High HER2 Research Articles

Molecular-based targeted therapies in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma refractory to atezolizumab/bevacizumab

The "French Medicine Genomic program 2025" has been designed to give patients with cancers that are refractory to systemic treatments access to off-label therapies adapted to their specific genomic profile. Herein, we reported the results of this program in patients with advanced hepatocellular carcinoma (HCC) and hepato-cholangiocarcinoma (H-CCK). In one center, all patients with HCC or H-CCK who progressed under atezolizumab/bevacizumab with available tumor frozen samples benefited from whole-genome/-exome and RNA sequencing. Targeted therapies were matched to genomic alterations following the recommendations of a molecular tumor board and radiological response and overall survival were assessed. Among 135 patients with HCC and H-CCK treated by atezolizumab/bevacizumab, 20 patients benefited from genomic analysis after progression (16 HCC; 4H-CCK). Nineteen patients had analyzable data, 70% were male, median age was 57 years, 65% had metastatic disease and 45% had vascular invasion. Among these 19 patients, 14 patients (76%) harbored at least one actionable genomic alteration and 9/14 received an adapted targeted therapy (45%). One patient with H-CCK showing CDK4 amplification was treated with palbociclib and achieved a partial radiological response for 16 months. Another patient with H-CCK, high HER2 overexpression and a high homologous recombination score was treated with trastuzumab/olaparib and had stable disease. One patient with an HCC and bi-allelic inactivation of TSC2 achieved a complete radiological response under everolimus. The remaining six treated patients (all HCC) had progressive disease, including three patients treated with trametinib, two with everolimus and one with olaparib. Molecular-based guided therapy is feasible in patients with HCC/H-CCK progressing under atezolizumab/bevacizumab and may be useful in a small subset of patients. The use of whole-genome/-exome and RNA sequencing in clinical practice has not been reported in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma. Herein, we performed a pilot study which suggested that whole-genome/-exome and RNA sequencing is feasible on tumor biopsies from patients refractory to atezolizumab/bevacizumab, with a small subset of patients exhibiting at least one actionable genomic alteration and receiving an adapted targeted therapy. This proof-of-concept study suggests that this clinical strategy could benefit a small subset of patients. Finally, validation of this approach will be required in a larger cohort of patients.

Clinical Utility of Pre-Therapeutic [18F]FDG PET/CT Imaging for Predicting Outcomes in Breast Cancer.

[18F]FDG PET/CT is used for staging and could also provide information associated with clinical outcomes. The objective of this study was to determine the clinical utility of biomarkers measured using [18F]FDG PET/CT to predict the absence of pathological complete response (no-pCR) and recurrence. In this retrospective study, we included patients with non-special-type breast carcinoma who underwent [18F]FDG PET/CT before neoadjuvant chemotherapy between 2011 and 2019. Clinicopathological data were collected. Tumor SUVmax and total metabolic tumor volume (TMTV) were measured from PET images. The association between biomarkers and no-pCR was studied using logistic regression. The cut-off value was determined using the area under the ROC Curve. To predict 3-year recurrence-free survival (RFS), we used a multivariable Cox model, and the cut-off value was determined using time-dependent ROC and predictiveness curves. Two hundred and eighty-six patients were included in the analysis. One hundred and twelve patients had a pCR (39.2%). The pCR rate was significantly higher in patients with a high nuclear grade (p < 0.01), HER2+ and TNBC subtypes (p < 0.01), high Ki67 (p < 0.01), and low TMTV (p < 0.01). A high TMTV value (>9.0 cm3) was significantly associated with no-pCR in the whole cohort (OR = 2.4, 95% CI: 1.3-4.2, p < 0.01). After a median follow-up of 4.5 years, 65 patients experienced recurrence and 39 patients died. High TMTV (>13.5 cm3) was associated with shorter RFS (HR = 4.0, 95% CI: 1.9-8.4, p < 0.01). High TMTV in pre-therapeutic imaging is associated with no-pCR and recurrence. It can help in identifying high-risk patients and be considered as an intensified or alternative adjuvant therapy for closely monitoring patients.

Clinicopathological significance of TUBB3 in upper tract urothelial carcinoma and possible application in urine cytology.

βIII-Tubulin, encoded by the TUBB3 gene, is a microtubule protein. We previously reported that TUBB3 is overexpressed in renal cell carcinoma. We investigated the clinicopathological significance of TUBB3 in upper tract urothelial carcinoma (UTUC) by immunohistochemistry. In normal tissue, TUBB3 expression was weak or absent. In contrast, TUBB3 overexpression was observed in urothelial carcinoma (UC) tissues in 51 (49%) of 103 UTUC cases. TUBB3 overexpression was associated with nodular/flat morphology, high-grade disease, high T stage, and a poor prognosis. Similar results were obtained in The Cancer Genome Atlas bladder cancer cohort. TUBB3 expression was also associated with high Ki-67 labeling index, CD44v9, HER2, EGFR, and p53 expression in UTUC. Among representative cancer-related molecules, TUBB3 was an independent predictor of progression-free survival and high-grade UC. Finally, using urine cytology samples, we analyzed TUBB3 expression by immunocytochemistry. TUBB3 expression was more frequently found in UC cells than in nonneoplastic cells. The diagnostic accuracy of urine cytology was improved when combined with TUBB3 immunostaining. The findings suggest the importance of TUBB3 in tumor progression and its potential application as a biomarker for high-grade disease and the prognosis of UC. Moreover, combination with TUBB3 immunostaining might improve the diagnostic accuracy of urine cytology.

