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Background: The emergence of anti-HER2 antibody–drug conjugates (ADCs) gave rise to the concept of HER2-low breast cancer (BC). HER2-low BC, which refers to a subgroup of HER2-negative BC with relatively higher HER2 expression (defined as 1+ or 2+ by immunohistochemistry (IHC) staining, without ERBB2 amplification), represents a rather large part of all BCs. However, the molecular nature and internal heterogeneity of HER2-low breast cancer remain obscure, and little is known about the ethnic differences of HER2-low BC. These limitations prevent us from a more precise patient selection and better drug combination strategies in the era of ADCs. To provide a comprehensive and intensive landscape of HER2-low BCs, we characterized HER2-low BCs both clinically and molecularly, which may help clinicians to achieve a more precise clinical management of these patients. Patients and methods: We established a HER2-low BC cohort (N=441) in early-stage Chinese patients and included HER2-0 (N=114) and HER2-positive (N=181) tumors as auxiliary cohorts to characterize HER2-low breast cancers both clinically and molecularly. Whole-exome sequencing, copy number variation assays, RNA sequencing and isobaric quantitative proteomics were conducted to obtain multiomics data. We compared the clinicopathological and molecular features between HER2-low tumors and other HER2 status subgroups stratified and not stratified hormone receptor (HR) status to clarify the distinctness of HER2-low BCs. And we analyzed the internal heterogeneity and ethnic difference of HER2-low BCs by characterizing a distinct subgroup of patients with unique driving mechanisms. Results: HER2-low BCs showed different molecular manifestations from HER2-0 BCs in different HR subgroups. In the HR-negative subgroup, HER2-low BCs consisted of more non-basal-like subtypes than HER2-0 tumors (40.0% vs. 9.1%, P = 0.002), which was an East Asian-specific phenomenon absent in Western cohorts. Also, HR-negative HER2-low BCs showed significant internal molecular heterogeneity, of which basal-like tumors closely mimicked HER2-0 BCs, whereas non-basal-like tumors were similar to HER2-positive BCs. These non-basal-like tumors were mostly categorized as HER2-enriched and luminal androgen receptor (LAR) subtypes. These molecularly distinct tumors might be driven by frequent mutation in PIK3CA and overexpression of FGFR4 and PTK6, which may also serve as therapeutic targets. These results have also been proved in a triple negative breast cancer cohort we reported previously. In contrast, in the HR-positive subgroup, HER2-low BCs showed no large-scale molecular difference from HER2-0 BCs or internal heterogeneity. However, HER2-low patients showed significantly better distant metastasis-free survival than HER2-0 patients (P = 0.029), which might be attributed to the lower loss/deletion levels of 17q11.12 and 17q21.31 in HER2-low breast cancers, in which genes including NF1 and BRCA1 are located. Conclusions: We reported the largest single-center multiomics HER2-low BC cohort in East Asian hitherto, and revealed its molecular nature, internal heterogeneity and ethnic difference. Compared with HR-positive diseases, HER2-low BCs in the HR-negative subgroup were more likely to be a molecularly distinct entity from HER2-0 tumors. Furthermore, HR-negative HER2-low BC also accommodates higher internal heterogeneity, which was ethnicity-specific in our East Asian cohort and may infer a different treatment response. Our work emphasized the need of a more precise stratification within HER2-low BCs and across ethnic groups, which has also been inferred by the results in the subgroup analysis of DESTINY-Breast04 trial. Citation Format: Lei-Jie Dai, Ding Ma, Yi Xiao, Xi Jin, Song-Yang Wu, Ya-Xin Zhao, Han Wang, Wen-Tao Yang, Yi-Zhou Jiang, Zhi-Ming Shao. HER2-09 Multiomics Profiling Characterizes Distinct HER2-low Breast Cancer Subgroups in the East Asian Population [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 HER2-09.

PDF - 59KB, Quantitative RT-PCR analyses for Pim-1, Pim-2, and Pim-3.

PDF - 28KB, Supplemental Table 1. Primer sequences for PCR to generate the coding region of the gene below. Supplemental Table 2. Primer sequences for quantitative RT-PCR. Supplemental Table 3. The proteins interact with TCTP and interaction region.

