AXL – a new player in resistance to HER2 blockade

Cancer; HER2 disease; ResistanceCàncer; Malaltia HER2; ResistènciaCáncer; Enfermedad HER2; ResistenciaHER2 is a driver in solid tumors, mainly breast, oesophageal and gastric cancer, through activation of oncogenic signaling pathways such as PI3K or MAPK. HER2 overexpression associates with aggressive disease and poor prognosis. Despite targeted anti-HER2 therapy has improved outcomes and is the current standard of care, resistance emerge in some patients, requiring additional therapeutic strategies. Several mechanisms, including the upregulation of receptors tyrosine kinases such as AXL, are involved in resistance. AXL signaling leads to cancer cell proliferation, survival, migration, invasion and angiogenesis and correlates with poor prognosis. In addition, AXL overexpression accompanied by a mesenchymal phenotype result in resistance to chemotherapy and targeted therapies. Preclinical studies show that AXL drives anti-HER2 resistance and metastasis through dimerization with HER2 and activation of downstream pathways in breast cancer. Moreover, AXL inhibition restores response to HER2 blockade in vitro and in vivo. Limited data in gastric and oesophageal cancer also support these evidences. Furthermore, AXL shows a strong value as a prognostic and predictive biomarker in HER2+ breast cancer patients, adding a remarkable translational relevance. Therefore, current studies enforce the potential of co-targeting AXL and HER2 to overcome resistance and supports the use of AXL inhibitors in the clinic

MicroRNA-33b Suppresses Epithelial-Mesenchymal Transition Repressing the MYC-EZH2 Pathway in HER2+ Breast Carcinoma

Downregulation of miR-33b has been documented in many types of cancers and is being involved in proliferation, migration, and epithelial-mesenchymal transition (EMT). Furthermore, the enhancer of zeste homolog 2-gene (EZH2) is a master regulator of controlling the stem cell differentiation and the cell proliferation processes. We aim to evaluate the implication of miR-33b in the EMT pathway in HER2+ breast cancer (BC) and to analyze the role of EZH2 in this process as well as the interaction between them. miR-33b is downregulated in HER2+ BC cells vs healthy controls, where EZH2 has an opposite expression in vitro and in patients' samples. The upregulation of miR-33b suppressed proliferation, induced apoptosis, reduced invasion, migration and regulated EMT by an increase of E-cadherin and a decrease of ß-catenin and vimentin. The silencing of EZH2 mimicked the impact of miR-33b overexpression. Furthermore, the inhibition of miR-33b induces cell proliferation, invasion, migration, EMT, and EZH2 expression in non-tumorigenic cells. Importantly, the Kaplan-Meier analysis showed a significant association between high miR-33b expression and better overall survival. These results suggest miR-33b as a suppressive miRNA that could inhibit tumor metastasis and invasion in HER2+ BC partly by impeding EMT through the repression of the MYC-EZH2 loop

SOLTI-1805 TOT-HER3 Study Concept: A Window-of-Opportunity Trial of Patritumab Deruxtecan, a HER3 Directed Antibody Drug Conjugate, in Patients With Early Breast Cancer

Background: Preclinical data support a key role for the human epidermal growth factor receptor 3 (HER3) pathway in hormone receptor (HR)-positive breast cancer. Recently, new HER3 directed antibody drug conjugates have shown activity in breast cancer. Given the need to better understand the molecular biology, tumor microenvironment, and mechanisms of drug resistance in breast cancer, we designed this window-of-opportunity study with the HER3 directed antibody drug conjugate patritumab deruxtecan (HER3-DXd; U3-1402). Trial Design: Based on these data, a prospective, multicenter, single-arm, window-of-opportunity study was designed to evaluate the biological effect of patritumab deruxtecan in the treatment of naïve patients with HR-positive/HER2-negative early breast cancer whose primary tumors are ≥1 cm by ultrasound evaluation. Patients will be enrolled in four cohorts according to the mRNA-based ERBB3 expression by central assessment. The primary endpoint is a CelTIL score after one single dose. A translational research plan is also included to provide biological information and to evaluate secondary and exploratory objectives of the study

The GATA3 X308_Splice breast cancer mutation is a hormone context-dependent oncogenic driver

As the catalog of oncogenic driver mutations is expanding, it becomes clear that alterations in a given gene might have different functions and should not be lumped into one class. The transcription factor GATA3 is a paradigm of this. We investigated the functions of the most common GATA3 mutation (X308_Splice) and five additional mutations, which converge into a neoprotein that we called “neoGATA3,” associated with excellent prognosis in patients. Analysis of available molecular data from >3000 breast cancer patients revealed a dysregulation of the ER-dependent transcriptional response in tumors carrying neoGATA3-generating mutations. Mechanistic studies in vitro showed that neoGATA3 interferes with the transcriptional programs controlled by estrogen and progesterone receptors, without fully abrogating them. ChIP-Seq analysis indicated that ER binding is reduced in neoGATA3-expressing cells, especially at distal regions, suggesting that neoGATA3 interferes with the fine tuning of ER-dependent gene expression. This has opposite outputs in distinct hormonal context, having pro- or anti-proliferative effects, depending on the estrogen/progesterone ratio. Our data call for functional analyses of putative cancer drivers to guide clinical application.Institute of Cancer Research of the Medical University Vienna and by the grant P27361-B23 from the Austrian Science Grant (FWF), FXR was supported by SAF2011-29530 and SAF2015-70553-R grants from Ministerio de Economía y Competitividad (Madrid, Spain) (co-funded by the ERDF-EU), Fundación Científica de la Asociación Española Contra el Cáncer. CNIO is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-051