Low co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET in urothelial carcinoma is associated with discordant expression between primary and metastatic sites

Novel regimens targeting immune checkpoints and the cMET or HER2 pathways are under investigation in metastatic urothelial carcinoma (mUC) though co-expression of these molecular targets has not been defined. We sought to characterize the protein co-expression rates of PD-L1, cMET and HER2 in primary and metastatic mUC lesions and agreement rates in paired biopsies.We assessed PD-L1, cMET and HER2 protein expression by immunohistochemistry (IHC) in archival mUC samples identified from an institutional database (n = 143). Correlation of expression between primary and metastatic biopsies was performed in patients with available paired biopsies (n = 79). Protein expression levels by predefined thresholds were measured, and Cohen's kappa statistics (κ) were utilized to assess the agreement in expression between paired primary and metastatic samples.In primary tumors (n = 85), high expression of PD-L1, cMET, and HER2 was observed in 14.1%, 34.1%, and 12.9%, respectively. In metastatic samples (n = 143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between paired specimens (n = 79) were PD-L1: 79.7% (κ = 0.09), cMET: 69.6% (κ = 0.35), HER2: 84.8% (κ = 0.17). High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n = 7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in 3.8% (n = 3) of primary samples and no metastatic samples. The overall co-expression agreement between paired samples was 55.7% (κ = 0.22) for PD-L1/cMET and 67.1% (κ = 0.06) for PD-L1/HER2, but agreement for high co-expression between paired samples was very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2).Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort. Agreement of high co-expression between primary and metastatic sites is rare. Biomarker-based strategies used in selection of patients for contemporary trials testing combinations of immune checkpoint inhibitors with either cMET or HER2-targeted agents should account for discordant biomarker expression between primary and metastatic sites.

Clinicopathological characteristics and prognostic analysis of breast cancer with a hormone receptor status of ER(-)/PR(+).

It is unknown whether ER(-)/PR(+) breast cancer is an independent breast cancer subtype, how it differs from other subtypes, and what its significance is regarding treatment and prognosis. This study compared ER(-)/PR(+) breast cancer with other subtypes to better understand the biological characteristics and prognosis of ER(-)/PR(+) breast cancer, to guide clinical treatment and establish a theoretical foundation. We retrospectively analyzed data for patients diagnosed with breast cancer in the Surveillance, Epidemiology, and End Results (SEER) database. The clinicopathological characteristics of ER(-)/PR(+) breast cancer, including age, tumor size, lymph node status, HER-2 status, pathological type and histological grade, were compared with other types of breast cancer. A risk scoring system was developed based on independent risk factors influencing prognosis to predict the patient's prognosis, and a nomogram model was created to predict the patient's survival rate. Receiver operating characteristic curve (ROC) and calibration curve was used to evaluate the predictive performance of the nomogram. The rates of T3-4, lymph node positivity, HER-2 positivity, infiltrating non-special pathological type, and G3 were significantly higher in ER(-)/PR(+) than in ER(+)/PR(+) cancer (p <0.001). ER(-)/PR(+) was similar to biological activity of ER(-)/PR(-) type. ER(-)/PR(+)/HER-2(+) patients had a better survival prognosis than ER(-)/PR(+) HER-2(-) patients (p<0.05). The prognosis of ER-/PR+ breast cancer was significantly associated with age, HER-2 status, and T stage. ER(-)/PR(+) breast cancer is more similar to ER(-)/PR(-) breast cancer than other breast cancer subtypes, with an early age of onset, a high proportion of infiltrating non-special types, a high histological grade, and a high HER-2 positivity rate. Whether HER-2 positivity can improve the prognosis of ER(-)/PR(+)breast cancer is worth further discussion. The risk scoring system we developed can effectively distinguish between high-risk and low-risk patients. The nomogram we created had a concordance index of 0.736, and the calibration curve showed good agreement between the predicted and observed outcomes.

Preclinical and clinical activity of DZD1516, a full blood–brain barrier-penetrant, highly selective HER2 inhibitor

BackgroundPatients with HER2-positive metastatic breast cancer (MBC) are at high risk of developing central nervous system (CNS) metastases. A potent and selective HER2 inhibitor with good blood–brain barrier (BBB) penetration is highly desirable.MethodsThe design and structure–activity relationship of DZD1516 was described. The potency and selectivity of DZD1516 were determined by enzymatic and cellular assays. The antitumor activity of DZD1516 monotherapy or in combination with HER2 antibody–drug conjugate was assessed in CNS and subcutaneous xenograft mouse models. A phase 1 first-in-human study evaluated the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DZD1516 in patients with HER2+ MBC who relapsed from standard of care.ResultsDZD1516 showed good selectivity against HER2 over wild-type EGFR in vitro and potent antitumor activity in vivo. Twenty-three patients were enrolled and received DZD1516 monotherapy treatment across six dose levels (25–300 mg, twice daily). Dose-limiting toxicities were reported at 300 mg, and thus 250 mg was defined as the maximum tolerated dose. The most common adverse events included headache, vomiting, and hemoglobin decreased. No diarrhea or skin rash was observed at ≤ 250 mg. The mean Kp,uu,CSF was 2.1 for DZD1516 and 0.76 for its active metabolite DZ2678. With median seven lines of prior systemic therapy, the best antitumor efficacy in intracranial, extracranial and overall lesions was stable disease.ConclusionsDZD1516 provides positive proof of concept for an optimal HER2 inhibitor with high BBB penetration and HER2 selectivity. Further clinical evaluation of DZD1516 is warranted, with the RP2D being 250 mg BID.Clinicaltrials.gov identifier NCT04509596. Registered on August 12, 2020; Chinadrugtrial: CTR20202424 Registered on December 18, 2020.

