Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer.

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Spatial analyses of immune cell infiltration in cancer: current methods and future directions: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.

Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector-based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well-described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland.

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

High VEGFR3 Expression Reduces Doxorubicin Efficacy in Triple-Negative Breast Cancer.

Due to the lack of specific targets, cytotoxic chemotherapy still represents the common standard treatment for triple-negative breast patients. Despite the harmful effect of chemotherapy on tumor cells, there is evidence that treatment could modulate the tumor microenvironment in a way favoring the propagation of the tumor. In addition, the lymphangiogenesis process and its factors could be involved in this counter-therapeutic event. In our study, we have evaluated the expression of the main lymphangiogenic receptor VEGFR3 in two triple-negative breast cancer in vitro models, resistant or not to doxorubicin treatment. The expression of the receptor, at mRNA and protein levels, was higher in doxorubicin-resistant cells than in parental cells. In addition, we confirmed the upregulation of VEGFR3 levels after a short treatment with doxorubicin. Furthermore, VEGFR3 silencing reduced cell proliferation and migration capacities in both cell lines. Interestingly, high VEGFR3 expression was significantly positively correlated with worse survival in patients treated with chemotherapy. Furthermore, we have found that patients with high expression of VEGFR3 present shorter relapse-free survival than patients with low levels of the receptor. In conclusion, elevated VEGFR3 levels correlate with poor survival in patients and with reduced doxorubicin treatment efficacy in vitro. Our results suggest that the levels of this receptor could be a potential marker of meager doxorubicin response. Consequently, our results suggest that the combination of chemotherapy and VEGFR3 blockage could be a potentially useful therapeutic strategy for the treatment of triple-negative breast cancer.

Is Axillary Assessment of Ductal Carcinoma In Situ of the Breast Necessary in All Cases?

BACKGROUND: Staging of the axilla in women with ductal carcinoma in situ (DCIS) is a point of controversy. We aimed to assess whether there is a group of patients in whom axillary assessment can be avoided and whether the likelihood of underdiagnosis of infiltrating carcinoma is sufficient to justify this evaluation. MATERIALS AND METHODS: This was a multicenter, prospective, observational study of patients who were operated on between 2008 and 2018 in three Spanish hospitals, with a diagnosis by radiological or excisional biopsy of DCIS and clinically and radiologically negative axilla. RESULTS: A total of 530 patients with a preoperative diagnosis of DCIS were studied. An axillary assessment was performed in 77% of the patients. In 397 patients, selective sentinel lymph node biopsy was performed. Axillary involvement was found in 7.2% of all patients, which dropped to 2.15% if we only included DCIS diagnosed after a definitive anatomical pathology analysis. Underdiagnosis was correlated with the type of biopsy performed: the risk was 1.34 times as high if the biopsy was performed with a core needle. The risk of lymph node metastasis was higher when there was lymphovascular invasion and when mastectomy was performed. CONCLUSIONS: We propose an axilla management algorithm in patients with a preoperative diagnosis of DCIS. The patients who would benefit from sentinel lymph node biopsy would be those who are not candidates for breast-conserving surgery, those with a BIRADS 5 lesion biopsied by core-needle biopsy, and those whose definitive diagnosis is lymphovascular invasion.

Histological Grade and Tumor Stage Are Correlated with Expression of Receptor Activator of Nuclear Factor Kappa b (Rank) in Epithelial Ovarian Cancers.

The receptor activator of nuclear factor kappa B (RANK) is becoming recognized as a master regulator of tumorigenesis, yet its role in gynecological cancers remains mostly unexplored. We investigated whether there is a gradation of RANK protein and mRNA expression in epithelial ovarian cancer (EOC) according to malignancy and tumor staging. Immunohistochemical expression of RANK was examined in a cohort of 135 (benign n = 29, borderline n= 23 and malignant n = 83) EOCs. Wild type and truncated RANK mRNA isoform quantification was performed in a cohort of 168 (benign n = 26, borderline n = 13 and malignant n = 129) EOCs. RANK protein and mRNA values were increased in malignant vs. benign or borderline conditions across serous, mucinous and endometrioid cancer subtypes. Additionally, a trend of increased RANK values with staging was observed for the mucinous and serous histotype. Thus, increased expression of RANK appears associated with the evolution of disease to the onset of malignancy in EOC. Moreover, in some EOC histotypes, RANK expression is additionally associated with clinicopathological markers of tumor aggressiveness, suggesting a role in further progression of tumor activity.