Translationally controlled tumor protein (TCTP/TPT1) was identified from a yeast 2-hybrid screen and shown to interact with Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase activity. TCTP was aberrantly expressed in human pancreatic cancer cells and malignant ductal epithelial cells, but not in normal pancreatic duct epithelial cells adjacent to tumor foci of human pancreatic cancer tissue. Moreover, TCTP colocalized with Pim-3 both in human pancreatic cancer cells and in clinical tissues. Mapping studies revealed that the interaction between Pim-3 and TCTP occurred through the C-terminal region of Pim-3 and N-terminal region of TCTP. Although Pim-3 had no effect on TCTP expression or phosphorylation, overexpression of TCTP increased the amount of Pim-3 in a dose-dependent manner. Interestingly, RNAi-mediated ablation of TCTP expression reduced Pim-3 protein but not mRNA, through a mechanism involving the ubiquitin–proteasome degradation system. As a consequence of Pim-3 instability and subsequent degradation, tumor growth in vitro and in vivo was inhibited by arresting cell-cycle progression and enhancing apoptosis. Furthermore, TCTP and Pim-3 expression were significantly correlated in pancreatic adenocarcinoma specimens, and patients with highly expressed TCTP and Pim-3 presented with a more advanced tumor stage. These observations indicate that TCTP enhances Pim-3 stability to simultaneously promote and prevent cell-cycle progression and apoptosis, respectively. Hence, TCTP and Pim-3 serve a pivotal role in human pancreatic cancer with important ramifications for clinical diagnostic and therapeutic implications.Implications: The present study provides a new idea and experimental evidence for recognizing TCTP/Pim-3 pathway as a target for therapy in human pancreatic cancer. Mol Cancer Res; 11(12); 1508–20. ©2013 AACR.

PDF - 30KB, Supplementary Materials an Methods including antibody, patient and tissue samples, and scoring of TCTP, and a Pim-3 in human pancreatic cancer tissues section.

Supplementary Methods

Purpose: Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance.Experimental Design: ErbB2-overexpressing and ErbB2-low-expressing breast cancer cell lines were used to compare their mitotic exit rate, survivin expression level, and apoptosis level in response to Taxol. Survivin was then down-regulated by antisense oligonucleotides to evaluate its contribution to mitotic exit and Taxol resistance in ErbB2-overexpressing breast cancer cells. At last, specific PI3K/Akt and Src inhibitors were used to investigate the involvement of these two pathways in ErbB2-mediated survivin up-regulation and Taxol resistance.Results: We found that ErbB2-overexpressing cells expressed higher levels of survivin in multiple breast cancer cell lines and patient samples. ErbB2-overexpressing cells exited M phase faster than ErbB2-low-expressing cells, which correlated with the increased resistance to Taxol-induced apoptosis. Down-regulation of survivin by antisense oligonucleotide delayed mitotic exit of ErbB2-overexpressing cells and also sensitized ErbB2-overexpressing cells to Taxol-induced apoptosis. Moreover, ErbB2 up-regulated survivin at translational level and PI3K/Akt and Src activation are involved. In addition, combination treatment of Taxol with PI3K/Akt and Src inhibitor led to increased apoptosis in ErbB2-overexpressing breast cancer cells than single treatment.Conclusions: Survivin up-regulation by ErbB2 is a critical event in ErbB2-mediated faster mitotic exit and contributes to Taxol resistance.

Supplementary Figure 1. Workflow of our research. Supplementary Figure 2. Estimation of the optimal clustering numbers of triple-negative breast cancer microenvironment phenotypes. Supplementary Figure 3. Validation of microenvironment phenotypes clustering in METABRIC cohort. Supplementary Figure 4. Validation of microenvironment phenotypes clustering in TCGA cohort. Supplementary Figure 5. Comparison of potential molecules involved in the initiation of innate immunity among microenvironment clusters in FUSCCTNBC cohort. Supplementary Figure 6. SNV and indel neoantigen load of the three microenvironment clusters in triple-negative breast cancer. Supplementary Figure 7. Chromosome instability of the three microenvironment clusters in triple-negative breast cancer. Supplementary Figure 8. Cancer testis antigen landscape of triple-negative breast cancer. Supplementary Figure 9. Gene set enrichment analysis of enriched pathways in each cluster. Supplementary Figure 10. Batch effect evaluation after "Combat" of RNA-seq and HTA microarray datasets. Supplementary Figure 11. Process and validation of mRNA clustering.