T cells expressing a HER2-specific chimeric antigen receptor as treatment for breast cancer.

Human endothelial growth factor receptor-2 (HER2) is a leucine kinase receptor that is closely related to cell growth and differentiation. It is very weakly expressed in a few epithelial cells in normal tissue. Abnormal expression of HER2 usually leads to sustained activation of downstream signaling pathways, enabling epithelial cell growth, proliferation, and differentiation; this disturbs normal physiological processes and causes tumor formation. Overexpression of HER2 is related to the occurrence and development of breast cancer. HER2 has become a well-established immunotherapy target for breast cancer. We chose to construct a second-generation CAR targeting HER 2 to test whether it kills breast cancer. We constructed a second-generation CAR molecule targeting HER2, and we generated cells expressing this second-generation CAR through lentivirus infection of T lymphocytes. LDH assay and flow cytometry were perform to detect the effect of cells and animal models. The result indicated that the CARHER2 T cells could selectively kill cells with high Her2 expression. The PBMC-activated/CARHer2 cells had stronger in vivo tumor suppressive activity than PBMC-activated cells, and administration of PBMC-activated/CARHer2 cells significantly improved the survival of tumor-bearing mice, and induced the production of more Th1 cytokines in tumor-bearing NSG mice. We prove that the generated T cells carrying the second-generation CARHer2 molecule could effectively guide immune effector cells to identify and kill HER2-positive tumor cells and inhibit tumors in model mice.

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T.

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising.Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence.HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy.HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl.HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.

Clinicopathologic features of early stage HER2-low breast cancer.

e12545 Background: Breast cancer (BC) is the leading cause of cancer deaths in women worldwide. Traditionally, BC is divided into 3 categories: hormone receptor positive (HR+) if tumors overexpress either estrogen or progesterone receptors, HER2-positive (Pos) if tumors overexpress the human epidermal growth factor receptor protein (HER2), and triple negative if tumors lack overexpression of HER2, ER, or PR. 15% of all BC are HER2-Pos, which has poorer outcomes when compared to HER2-Neg. Early HER2 targeted therapies only showed benefit for treating tumors with high enough HER2 overexpression. Thus, HER2-Pos refers to tumors which stained 3+ for HER2 on immunohistochemistry (IHC) or 2+ with a positive result on amplification of the HER2 gene by fluorescence in-situ hybridization (FISH). In the recent DESTINY-Breast04 study, the drug trastuzumab deruxtecan showed benefit in treating metastatic BC patients with low levels of HER2 overexpression. HER2-Low, now a clinically relevant group, is defined as 1+ by IHC or 2+ and FISH negative. However, early stage HER2-Low BC is treated the same as HER2-Neg by the current standard of care. Large registry trials have described the HER2-Low patient population, mostly in the metastatic setting. Our study aims to describe the prevalence, pathological and clinical aspects of HER2-Low when compared to HER2-Neg BC in early stage BC patients within a community practice setting in the United States. Methods: This retrospective study analyzed 1,106 patients of which 120 were excluded due to being HER2-Pos. 986 HER2-Neg and HER2-Low patients of stages I-III were included. Patients had diagnosis dates between January 1st, 2015 and December 31st, 2022. Categorical and continuous analyses (two-proportion Z test) were performed to describe differences between HER2-Low and HER2-Neg patients at the time of diagnosis in age, tumor histology and grade, and need for chemotherapy. Results: 49% of patients (n = 1,106) were found to be HER2-Low. 91% of HER2-low patients were HR+, compared to 87% of HER2-Neg who were HR+ (p = 0.04). HER2-Low patients were more likely than HER2-Neg to have ductal carcinoma (82% vs. 75%, p = 0.01) and to be classified as grade 2 (58% vs. 48%, p = 0.001). The median age of patients with HER2-Low was 69 compared to 70 in patients with HER2-Neg disease. 36% of HER2-Low patients needed chemotherapy (neoadjuvant or adjuvant) compared to 35% who were HER2-Neg (p = 0.74). Conclusions: Our study revealed that HER2-Low patients were more likely than HER2-Neg patients to be HR+, grade 2, and to have ductal histology. In this early stage BC population, there was no difference in need for chemotherapy between HER2-Low and HER2-Neg patients. Future studies with a more racially diverse patient population looking at parameters of progression-free survival and overall survival in HER2-Low early stage BC population should be pursued.