Role of SALL4 in HER2+ Breast Cancer Progression: Regulating PI3K/AKT Pathway.

Treatment for the HER2+ breast cancer subtype is still unsatisfactory, despite breakthroughs in research. The discovery of various new molecular mechanisms of transcription factors may help to make treatment regimens more effective. The transcription factor SALL4 has been related to aggressiveness and resistance therapy in cancer. Its molecular mechanisms and involvement in various signaling pathways are unknown in the HER2+ breast cancer subtype. In this study, we have evaluated the implication of SALL4 in the HER2+ subtype through its expression in patients' samples and gain and loss of function in HER2+ cell lines. We found higher SALL4 expression in breast cancer tissues compared to healthy tissue. Interestingly, high SALL4 expression was associated with disease relapse and poor patient survival. In HER2+ cell lines, transient overexpression of SALL4 modulates PI3K/AKT signaling through regulating PTEN expression and BCL2, which increases cell survival and proliferation while reducing the efficacy of trastuzumab. SALL4 has also been observed to regulate the epithelial-mesenchymal transition and stemness features. SALL4 overexpression significantly reduced the epithelial markers E-cadherin, while it increased the mesenchymal markers ß-catenin, vimentin and fibronectin. Furthermore, it has been also observed an increased expression of MYC, an essential transcription factor for regulating epithelial-mesenchymal transition and/or cancer stem cells. Our study demonstrates, for the first time, the importance of SALL4 in the HER2+ subtype and partial regulation of trastuzumab sensitivity. It provides a viable molecular mechanism-driven therapeutic strategy for an important subset of HER2-overexpressing patients whose malignancies are mediated by SALL4 expression.

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study.

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Sentinel lymph node BIOPSY after neoadjuvant therapy in breast cancer patients with lymph node involvement at diagnosis. Could wire localization of clipped node improve our results?

INTRODUCTION: Sentinel lymph node biopsy (SLNB) after neoadjuvant therapy (NAT) in node-positive (N+) breast cancer patients at diagnosis remains a controversial issue, with no consensus on implementation or safety. OBJECTIVES: We sought to assess the accuracy of SLNB after NAT in biopsy-proven N+ cases at diagnosis and the efficacy and accuracy of wire localization of the clipped node to improve results. MATERIAL AND METHODS: A cross-sectional diagnostic technique validation study in N+ patients following NAT was performed. The biopsy-proven affected lymph node was clipped at diagnosis. SLNB and axillary lymph node dissection (ALND) were performed in cases of clinical-radiological lymph node response after NAT. For the purposes of our study we added wire localization of the clipped node. RESULTS: 103 patients were included (mean age, 54.4 years [± 12.7]). Wire marking was performed in 28 cases. The overall identification rate (IR) of SLN was 81.6%. The median number of nodes removed was 2 (range 2). The overall false negative rate (FNR) was 6.1%. Sensitivity and overall accuracy were 93.9% and 95.2%, respectively (area under curve 0.97). In the double-marked (clip and wire) group the FNR decreased to 0% and accuracy was 100%. Axillary pathologic complete response was observed in 24.3% of cases. CONCLUSIONS: SLNB is useful in node-positive patients at diagnosis who respond to NAT. Combining this with preoperative wire localization of the biopsied lymph node reduces the FNR without increasing the number of complications.

Neoadjuvant Management of Early Breast Cancer: A Clinical and Investigational Position Statement.

BACKGROUND: Neoadjuvant treatment is increasingly one of the preferred therapeutic options for early breast cancer and may have some unique outcomes, such as identifying predictive and prognostic factors of response or increasing the knowledge of individual tumor biology. DESIGN: A panel of experts from different specialties reviewed published clinical studies on the neoadjuvant management of breast cancer. Recommendations were made that emphasized the clinical multidisciplinary management and the investigational leverage in early breast cancer. RESULTS: Neoadjuvant therapy has equivalent efficacy to adjuvant therapy, and it has some additional benefits that include increasing breast conservation, assessing tumor response, establishing prognosis based on the pathological response, and providing a "second opportunity" for nonresponding patients. Achieving pathological complete remission because of neoadjuvant therapy has been correlated with long-term clinical benefit, particularly in HER2-positive and triple-negative breast cancer. In addition, the neoadjuvant setting is a powerful model for the development of new drugs and the identification of prognostic markers. Finally, neoadjuvant therapy has proven to be cost-effective by reducing nondrug costs, avoiding radical surgery, and reducing hospital stays when compared with other treatment approaches. CONCLUSION: Neoadjuvant therapy has clinical benefits in early breast cancer and provides in vivo information of individual breast cancer biology while allowing the investigation of new treatment approaches. Access to neoadjuvant therapy should be an option available to all patients with breast cancer through multidisciplinary tumor management. IMPLICATIONS FOR PRACTICE: Neoadjuvant treatment should be strongly considered as a therapeutic option for localized breast cancer and is a powerful tool for understanding breast cancer biology and investigating new treatment approaches.