Clinical, pathological and PAM50 gene expression features of HER2-low breast cancer

Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression

HOPE (SOLTI-1903) breast cancer study: real-world, patient-centric, clinical practice study to assess the impact of genomic data on next treatment decision-choice in patients with locally advanced or metastatic breast cancer

Background Metastatic breast cancer (mBC) causes nearly all BC-related deaths. Next-generation sequencing (NGS) technologies allow for the application of personalized medicine using targeted therapies that could improve patients' outcomes. However, NGS is not routinely used in the clinical practice and its cost induces access-inequity among patients. We hypothesized that promoting active patient participation in the management of their disease offering access to NGS testing and to the subsequent medical interpretation and recommendations provided by a multidisciplinary molecular advisory board (MAB) could contribute to progressively overcome this challenge. We designed HOPE (SOLTI-1903) breast cancer trial, a study where patients voluntarily lead their inclusion through a digital tool (DT). The main objectives of HOPE study are to empower mBC patients, gather real-world data on the use of molecular information in the management of mBC and to generate evidence to assess the clinical utility for healthcare systems.Trial design After self-registration through the DT, the study team validates eligibility criteria and assists patients with mBC in the subsequent steps. Patients get access to the information sheet and sign the informed consent form through an advanced digital signature. Afterwards, they provide the most recent (preferably) metastatic archival tumor sample for DNA-sequencing and a blood sample obtained at the time of disease progression for ctDNA analysis. Paired results are reviewed by the MAB, considering patient's medical history. The MAB provides a further interpretation of molecular results and potential treatment recommendations, including ongoing clinical trials and further (germline) genetic testing. Participants self-document their treatment and disease evolution for the next 2 years. Patients are encouraged to involve their physicians in the study. HOPE also includes a patient empowerment program with educational workshops and videos about mBC and precision medicine in oncology. The primary endpoint of the study was to describe the feasibility of a patient-centric precision oncology program in mBC patients when a comprehensive genomic profile is available to decide on a subsequent line of treatment

A Pathology-Based Combined Model to Identify PAM50 Non-luminal Intrinsic Disease in Hormone Receptor-Positive HER2-Negative Breast Cancer

Background: In hormone receptor-positive (HR+)/HER2-negative breast cancer, the HER2-enriched and Basal-like intrinsic subtypes are associated with poor outcome, low response to anti-estrogen therapy and high response to chemotherapy. To date, no validated biomarker exists to identify both molecular entities other than gene expression.Methods: PAM50 subtyping and immunohistochemical data were obtained from 8 independent studies of 1,416 HR+/HER2-negative early breast tumors. A non-luminal disease score (NOLUS) from 0 to 100, based on percentage of estrogen receptor (ER), progesterone receptor (PR) and Ki67 tumor cells, was derived in a combined cohort of 5 studies (training dataset) and tested in a combined cohort of 3 studies. The performance of NOLUS was estimated using Area Under the ROC Curve (AUC).Results: In the training dataset (n = 903) and compared to luminal disease, non-luminal disease had lower percentage of ER-positive cells (median 65.2 vs. 86.2%, p < 0.01) and PR-positive cells (33.2 vs. 56.4%, p < 0.01) and higher percentage of Ki67-positive cells (18.2 vs. 13.1%, p = 0.01). A NOLUS formula was derived: −0.45*ER −0.28*PR +0.27*Ki67 + 73.02. The proportion of non-luminal tumors in NOLUS-positive (≥51.38) and NOLUS-negative (<51.38) groups was 52.6 and 8.7%, respectively. In the testing dataset (n = 514), NOLUS was found significantly associated with non-luminal disease (p < 0.01) with an AUC 0.902. The proportion of non-luminal tumors in NOLUS-positive and NOLUS-negative groups was 76.9% (56.4–91.0%) and 2.6% (1.4–4.5%), respectively. The sensitivity and specificity of the pre-specified cutoff was 59.3 and 98.7%, respectively.Conclusions: In the absence of gene expression data, NOLUS can help identify non-luminal disease within HR+/HER2-negative breast cancer

Nuevas vías moleculares implicadas en los mecanismos de resistencia primaria y adquirida en cáncer de mama HER2 positivo