The changes of HER3 expression in head and neck cancer patients treated with induction chemotherapy.

e18004 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is broadly expressed in various types of cancer and is associated with poor prognosis. Recent evidence supports HER3 plays a role in acquired resistance to chemotherapy. The change of HER3 expression by chemotherapy in head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods: We evaluated 95 patients with locally advanced HNSCC who had received induction chemotherapy (ICT) between 2009 and 2019. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. H-score of membrane staining was evaluated. Results: Median age of patients was 64 years, and 85% were male. Primary locations included oropharynx (54.8%), hypopharynx (21.5%), oral cavity (17.2%), and salivary gland (6.5%). Mean H-score of HER3 in tumor tissues was 77.6 ± 7.9. H-score of HER3 in HPV-positive tumors was higher than HPV-negative tumors (p = 0.019), which was more pronounced in oropharynx and hypopharynx tumors. While, H-score of HER3 in adjacent normal tissues of these tumors did not significantly differ. Compared with the non-smokers, tumor tissues of current or ex-smokers demonstrated higher H-score of HER3 (p = 0.033), independent of tumor location. In contrast, in adjacent normal tissues, no significant differences of H-score of HER3 were observed depending on the smoking status. The H-score higher than 100 was defined as HER3-high group. Among oropharynx and hypopharynx tumors with positive correlation between H-score of HER3 and HPV status (n = 56), HER3-high group was significantly correlated with HPV positivity (p = 0.009) and good response to ICT (p = 0.035). Good clinical response was also strongly related to HPV-positivity (p = 0.001). In addition, there was a tendency for longer overall survival in HER3-high group than low HER3 expression group (93 vs. 70 months, p = 0.053). The paired tumor samples collected at pre- and post-ICT (n = 21) were analyzed. HER3 expressions in adjacent normal tissue were higher at post-ICT compared to pre-ICT (p = 0.003), and these results were more pronounced in the non-smoking group, but no differences were found according to HPV-status. Conclusions: Taken together, our results showed that H-score of HER3 was significantly higher in both HPV-positive and smoking group. Interestingly, high HER3 expression in oropharynx and hypopharynx tumors was associated with HPV positivity and good clinical response to ICT. Additional analysis of tumor immune microenvironment in tumor tissues are underway.

Nomograms for predicting survival in patients with HER2 negative breast cancer.

e12528 Background: It is increasingly recognized that there is considerable heterogeneity in HER2-negative breast cancer. The clinicopathological characteristics differed between HER2-low and HER2-0 breast cancer evaluated by immunohistochemistry (IHC) or in situ hybridization (ISH). HER2-low breast cancer may benefit from novel antibody-drug conjugates. However, the immunohistochemical agreement of HER2-low is poor. We aimed to construct nomograms to predict the survival of HER2-negative breast cancer and to evaluate the effects of HER2 on survival at the RNA transcript level. Methods: A retrospective analysis was performed on breast cancer patients who were evaluated HER2-negative by IHC or ISH in the TCGA database. Univariate and multivariate cox regression analysis was used to evaluate whether the HER2 RNA transcript level and clinical parameters were independent predictors for overall survival (OS), progression-free survival (PFS), and Disease-Specific Survival (DSS), and nomograms were generated for the prediction of 1-, 2-, 3-, 5-year OS, PFS, and DSS based on significant parameters. Survival curves were drawn using the Kaplan-Meier methods to compare mortality outcomes according to HER2 RNA transcript level (75% vs. 25%). Results: A total of 769 HER2-negative patients were included in the analysis. The median age of patients was 56 years (range 26-30), and the majority were White (80.3%). Univariate and multivariate cox regression analysis showed that age, pN_stage, and pM_stage were independent predictors for OS, but HER2 was not. The C-index of a nomogram based on these three parameters was 0.723 (95%CI, 0.6555-1). HER2, pT_stage, pN_stage, and pM_stage were analyzed as independent predictors of PFS. HER2, pN_stage, and pM_stage were independent predictors of DSS. The C-indexes of established nomograms were 0.734 (95%CI, 0.672-1) and 0.775 (95%CI, 0.686-1) respectively. PFS and DSS were significantly better for patients with high HER2 RNA transcript levels (PFS, log-rank p = 0.00446, HR = 0.352; DSS, log-rank p = 0.0119, HR = 0.247). Conclusions: In the TCGA database, the HER2 RNA transcript level was an independent predictor for PFS and DSS in HER2-negative breast cancer. Stratification of patients at the RNA level can help clinicians optimize treatment for HER2-negative breast cancer.

Deep multi-omic analyses to identify targetable pathways in desmoplastic small round cell tumor (DSRCT) with opportunities for clinical intervention.