Receptor Activator of Nuclear Factor Kappa B (RANK) and Clinicopathological Variables in Endometrial Cancer: A Study at Protein and Gene Level.

The system integrated by the receptor activator of nuclear factor kappa B (RANK) and its ligand, RANKL, modulates the role of hormones in the genesis and progression of breast tumors. We investigated whether the expression of RANK was related with clinicopathological features of primary endometrial tumors. Immunohistochemistry was used in an endometrial cancer tissue array containing samples from 36 tumors. The amount of RANK mRNA was examined in a tissue scan cDNA array containing cDNA from 40 tumors. Normal endometrium was examined for comparison. Immunohistochemical analyses showed that RANK expression was higher in malignant than in normal endometrium (p < 0.05). RANK expression was related to histological grade (Pearson correlation index = 0.484, p < 0.001), but not to tumor stage or to age of the women. The gene expression was similar in malignant and normal endometrium. The study of RANK isoforms confirmed that the overall relative abundance of the three clearly identified transcripts was similar in normal and pathological endometrium. RANK protein expression increased from normal to malignant endometrium, and the expression level was related with tumor grade but not with stage or the age of subjects in endometrial cancer. In contrast, similar comparisons showed no change in RANK gene expression.

Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma.

BACKGROUND: NF-κB signalling appears deregulated in breast tumours. The purpose of this study was to determine whether the non-canonical NF-κB pathway, is activated in oestrogen receptor positive (ER+) breast cancer, to identify any correlation between its activity and the clinico-pathological phenotype and to explore whether NF-κB2 and RelB subunits and/or any of their target genes might be used as a predictive marker. METHODS: Two independent cohorts of ER+ early breast cancer patients treated with adjuvant endocrine therapy were included in the study. Activation of RelB and NF-κB2 subunits was determined in a training set of 121 patients by measuring DNA-binding activities in nuclear extracts from fresh frozen specimens by an ELISA-based assay. Samples of 15 ER- breast cancer patients were also included in the study. In a large validation cohort of 207 patients, nuclear immunostaining of RelB and NF-κB2 on formalin-fixed paraffin-embedded specimens was performed. Statistical correlation within clinico-pathological factors, disease-free survival (DFS) and overall survival (OS) was evaluated. Publicly available gene expression and survival data have been interrogated aimed to identify target genes. RESULTS: Activation of NF-κB2 and RelB was found in 53.7 and 49.2% of the 121 ER+ tumours analysed, with similar levels to ER- breast tumours analysed in parallel for comparisons. In the validation cohort, we obtained a similar proportion of cases with activation of NF-κB2 and RelB (59.9 and 32.4%), with a 39.6% of co-activation. Multiplexing immunofluorescence in breast cancer tissue confirmed an inverse spatial distribution of ER with NF-κB2 and RelB nuclear expression in tumour cells. Interestingly, NF-κB2 and RelB mRNA expression was inversely correlated with ER gene (ESR1) levels (P<0.001, both) and its activation was significantly associated with worse DFS (P=0.005 and P=0.035, respectively) in ER+ breast cancer. Moreover, the co-activation of both subunits showed a stronger association with early relapse (P=0.002) and OS (P=0.001). Finally, higher expression of the non-canonical NF-κB target gene myoglobin was associated with a poor outcome in ER+ breast cancer (DFS, P<0.05). CONCLUSIONS: The non-canonical NF-κB pathway activation is inversely associated with oestrogen receptor expression in ER+ breast cancer and predicts poor survival in this subgroup. The myoglobin gene expression has been identified as a possible surrogate marker of the non-canonical NF-κB pathway activation in these tumours.

Clinical implications of routine genomic mutation sequencing in PIK3CA/AKT1 and KRAS/NRAS/BRAF in metastatic breast cancer.