Supplementary Data from Famitinib with Camrelizumab and Nab-Paclitaxel for Advanced Immunomodulatory Triple-Negative Breast Cancer (FUTURE-C-Plus): An Open-Label, Single-Arm, Phase II Trial

Supplementary Table 1. The compendium of microenvironment cell subtypes in triple-negative breast cancer. Supplementary Table 2. Correlation of estimated microenvironment cell numbers between our compendium and CIBERSORT or MCP-counter. Supplementary Table 3. Clinicopathological characteristics of three microenvironment phenotypes in FUSCC, METABRIC and TCGA cohort. Supplementary Table 4. Prognostic value of each cell subset by univariate Cox proportional hazards model for relapse free survival. Supplementary Table 5. The signatures of ten oncogenic pathways. Supplementary Table 6. Comparison of gene mutation frequency among clusters. Supplementary Table 7. Comparison of somatic copy number alterations among clusters. Supplementary Table 8. GO and KEGG annotation of genes in cluster-specific copy number variation peaks.

Supplementary Table from Famitinib with Camrelizumab and Nab-Paclitaxel for Advanced Immunomodulatory Triple-Negative Breast Cancer (FUTURE-C-Plus): An Open-Label, Single-Arm, Phase II Trial

Supplementary Data from Mitotic Deregulation by Survivin in ErbB2-Overexpressing Breast Cancer Cells Contributes to Taxol Resistance

Supplementary Figure from Famitinib with Camrelizumab and Nab-Paclitaxel for Advanced Immunomodulatory Triple-Negative Breast Cancer (FUTURE-C-Plus): An Open-Label, Single-Arm, Phase II Trial

Purpose:Camrelizumab, an mAb against programmed cell death protein 1 (PD-1), plus nab-paclitaxel exhibited promising antitumor activity in refractory metastatic immunomodulatory triple-negative breast cancer (TNBC). Famitinib is a tyrosine kinase inhibitor targeting VEGFR2, PDGFR, and c-kit. We aimed to assess the efficacy and safety of a novel combination of famitinib, camrelizumab, and nab-paclitaxel in advanced immunomodulatory TNBC.Patients and Methods:This open-label, single-arm, phase II study enrolled patients with previously untreated, advanced, immunomodulatory TNBC (CD8 IHC staining ≥10%). Eligible patients received 20 mg of oral famitinib on days 1 to 28, 200 mg of i.v. camrelizumab on days 1 and 15, and i.v. nab-paclitaxel 100 mg/m2 on days 1, 8, and 15 in 4-week cycles. The primary endpoint was objective response rate (ORR), as assessed by investigators per RECIST v1.1. Key secondary endpoints were progression-free survival (PFS), overall survival (OS), duration of response (DOR), safety, and exploratory biomarkers.Results:Forty-eight patients were enrolled and treated. Median follow-up was 17.0 months (range, 8.7–24.3). Confirmed ORR was 81.3% [95% confidence interval (CI), 70.2–92.3], with five complete and 34 partial responses. Median PFS was 13.6 months (95% CI, 8.4–18.8), and median DOR was 14.9 months [95% CI, not estimable (NE)–NE]. Median OS was not reached. No treatment-related deaths were reported. Among 30 patients with IHC, 13 (43.3%) were programmed death-ligand 1 (PD-L1)–negative, and PD-L1 was associated with favorable response. PKD1 and KAT6A somatic mutations were associated with therapy response.Conclusions:The triplet regimen was efficacious and well tolerated in previously untreated, advanced, immunomodulatory TNBC. The randomized controlled FUTURE-SUPER trial is under way to validate our findings.See related commentary by Salgado and Loi, p. 2728

Purpose:The tumor microenvironment has a profound impact on prognosis and immunotherapy. However, the landscape of the triple-negative breast cancer (TNBC) microenvironment has not been fully understood.Experimental Design:Using the largest original multi-omics dataset of TNBC (n = 386), we conducted an extensive immunogenomic analysis to explore the heterogeneity and prognostic significance of the TNBC microenvironment. We further analyzed the potential immune escape mechanisms of TNBC.Results:The TNBC microenvironment phenotypes were classified into three heterogeneous clusters: cluster 1, the “immune-desert” cluster, with low microenvironment cell infiltration; cluster 2, the “innate immune-inactivated” cluster, with resting innate immune cells and nonimmune stromal cells infiltration; and cluster 3, the “immune-inflamed” cluster, with abundant adaptive and innate immune cells infiltration. The clustering result was validated internally with pathologic sections and externally with The Cancer Genome Atlas and METABRIC cohorts. The microenvironment clusters had significant prognostic efficacy. In terms of potential immune escape mechanisms, cluster 1 was characterized by an incapability to attract immune cells, and MYC amplification was correlated with low immune infiltration. In cluster 2, chemotaxis but inactivation of innate immunity and low tumor antigen burden might contribute to immune escape, and mutations in the PI3K-AKT pathway might be correlated with this effect. Cluster 3 featured high expression of immune checkpoint molecules.Conclusions:Our study represents a step toward personalized immunotherapy for patients with TNBC. Immune checkpoint inhibitors might be effective for “immune-inflamed” cluster, and the transformation of “cold tumors” into “hot tumors” should be considered for “immune-desert” and “innate immune-inactivated” clusters.