e22006 Background: DSRCT is an extremely rare and highly aggressive soft-tissue sarcoma affecting adolescents and young adults with male predominance. The 5-year overall survival is &lt; 25% despite intensive multi-modality treatment including aggressive surgical resection, radiotherapy, chemotherapy, and targeted therapy. Therefore, more effective and innovative therapeutic options are urgently needed. Recent advances in genomic, epigenomic, proteomic, and immune profiling have uncovered a large number of therapeutic targets that are transforming the field of cancer medicine. This study aimed to identify novel targetable pathways in DSRCT using a deep multi-omics-based platform. Methods: Three patients with biopsy proven metastatic DSRCT were enrolled under an IRB-approved observational study within the “Serial Measurements of Molecular and Architectural Responses to Therapy” (SMMART) program at OHSU. Biopsies taken at enrollment were analyzed with targeted DNA sequencing, whole transcriptome sequencing, immunohistochemistry (IHC), NanoString GeoMx digital spatial profiling (DSP) multiplexed proteomics, single cell cyclic immunofluorescence (Cyc-IF), and reverse phase protein array (RPPA). Results: Multiple clinically relevant targets were identified, including HER2, androgen receptor (AR), elevated DNA damage response, and MAPK and mTOR signaling pathways. All three DSRCTs expressed HER2: clinical IHC identified HER2 low (1+) to HER2 equivocal (2+) expression; DSP analysis showed high HER2 and phospho-HER2 expression; and HER2 RNA expression was elevated compared to the TCGA sarcoma cohort. Two of the three patients displayed high AR protein expression, assessed by clinical IHC (90-100% positive cells) and DSP (top quartile compared to a 20 mixed-subtype breast cancer cohort). Strong evidence of DNA damage response was demonstrated at both RNA and protein levels, and RPPA revealed high PARP1, PAR, p-ATM, p-RPA32, and RAD51. Within cell signaling pathways, one DSRCT exhibited high total and phospho-ERK1/2 protein across all clinical and exploratory assays, while another patient displayed evidence of elevated mTOR pathway signaling by RPPA. Conclusions: Overall, this multi-omic analysis provided new insights into possible therapeutic targets for this rare and difficult to treat sarcoma subtype. Results from this study will be further validated in vivo using patient-derived xenograft (PDX) models, providing support for development of multiple early phase trials.

Prevalence of low HER2 expression in breast cancer patients: An Indian experience.

e12543 Background: With the recent advent of trastuzumab deruxtecan and encouraging results of the DESTINY-Breast-04 trial, low HER2 expression has emerged as a clinically significant therapeutic biomarker. The prevalence of low HER2 expression is unexplored in Indian population. Methods: This is a single center ambispective observational study conducted over a period of 4 years (1st January 2019 to 31st January 2023) at Dr. B.R.A, I.R.C.H, All India Institute of Medical Sciences (AIIMS), New Delhi, India. Consecutive female breast cancer patients registered during the study period with available clinical-pathological details were eligible for study inclusion. The primary objective of the study was to assess the prevalence of HER2 low breast cancer. HER2 low was defined as having HER expression by immunohistochemistry (IHC) of 1+ or 2+ with no amplification of the HER2gene detected with a fluorescent in situ hybridization (FISH) assay. Patients were divided into three categories: HER-2 high (IHC3+, IHC2+/FISH+), HER-2 low (IHC 1+, IHC2+/FISH-), and HER2-0 (HER2 negative, IHC:0). Staging description of the cases were done as per anatomic TNM classification according to AJCC 7th edition. Results: A total of 2200 female breast cancer were registered during the study period, of which 1932 patients had available clinical-pathological details. Among 1932 patients, 600 patients (31%) had high HER2 expression and remaining 1332 patients had HER2 low/HER2-0 expression. Of the 1332 cases, the median age was 49 years (range 18–91 years). The stage distribution was stage I, 94 (7.06%); stage II, 409 (30.70%); stage III, 528 (39.64%); and stage IV, 301 (22.59%). Eight hundred patients (60.06%) were hormone positive, and 532 (39.94%) cases were hormone negative. The prevalence of HER2 low expression was 798 (59.9%). It was observed in 640 (80%) of hormone positive cases and 158 (29.69%) hormone-negative cases (p &lt; 0.001). Rest 534 cases were HER2 0 [160 cases (20%) were hormone positive, and 374 cases (70.31%) were hormone negative]. Conclusions: This is the first study from India to document the prevalence of low HER2 expression in female breast cancers. We observed the prevalence to be around 60 % in our population, which is more in hormone receptor-positive breast cancer. The estimated prevalence is higher than that observed in Western population, and it underscores the need to systematically categorize these patients for therapeutic decision making at presentation.

Molecular and clinical correlates of HER3 expression highlights its potential role as a therapeutic target in melanoma

Overexpression of the epidermal growth factor receptor family member HER3 (erbB3) has been implicated in several types of cancer and recently drugs targeting HER3 have shown promising clinical activity. In melanoma, HER3 overexpression has been linked to both metastasis formation and resistance to drug therapy in cell culture models. Here, we sought to characterise the expression of HER3 in 187 melanoma biopsies (149 cutaneous, 38 mucosal) using immunohistochemistry, as well as to analyse the association between HER3 expression and molecular, clinical and pathological variables. A subset of the cutaneous melanoma specimens was taken prior to treatment with immune checkpoint blockade therapy (pre-ICB) (n=79). HER3 expression (≥1+) was observed in 136 of 187 samples (∼73%). HER3 expression was found to be markedly lower in the mucosal melanomas, with 17 of the 38 tumours (∼45%) demonstrating no HER3 expression. In cutaneous melanomas, there was a negative association between HER3 expression and mutational load, a positive association with NRAS mutational status, and a trend of negative association with PD-L1 expression. In the pre-ICB cohort, an association was found between high HER3 expression (≥2+) and overall survival after anti-PD-1-based immunotherapy. Overall, our results indicate that HER3 is a promising therapeutic avenue in cutaneous melanoma worthy of further clinical evaluation.