BACKGROUND: There is increasing interest in the molecular profiling of tumour tissues in order to investigate alternative breast cancer (BC) therapies. However, the impact of genomic screening for druggable mutations with targeted gene panel sequencing (TGPS) in routine practice remains controversial. METHODS: This is a retrospective analysis of data from a genomic screening programme at our institution, in which we performed simplified TGPS for mutations in PIK3CA, AKT1, KRAS, NRAS, and BRAF in order to select patients for targeted therapy clinical trials. The genomes of archived samples of primary (PT) and/or metastatic (MT) tumours from advanced BC patients were analysed with MassARRAY technology (Sequenom MassARRAY, OncoCarta v1.0). The level of PTEN expression was assessed by immunohistochemistry. The primary endpoint was to identify the proportion of BC patients with PI3 K and MAPK alterations who were included in clinical trials using targeted therapies against these pathways. RESULTS: Two hundred and fifteen metastatic BC patients (65 PT and 168 MT) were included. Fifty-two patients (24.19 %) were enrolled in tailored clinical trials, of whom 29 (55.77, 13.49 % of all patients screened) harboured mutations targeted by the study drug. Moreover, 12 wild-type patients out of the 215 (5.58 %) were included in the clinical trials for which mutation analysis was an inclusion criteria. All the patients received drugs targeting the PI3K-AKT pathway and only two were given combinations directed against the PI3K and MAPK pathways. PI3KCA mutations were present in 33.7 % (61/181) of the patients, 45.83 % in PTs and 29.32 % in MTs. AKT1 mutations were detected in 5.48 % (8/146) of patients and PTEN loss in 34.67 % (52/150). KRAS, NRAS, and BRAF mutations were present in 12.06, 5.67, and 3.18 % of patients, respectively. CONCLUSIONS: Genomic screening with a simplified TGPS is feasible, and was used to identify 13.49 % of patients who were included in clinical trials using targeted therapy against the mutations they harboured; PI3KCA mutations were the most frequent aberration in our series.

Overexpression of the truncated form of High Mobility Group A proteins (HMGA2) in human myometrial cells induces leiomyoma-like tissue formation.

The pathogenesis of uterine leiomyomas, the most common benign tumor in women, is still unknown. This lack of basic knowledge limits the development of novel non-invasive therapies. Our group has previously demonstrated that leiomyoma side population (SP) cells are present in tumor lesions and act like putative tumor-initiating stem cells in human leiomyoma. Moreover, accumulated evidence demonstrates that these benign tumors of mesenchymal origin are characterized by rearrangements of the High Mobility Group A proteins (HMGA). In this work, we tested the hypothesis that leiomyoma development may be due to overexpression of HMGA2 (encoding high mobility group AT-hook2) in myometrial stem cells using in vitro and in vivo approaches. Our work demonstrates that the truncated/short form of HMGA2 induces myometrial cell transformation toward putative tumor-initiating leiomyoma cells and opens up new possibilities to understand the origin of leiomyomas and the development of new therapeutic approaches.

Biphasic dermatofibrosarcoma protuberans with a labyrinthine plexiform high-grade fibrosarcomatous transformation.

Several variants of dermatofibrosarcoma protuberans, a low-grade superficial sarcoma, are well recognized. The most prognostically important is the fibrosarcomatous variant. We report a case of biphasic dermatofibrosarcoma protuberans in which the high-grade component exhibited a previously undescribed plexiform pattern. A clinicopathological study complemented with immunohistochemical, ultrastructural, reverse transcription polymerase chain reaction and fluorescence in situ hybridization analyses of this unique case. Histopathologically, a conventional low-grade dermatofibrosarcoma protuberans was admixed with intratumoral high-grade areas showing a striking labyrinthine plexiform pattern characterized by a higher cellularity of larger and slightly atypical tumor cells. CD34 expression was present in both components, while Ki-67 immunostaining was significantly higher in the plexiform high-grade areas. Focal epithelial membrane antigen and claudin-1 immunostaining was present at the interphase between high- and low-grade areas. COL1A1-PDGFB fusion transcripts, with breakpoints at exon 25 of COL1A1 and exon 2 of PDGFB, were present in both components, being more numerous, as the extra copies of both genes, in the high-grade areas. A previously undescribed histopathologic pattern of high-grade sarcomatous transformation of dermatofibrosarcoma protuberans is reported: a biphasic tumor with a labyrinthine plexiform high-grade component.

MicroRNA profile in very young women with breast cancer.

