICIs since it can promote a pro-inflammatory tumor microenvironment. We investigated the use of a granzyme B targeted peptide (GZP) as a cancer theranostic agent, radiolabeled with 68 Ga ( 68 Ga-GZP) as a PET imaging agent and radiolabeled with 90 Y ( 90 Y-GZP) as a targeted radionuclide therapy agent for combinational therapy with ICI in murine models of colon cancer. Our results demonstrate that GZP increasingly accumulates in tumor tissue after ICI and that the combination of ICI with 90 Y-GZP promotes a dose-dependent response, achieving curative response in some settings and increased overall survival.

Purpose: Standard therapy for HER2+ breast cancers includes HER2 inhibition. While HER2 inhibitors have significantly improved therapeutic outcomes, many patients remain resistant to therapy. An important intrinsic resistance mechanism to HER2 inhibition in some breast cancers is dynamic upregulation of HER3. Increase in HER3 expression that occurs in response to HER2 inhibition allows for continued growth signaling through HER2/HER3 heterodimers, promoting tumor escape. We hypothesized that a non-invasive method to image changes in HER3 expression would be valuable to identify those breast cancers that dynamically upregulate HER3 in response to HER2 inhibition. We further hypothesized that this imaging method could identify those tumors that would benefit by additional HER3 knockdown., Procedures: In a panel of HER2+ breast cancer cell lines treated with the HER2 inhibitor lapatinib, we evaluate changes in HER3 expression and viability. Mouse HER2+ breast cancer models treated with lapatinib were imaged with a peptide-based HER3-specific PET imaging agent [ 68 Ga]HER3P1 to assess for dynamic changes in tumoral HER3 expression and uptake confirmed by biodistribution. Subsequently, HER2+ cell lines were treated with the HER2 inhibitor lapatinib as well HER3-specific siRNA to assess for changes in viability and correlate with HER3 expression upregulation. For all statistical comparisons, P<0.05 was considered statistically significant., Results: Lapatinib treatment of a panel of HER2+ breast cancer cell lines increased HER3 expression in the lapatinib-resistant cell line MDA-MB 453 but not the lapatinib-resistant cell-line HCC-1569. Evaluation of [ 68 Ga]HER3P1 uptake in mice implanted with the HER2+ breast cancer cell lines MDA-MB453 or HCC-1569 prior to and after treatment with lapatinib demonstrated a significant increase in MDA-MB453 tumors only, consistent with in vitro findings. The additional knockdown of HER3 increased therapeutic efficacy of lapatinib only in MDA-MB453 cells, but not in HCC-1569 cells., Conclusion: HER3 PET imaging can be used to visualize dynamic changes in HER3 expression that occur in HER2+ breast cancers with HER2 inhibitor treatment and identify those likely to benefit by the addition of combination HER3 and HER2 inhibition., (© 2021. World Molecular Imaging Society.)

Purpose: Cancer immunotherapy has markedly improved the prognosis of patients with a broad variety of malignancies. However, benefits are weighed against unique toxicities, with immune-related adverse events (irAE) that are frequent and potentially life-threatening. The diagnosis and management of these events are challenging due to heterogeneity of timing onset, multiplicity of affected organs, and lack of non-invasive monitoring techniques. We demonstrate the use of a granzyme B-targeted PET imaging agent (GZP) for irAE identification in a murine model., Experimental Design: We generated a model of immunotherapy-induced adverse events in Foxp3-DTR-GFP mice bearing MC38 tumors. GZP PET imaging was performed to evaluate organs non-invasively. We validated imaging with ex vivo analysis, correlating the establishment of these events with the presence of immune infiltrates and granzyme B upregulation in tissue. To demonstrate the clinical relevance of our findings, the presence of granzyme B was identified through immunofluorescence staining in tissue samples of patients with confirmed checkpoint inhibitor-associated adverse events., Results: GZP PET imaging revealed differential uptake in organs affected by irAEs, such as colon, spleen, and kidney, which significantly diminished after administration of the immunosuppressor dexamethasone. The presence of granzyme B and immune infiltrates were confirmed histologically and correlated with significantly higher uptake in PET imaging. The presence of granzyme B was also confirmed in samples from patients that presented with clinical irAEs., Conclusions: We demonstrate an interconnection between the establishment of irAEs and granzyme B presence and, for the first time, the visualization of those events through PET imaging., (©2021 American Association for Cancer Research.)