3MO HER2 expression and early response to patritumab deruxtecan (HER3-DXd) in early-stage hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC): A correlative analysis from SOLTI-TOT-HER3 trial

HER3-DXd and trastuzumab deruxtecan (T-DXd) are antibody-drug-conjugates (ADCs) directed against HER3 and HER2, respectively. Both drugs have shown efficacy in patients (pt) with HR+/HER2- BC. Baseline HER3 protein or mRNA levels do not predict efficacy from HER3-DXd. Here, we aimed to evaluate the association of early response to HER3-DXd with HER2 levels. TOT-HER3 (NCT04610528), a window-of-opportunity trial, evaluated a single dose of HER3-DXd in 77 pts with untreated HR+/HER2- early BC in Part A (6.4 mg/kg) and 17 in Part B (5.6 mg/kg). Primary objective was CelTIL score [a surrogate of response that combines tumor cellularity and tumor-infiltrating lymphocytes] variation between baseline and day 21 tumor samples. Here, CelTIL response was defined as an absolute increase of 20 at day 21. HER2 expression was determined by immunohistochemistry (IHC). RNA and DNA were purified from baseline FFPE tumor samples and analyzed using the nCounter and the VHIO-300 NGS platforms, respectively. Logistic regression models and area under the ROC Curve (AUC) estimated the performance of HER2 IHC, mRNA or copy-number (CN) levels with CelTIL response. In TOT-HER3 Part A, HER2 IHC status was 32% HER2 0, 38% HER2 1+ and 30% HER2 2+. Distribution of high CelTIL responders was 40% in HER2 0, 45% in HER2 1+ and 13% in HER2 2+ (p=0.034). High ERBB2 mRNA and RNA-based HER2 amplicon signatures were significantly associated with low CelTIL response (ERBB2: Odds Ratio [OR]=0.34, p<0.001, AUC=0.71; and HER2 amplicon: OR=0.20, p=0.005, AUC=0.74). The association of ERBB2 mRNA with CelTIL response was independent of HER2 IHC expression (p=0.002), PAM50 subtype (p=0.001) or proliferation (p=0.003). High HER2 CN signal was found associated with low CelTIL response (p=0.005). The association of HER2 IHC and ERBB2 mRNA with CelTIL response will be independently validated in TOT-HER3 Part B. Low HER2 IHC, mRNA or CN levels in early-stage HR+/HER2- BC are associated with high response to HER3-DXd. HER3-DXd might be highly active in HR+ BC with very low HER2 levels, thereby offering a new therapeutic paradigm to this subset of patients.

Engineering a HER2-CAR-NK Cell Secreting Soluble Programmed Cell Death Protein with Superior Antitumor Efficacy

A new therapy strategy for relapsing patients who have received trastuzumab treatment urgently needs to be explored. HER2-specific chimeric antigen receptor (CAR)-expressing NK cells are being rapidly developed for solid tumor therapy, as they have many advantages over HER2-CAR-T cells. Endogenous soluble PD-1 (sPD-1) from the PD-1 extracellular domain blocks PD-1/PD-L1 interaction to promote cancer immunology. Herein, we engineered a new HER2-CAR-NK cell that co-expresses sPD-1 (designed as sPD-1-CAR-NK cells) and assessed its cytotoxic activities toward various cancer cells, activation of immunity and sPD-1 release in vitro and in mouse models bearing breast cancer cells with high HER2 expression, with or without trastuzumab resistance. We demonstrated that sPD-1-CAR-NK cells were able to release bioactive sPD-1, thereby enhancing the cytolytic activities of HER2-CAR-NK cells against HER2 and PD-L1 highly expressing target cells accompanied by increases in the secretion of perforin, granzyme B and IFN-γ. In vivo, sPD-1-CAR-NK cells had superior immunological anticancer efficacy compared to HER2-CAR-NK cells, and they had advantages over HER2-CAR-NK cells in the intraperitoneal injection of sPD-1. Moreover, the infiltration and activation of NK and T cells into tumor tissue were increased in mice with sPD-1-CAR-NK cells. There was no significant change in the body temperature, organ tissue and body weight in all groups except for the group with the PD-1 injection. Together, these data indicate that HER2-specific sPD-1-CAR-NK cells can transport sPD-1 into cancer tissues with high HER2 expression, further improving the efficacy of HER-CAR-NK cells without obvious side effects. sPD-1-CAR-NK is a promising cytotherapeutic agent for patients bearing HER2-positive breast cancer, including those with trastuzumab resistance.