BACKGROUND: Breast cancer is rarely diagnosed in very young women (35 years old or younger), and it often presents with distinct clinical-pathological features related to a more aggressive phenotype and worse prognosis when diagnosed at this early age. A pending question is whether breast cancer in very young women arises from the deregulation of different underlying mechanisms, something that will make this disease an entity differentiated from breast cancer diagnosed in older patients. METHODS: We performed a comprehensive study of miRNA expression using miRNA Affymetrix2.0 array on paraffin-embedded tumour tissue of 42 breast cancer patients 35 years old or younger, 17 patients between 45 and 65 years old and 29 older than 65 years. Data were statistically analyzed by t-test and a hierarchical clustering via average linkage method was conducted. Results were validated by qRT-PCR. Putative targeted pathways were obtained using DIANA miRPath online software. RESULTS: The results show a differential and unique miRNA expression profile of 121 miRNAs (p-value <0.05), 96 of those with a FDR-value <0.05. Hierarchical clustering grouped the samples according to their age, but not by subtype nor by tumour characteristics. We were able to validate by qRT-PCR differences in the expression of 6 miRNAs: miR-1228*, miR-3196, miR-1275, miR-92b, miR-139 and miR-1207. Moreover, all of the miRNAs maintained the expression trend. The validated miRNAs pointed out pathways related to cell motility, invasion and proliferation. CONCLUSIONS: The study suggests that breast cancer in very young women appears as a distinct molecular signature. To our knowledge, this is the first time that a validated microRNA profile, distinctive to breast cancer in very young women, has been presented. The miRNA signature may be relevant to open an important field of research in order to elucidate the underlying mechanism in this particular disease, which in a more clinical setting, could potentially help to identify therapeutic targets in this particular set of patients.

Clinical Impact of New Treatment Strategies for HER2-Positive Metastatic Breast Cancer Patients with Resistance to Classical Anti-HER Therapies.

HER2-positive breast cancer accounts for 15-20% of all breast cancer cases. This subtype is characterized by an aggressive behavior and poor prognosis. Anti-HER2 therapies have considerably improved the natural course of the disease. Despite this, relapse still occurs in around 20% of patients due to primary or acquired treatment resistance, and metastasis remains an incurable disease. This article reviews the main mechanisms underlying resistance to anti-HER2 treatments, focusing on newer HER2-targeted therapies. The progress in anti-HER2 drugs includes the development of novel antibody-drug conjugates with improvements in the conjugation process and novel linkers and payloads. Moreover, trastuzumab deruxtecan has enhanced the efficacy of trastuzumab emtansine, and the new drug trastuzumab duocarmazine is currently undergoing clinical trials to assess its effect. The combination of anti-HER2 agents with other drugs is also being evaluated. The addition of immunotherapy checkpoint inhibitors shows some benefit in a subset of patients, indicating the need for useful biomarkers to properly stratify patients. Besides, CDK4/6 and tyrosine kinase inhibitors are also included in the design of new treatment strategies. Lapitinib, neratinib and tucatinib have been approved for HER2-positive metastasis patients, however clinical trials are currently ongoing to optimize combined strategies, to reduce toxicity, and to better define the useful setting. Clinical research should be strengthened along with the discovery and validation of new biomarkers, as well as a deeper understanding of drug resistance and action mechanisms.

miR-146a-5p Promotes Angiogenesis and Confers Trastuzumab Resistance in HER2+ Breast Cancer.

Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells' exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells.

Targeted immunotherapy against distinct cancer-associated fibroblasts overcomes treatment resistance in refractory HER2+ breast tumors.

About 50% of human epidermal growth factor receptor 2 (HER2)+ breast cancer patients do not benefit from HER2-targeted therapy and almost 20% of them relapse after treatment. Here, we conduct a detailed analysis of two independent cohorts of HER2+ breast cancer patients treated with trastuzumab to elucidate the mechanisms of resistance to anti-HER2 monoclonal antibodies. In addition, we develop a fully humanized immunocompetent model of HER2+ breast cancer recapitulating ex vivo the biological processes that associate with patients' response to treatment. Thanks to these two approaches, we uncover a population of TGF-beta-activated cancer-associated fibroblasts (CAF) specific from tumors resistant to therapy. The presence of this cellular subset related to previously described myofibroblastic (CAF-S1) and podoplanin+ CAF subtypes in breast cancer associates with low IL2 activity. Correspondingly, we find that stroma-targeted stimulation of IL2 pathway in unresponsive tumors restores trastuzumab anti-cancer efficiency. Overall, our study underscores the therapeutic potential of exploiting the tumor microenvironment to identify and overcome mechanisms of resistance to anti-cancer treatment.