Background: Since the serendipitous discovery of bovine α-lactalbumin made lethal to tumour cells (BAMLET)/human α-lactalbumin made lethal to tumour cells there has been an increased interest in the ability of the two components, oleic acid and α-lactalbumin, to form anti-cancer complexes. Here we have investigated the in-vitro efficacy of the BAMLET complex in killing oral cancer (OC) cells, determined the active component of the complex and investigated possible biological mechanisms., Materials and Methods: Two OC cell lines (±p53 mutation) and one dysplastic cell line were used as a model of progressive oral carcinogenesis. We performed cell viability assays with increasing BAMLET concentrations to determine the cytotoxic potential of the complex. We further analysed the individual components to determine their respective cytotoxicities. siRNA knockdown of p53 was used to determine its functional role in mediating sensitivity to BAMLET. Cell death mechanisms were investigated by flow cytometry, confocal microscopy and the lactate dehydrogenase assay., Results: Our results show that BAMLET is cytotoxic to the OC and dysplastic cell lines in a time and dose-dependent manner. The cytotoxic component was found to be oleic acid, which, can induce cytotoxicity even when not in complex. Our results indicate that the mechanism of cytotoxicity occurs through multiple simultaneous events including cell cycle arrest, autophagy like processes with a minor involvement of necrosis., Conclusion: Deciphering the mechanism of cytotoxicity will aid treatment modalities for OC. This study highlights the potential of BAMLET as a novel therapeutic strategy in oral dysplastic and cancerous cells., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Triple Negative Breast Cancer (TNBC) is an aggressive form of Breast Cancer (BC). Numerous kinase inhibitors (KI) targeting different pathway nodes have shown limited benefit in the clinical setting. In this study, we aim to characterize the extent of HER3 reliance and to define the effect of Neuregulin (NRG) isoforms in TNBCs. Basal and Claudin type TNBC cell lines were treated with a range of small molecule inhibitors, in the presence or absence of the HER3 ligand NRG. Single agent and combination therapy was also evaluated in human cancer cell lines through viability and biochemical assessment of the AKT/MAPK signaling pathway. We show that Basal (BT20, HCC-70, and MDA-MB-468) and Claudin type (MDA-MB-231, BT-549) TNBC cell lines displayed differential reliance on the HER family of receptors. Expression and dynamic HER3 upregulation was predominant in the Basal TNBC subtype. Furthermore, the presence of the natural ligand NRG showed potent signaling through the HER3-AKT pathway, significantly diminishing the efficacy of the AKT and PI3K inhibitors tested. We report that NRG augments the HER3 feedback mechanism for continued cell survival in TNBC. We demonstrate that combination strategies to effectively block the EGFR-HER3-AKT pathway are necessary to overcome compensatory mechanisms to NRG dependent and independent resistance mechanisms. Our findings suggests that the EGFR-HER3 heterodimer forms a major signaling hub and is a key player in tumorigenesis in Basal but not Claudin type TNBC tested. Thus, HER3 could potentially serve as a biomarker for identifying patients in which targeted therapy against the EGFR-HER3-AKT axis would be most valuable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sinevici, Ataeinia, Zehnder, Lin, Grove, Heidari and Mahmood.)

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann-Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Oral Cancer (OC) is a subset of head and neck cancer (HNC) with an annual worldwide incidence of 275,000 cases. OC remains a significant burden worldwide in terms of diagnosis, treatment and prognosis. Despite desirable outcomes in early diagnosed OCs and treatment advances most OCs are detected in advanced stages. The 5-year survival rate of early-stage disease is ∼80% and that of late-stage disease is only ∼20%. Recurrence and chemoresistance from a treatment point of view and pain and disfiguration are important factors contributing to the high morbidity and mortality of OC. Furthermore the process of oral carcinogenesis is complex and not yet fully understood. Consequently numerous potential biomarkers have been hypothesised though controversial results across the board hamper their clinical implementation. Of greatest advantage would be biomarkers signalling early events preceeding OC. Biomarker targets predominately involve deregulated molecular events that participate in cell signalling, growth, survival, motility, angiogenesis and cell cycle control but can also use changes in metabolic genes to discriminate healthy form disease state. Promising potential biomarkers include the growth signalling oncogenes, Epidermal Growth Factor Receptor and Cyclin D1, the anti-growth signalling components p53 and p21, apoptotic effectors such as Bcl-2 and also components involved in immortalisation, angiogenesis, invasion and metastasis processes. Translation of these potential biomakers to the patients is closer than ever though few issues remain to be resolved. Firstly large clinical trials are needed to validate their clinical applicability but also standardised methods of collection, storage and processing methods are needed to minimise variability., (Copyright © 2016 Elsevier Ltd. All rights reserved.)