Backgrounds: Since human epidermal growth factor receptor 2 (HER2)-positive breast cancers may have different HER2/CEP17 ratios and HER2 copy numbers, outcomes of HER2-positive breast cancer patients treated with anti-HER2 neoadjuvant chemotherapy (NACT) may be different. The aim of this study is to explore the relationship between different groups of HER2 fluorescence in situ hybridization (FISH) positive patterns and response to anti-HER2 NACT. Methods: 513 HER2-positive invasive breast cancers who received anti-HER2 NACT in Fudan University Shanghai Cancer Center, during January 2015 to September 2020, were collected. According to FISH results, 513 patients were divided into three groups. Group A: HER2/CEP17 < 2.0 and HER2 average copy number ≥6.0; Group B: HER2/CEP17≥2.0 and HER2 average copy number ≥4.0 and < 6.0; Group C: HER2/CEP17≥2.0 and HER2 average copy number ≥6.0. Clinicopathological characteristics and pathological complete response(pCR) rates of three groups were analyzed. Results: All 513 patients were treated with anti-HER2 NACT. The anti-HER2 treatment included trastuzumab in 463 (90.3%) patients, trastuzumab plus pertuzumab in 21 (4.1%) patients, trastuzumab plus lapatinib in 3 (0.6%) patients, and trastuzumab plus pyrotinib in 1 (0.2%) patient. 25 (4.9%) cases were unblinded in clinical trials, who were treated either with trastuzumab plus pertuzumab or with trastuzumab plus pyrotinib. Among 513 patients, 237 cases (46.2%)were luminal B (hormone receptor positive and HER2 positive) and 276 cases (53.8%) were hormone receptor negative and HER2 overexpressed (HER2 overexpression type). According to IHC results, cases with HER2 1+,2+ and 3+ were 8 (1.6%), 123 (24.0%) and 382(74.5%), respectively. Among them, 0.0%, 25.2%, and 48.7% achieved pCR (p Citation Format: Hong Lv, Qian-Ming Bai, Yin Liu, Zhong-Hua Wang, Ruo-Hong Shui, Hong-Fen Lu, Xiao-Li Xu, Bao-Hua Yu, Xiao-Yu Tu, Rui Bi, Yu-Fan Cheng, Xiao-Yan Zhou, Zhi-Min Shao, Wen-Tao Yang. Response to anti-HER2 neoadjuvant chemotherapy in invasive breast cancers with different HER2 FISH-positive patterns [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 P2-13-11.

Purpose: Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance. Experimental Design: ErbB2-overexpressing and ErbB2-low-expressing breast cancer cell lines were used to compare their mitotic exit rate, survivin expression level, and apoptosis level in response to Taxol. Survivin was then down-regulated by antisense oligonucleotides to evaluate its contribution to mitotic exit and Taxol resistance in ErbB2-overexpressing breast cancer cells. At last, specific PI3K/Akt and Src inhibitors were used to investigate the involvement of these two pathways in ErbB2-mediated survivin up-regulation and Taxol resistance. Results: We found that ErbB2-overexpressing cells expressed higher levels of survivin in multiple breast cancer cell lines and patient samples. ErbB2-overexpressing cells exited M phase faster than ErbB2-low-expressing cells, which correlated with the increased resistance to Taxol-induced apoptosis. Down-regulation of survivin by antisense oligonucleotide delayed mitotic exit of ErbB2-overexpressing cells and also sensitized ErbB2-overexpressing cells to Taxol-induced apoptosis. Moreover, ErbB2 up-regulated survivin at translational level and PI3K/Akt and Src activation are involved. In addition, combination treatment of Taxol with PI3K/Akt and Src inhibitor led to increased apoptosis in ErbB2-overexpressing breast cancer cells than single treatment. Conclusions: Survivin up-regulation by ErbB2 is a critical event in ErbB2-mediated faster mitotic exit and contributes to Taxol resistance.