Abstract 968: Evaluation of Her2 RNA expression as a potential predictive biomarker for anti-Her2 therapy

Abstract Trastuzumab deruxtecan (Enhertu) is effective in "HER2 Low" breast cancer, defined by 1+ or 2+ expression by immunohistochemistry (IHC). Interest has now turned toward defining a sub-population of IHC 0+ tumors that may have HER2 expression below the limit of IHC detection/quantification and may thus also be responsive. We previously validated a high dynamic range HER2 RNA expression assay run as part of our comprehensive genomic profiling test, StrataNGS. Herein, we evaluated the HER2 RNA expression data together with copy number and clinical outcome data from the Strata Clinical Molecular Database (SCMD) in advanced breast cancer (n = 3,063) and other advanced solid tumors (n = 26,715). As expected, HER2 gene expression was significantly higher in tumors with DNA amplification (&amp;gt;=6 copies; median: 13.9 vs. 10.0 in log2 units; p &amp;lt; 1e-100). Despite similar copy number levels in amplified breast vs. other cancers (median: 21.8 vs. 19.8 copies), HER2 expression levels were ~2-fold higher (median: 14.5 vs. 13.5; p = 1.3e-10). Similarly, HER2 expression levels were higher in non-amplified breast vs. other cancers (median: 10.7 vs. 9.9; p&amp;lt;1e-100), suggesting that DNA amplification and cell lineage affect HER2 expression. Using our previously validated HER2 threshold, among 75 eligible SCMD breast cancer patients treated with 1st or 2nd line systemic trastuzumab or pertuzumab containing therapy, HER2 RNA High patients (n=46, 59%) had significantly longer time to next therapy (TTNT) compared to HER2 RNA Not High patients (median TTNT 26.9 vs. 5.6 months, adjusted hazard ratio 0.31, p=0.005 when adjusted for 1st vs. 2nd line, pertuzumab inclusion, and inclusion of chemotherapy or hormonal therapy). In patients with available IHC data (n = 388), HER2 RNA expression trended with IHC across the 0-3+ range, however, while 3+ tumors had distinctly high RNA expression (median: 14.4), 0-2+ tumors had lower expression with overlapping distributions (median: 10.5, 10.9, 11.5, respectively), suggesting that 0-2+ tumors do not represent distinct biological groups, but rather a continuum of low expression. We defined a HER2 RNA Low threshold (&amp;gt;10.6), corresponding to the top 75% of IHC 1-2+ breast cancers. Importantly, at this threshold, nearly half (44.1%) of 0+ breast cancers were also classified as HER2 RNA Low. Additionally, 25.8% of all non-breast solid tumors were classified as HER2 RNA Low. Given that HER2 RNA High predicted benefit from 1st generation anti-HER2 therapies, future studies should consider HER2 RNA Low as an alternative biomarker to Her2 IHC Low, with the opportunity to further expand trastuzumab deruxtecan use into the IHC 0+ breast cancer population and potentially to additional solid tumors. Citation Format: Laura E. Lamb, Nickolay A. Khazanov, Daniel H. Hovelson, Kat Kwiatkowski, D. Bryan Johnson, Daniel R. Rhodes, Scott A. Tomlins. Evaluation of Her2 RNA expression as a potential predictive biomarker for anti-Her2 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 968.

Abstract 6337: Tri-specific CD3 x HER2 x HER2 antibodies in 1:2 format recognizing two different epitopes on HER2 demonstrate selective activity on human cancer cells with high HER2 expression

Abstract Human epidermal growth factor receptor 2 (HER2) is a membrane tyrosine kinase that was found to be amplified and overexpressed in various human cancers including breast cancer. Monoclonal antibodies targeting HER2 such as trastuzumab and pertuzumab provide good therapeutic benefits in patients with high HER2 expression tumors, however, not all patients respond to the treatment, and many suffer recurrence. There is still a large unmet need for targeted therapies to treat HER2 high expression tumors. T cell engaging antibodies that direct T cells to kill cancer cells by simultaneously binding to CD3 on T cells and tumor-associated antigens (TAA) on cancer cells have emerged as a promising new approach. Such therapeutics are potent, but safe use of them depends on selectivity of the TAA engaged. Though HER2 is highly overexpressed in ~ 20% of breast cancer, expression of HER2 is not restricted to cancer cells and present at low level in normal tissues. The capability of selectively targeting cancer cells over HER2 low expressing normal tissues is critical to the safety of CD3 x HER2 antibodies. It is noteworthy to mention that unlike antibody-drug conjugate (ADC) therapeutics, CD3 x HER2 antibodies might not be a good approach to target HER2 low/neg expression tumors due to the difference in mechanism of action. CMG6A19 is a 150 kD IgG-like tri-specific CD3 x HER2 x HER2 T cell-engaging antibody generated on Chimagen’s TRIAD platform. It is capable of simultaneously recognizing and binding to two different epitopes on HER2 proteins. Multiple human cell lines with different levels of HER2 expression were used as targets in the antibody-mediated hPBMC killing assays in vitro and in vivo, among which SK-BR-3, KPL-4 and BT474 have high HER2 expression as seen in HER2 positive cancers, HT55 and JIMT1 have medium level of HER2 expression, and PC3 and MCF7 have HER2 expression level that is close to in normal tissues. Conventional 1:1 IgG-like CD3 x HER2 bispecific antibodies were used as control. Results showed that CMG6A19 demonstrated higher tumor inhibition effect compared to control antibodies in SK-BR-3/KPL-4/BT474 models, meanwhile remarkably weaker effect in PC3/MCF7 models suggesting better safety in HER2 low expressing tissues. This selectivity of reactivity towards HER2 high expressing cells is likely due to the unique orientation of the antibody which allows better T cell activation when antigen abundance is high. Preliminary preclinical study in cynomolgus monkeys suggested that the antibody was well tolerated in monkeys at the dosage of 20 mpk and had a half-life that is comparable to conventional antibodies. CD3 x HER2 x HER2 tri-specific antibodies selectively showed potent anti-tumor activity on HER2 high expressing cells and good safety on HER2 low expressing cells. Current findings warrant further development in human. Citation Format: Jie Zhang, Xiaoqing Wang, Zhenhao Zhou. Tri-specific CD3 x HER2 x HER2 antibodies in 1:2 format recognizing two different epitopes on HER2 demonstrate selective activity on human cancer cells with high HER2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6337.

Treatment options for molecular subtypes of endometrial cancer in 2023.

This article reviews treatment strategies in endometrial cancer by molecular subtype. The Cancer Genome Atlas (TCGA) classifies four molecular subtypes of endometrial cancer - mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H), copy number high (CNH)/p53abn, copy number low (CNL)/no specific molecular profile (NSMP), and POLEmut - which are validated and highly prognostic. Treatment consideration by subtype is now recommended. FDA-approved immune checkpoint inhibitors (ICIs) include pembrolizumab and dostarlimab for previously treated dMMR/MSI-H EC, and pembrolizumab/lenvatinib for mismatch repair-proficient/microsatellite-stable endometrial cancer, including CNH/p53abn and CNL/NSMP. ICIs are being studied as first-line therapy in advanced/recurrent endometrial cancer by MMR status, as well as in combination with other targeted agents. Trastuzumab is NCCN compendium listed for HER2-positive serous endometrial cancer, which are primarily p53-abnormal. Antibody-drug conjugates targeting low and high HER2 levels show promise in breast cancer, and are beginning to be studied in endometrial cancer. In addition to hormonal therapy, maintenance therapy with selinexor (XPO1-inhibitor) showed potential benefit in p53 -wildtype endometrial cancer and is being investigated prospectively. Multiple prospective trials are evaluating de-escalation of care for POLEmut endometrial cancer given favorable survival regardless of adjuvant therapy. Molecular subtyping has important prognostic and therapeutic implications and should be guiding patient management and clinical trial design in endometrial cancer.

Abstract P4-07-23: Results of a Phase 2 Trial of Combination Immunotherapy with Concurrent Nelipepimut-S + GM-CSF and Trastuzumab in High-risk HER2+ Breast Cancer Patients

Abstract INTRODUCTION: The HER2-targeted peptide vaccine nelipepimut-S + GM-CSF (NeuVax) has been shown to be safe, immunogenic, and potentially synergistic with trastuzumab. Here we present the results of a randomized phase 2 trial assessing the ability of nelipepimut-S/GM-CSF versus GM-CSF alone, added to the standard adjuvant Trastuzumab, to prevent recurrences in high-risk HER2-positive breast cancer patients. METHODS: The study was a multi-center, prospective, randomized, controlled, single-blinded, phase 2 trial. Enrolled patients had high risk HER2+ breast cancer defined by the presence of residual disease post neoadjuvant therapy or by the presence of positive lymph nodes after upfront surgery. Eligible patients had completed an approved trastuzumab-chemotherapy containing regimen and they were receiving adjuvant Trastuzumab monotherapy. Enrollment was limited to patients with HLA-A2, A3, A24, and/or A26 alleles. Patients received intradermal injections of nelipepimut-S + GM-CSF or placebo + GM-CSF every three weeks for six total vaccinations with concurrent, standard monotherapy with iv trastuzumab. After completion of the primary vaccine series, booster inoculations were administered every six months for four doses. The primary outcome measure was invasive disease-free survival (iDFS) at 36 months. Secondary outcome measures were distant recurrence-free survival (DRFS), toxicity assessment, and evaluation of immune response. RESULTS: 100 patients were enrolled and randomized 1:1 to nelipepimut-S/GM-CSF or GM-CSF alone. There were no significant clinicopathologic differences between the groups. There was no difference in related local (p=0.49) or systemic toxicities (p=0.41). Kaplan-Meier estimates of iDFS at 36 months were 79% in the nelipepimut-S arm and 92% in the placebo arm (log rank, p=0.11). DRFS at 36 months was estimated to be 90% in the nelipepimut-S arm and 95% in the placebo arm (log rank, p=0.40). Delayed type hypersensitivity (DTH) response to nelipepimut-S was measured and considered positive if there was more than 5 mm induration after 48 hours. DTH response converted from negative to positive in 11% of patients in the vaccine group versus 5% of patients in the placebo group (p=0.36). In both groups, iDFS at 36 months was 100% for those with a positive DTH response post-inoculation and 88% for those with a negative DTH response post-inoculation (log rank, p=0.29). CONCLUSION: Combination immunotherapy with concurrent nelipepimut-S + GM-CSF and trastuzumab is safe, however there was no difference in iDFS or DRFS among high-risk HER2+ breast cancer patients who received nelipepimut-S + GM-CSF compared to GM-CSF alone. We observed a trend towards improved iDFS in patients with a positive DTH response to nelipepimut-S, though it was not statistically significant. Citation Format: Ankur Tiwari, Guy Clifton, Carmen Calfa, Gheath Alatrash, Jarrod Holmes, Isabelle Bedrosian, George Peoples, Elizabeth A. Mittendorf. Results of a Phase 2 Trial of Combination Immunotherapy with Concurrent Nelipepimut-S + GM-CSF and Trastuzumab in High-risk HER2+ Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-23.