El cáncer de mama es el tumor más frecuente en la mujer, representa aproximadamente el 28% de todas las neoplasias y es la principal causa de muerte por cáncer en las mujeres europeas. A pesar de los avances en el tratamiento y en el diagnóstico temprano, sigue siendo un importante problema de salud pública. El 20% de las pacientes diagnosticadas de cáncer de mama presentan una recaída. La enfermedad en estadios avanzados sigue siendo incurable con una supervivencia media de 2-3 años y una supervivencia global de 25% a los 5 años. El cáncer de mama es una enfermedad heterogénea que podemos clasificar en: luminal A, luminal B, HER2 amplificado o triple negativo en función de criterios anatomopatológicos. Evaluamos la expresión de receptores hormonales (receptores estrógeno [RE], receptores progesterona [RP]), índice de proliferación [Ki67] y la amplificación del gen HER2. Actualmente, disponemos también de la clasificación de los subtipos intrínsecos moleculares en función la expresión génica: Luminal A,Luminal B, HER2-enriched [HER2-E], Basal-like, Claudin-low y Normal-like. HER2 es uno de los oncogenes más importantes en cáncer de mama, se encuentra amplificado en aproximadamente 20% de las pacientes. Su sobreexpresión es un factor de mal pronóstico clínico asociado con más recaídas y peor supervivencia. Sin embargo, desde el desarrollo de trastuzumab, un anticuerpo monoclonal contra HER2, la sobreexpresión de dicha proteína se ha convertido también en un factor predictivo de respuesta y ha mejorado la supervivencia en este subgrupo de pacientes. Trastuzumab ha mostrado beneficio para las pacientes con cáncer de mama HER2+ tanto en enfermedad temprana como en avanzada. Sin embargo, existe un porcentaje de pacientes que presentan resistencia primaria o secundaria al tratamiento. Lo que pone de manifiesto la necesidad de identificar marcadores predictivos de respuesta/resistencia para identificar que pacientes se van a beneficiar del tratamiento y poder desarrollar nuevas estrategias para superar dichas resistencias. Uno de los mecanismos implicados en la resistencia es la transición epitelio-mesénquima, por lo que planteamos este mecanismo como fenómeno responsable de la resistencia a trastuzumab en cáncer de mama HER2 positivo. Para ello, estudiamos AXL y MET, dos receptores transmembrana tirosina quinasa cuya función está relacionada con este fenotipo. Ambos son receptores con potencial oncogénico que han sido descritos como biomarcadores de resistencia a diferentes fármacos en diferentes tipos de tumores. Disponemos de dos modelos celulares de resistencia, las HCC1954 con resistencia primaria a trastuzumab y mutación activadora en PIK3CA y las AU565-R con resistencia adquirida. La línea con resistencia adquirida se ha obtenido a través de la exposición mantenida a trastuzumab de su parenteral sensible (AU565-S). En ambos modelos preclínicos, de resistencia a trastuzumab tanto primaria como secundaria, no depende de cambios en la expresión del oncogen HER2. Al evaluar su expresión por IHC y por WB no encontramos diferencias entre la línea celular con resistencia adquirida (AU565-R) y su parental (AU565-S), manteniendo la amplificación de HER2 y la negatividad a la expresión de receptores hormonales. Además, la línea HCC1954 con resistencia primaria también mantiene la sobreexpresión de HER2. Al analizar la expresión de AXL y MET en nuestras líneas celulares con resistencia a trastuzumab, observamos la sobreexpresión de ambos con respecto a la línea sensible. Dicha sobreexpresión fue estable y consistente. Hubo una mayor transcripción de los genes con un aumento a nivel de RNA y una mayor traducción con un aumento de la síntesis de proteína. Esto es indicativo de que las células con la sobreexpresión de AXL y MET podrían adquirir propiedades mesenquimal-like que contribuyeran a la resistencia y que por tanto podrían constituir nuevas dianas terapéuticas, convirtiéndose en una estrategia razonable a explorar. Se ha descrito como MET contribuye a la resistencia a trastuzumab y está relacionado con peor pronóstico en las pacientes, además en estudios preclínicos, su inhibición restaura la sensibilidad. Por otro lado, la expresión de AXL se ha relacionado con la resistencia adquirida a lapatinib y como su inhibición restaura la sensibilidad en modelos in vitro, pero hasta la fecha no se ha descrito como mecanismo de resistencia a trastuzumab. Este hecho nos llevó a focalizarnos en el estudio de AXL como posible mecanismo responsable de la resistencia. El gen AXL pertenece a la firma Claudin-low de los subtipos intrínsecos de PAM50. Cuando exploramos su expresión a nivel de RNA en bases de datos públicas, observamos como está up-regulado en los subtipos no-Luminales con respecto a los Luminales, siendo predominante en el subtipo Claudin-low. Además presenta una correlación positiva y/o co-expresión de genes relacionados con la transición epitelio-mesénquima. Postulamos el receptor AXL como una nueva posible diana terapéutica en cáncer de mama HER2 positivo resistente a trastuzumab. Para ello utilizamos TP-0903, un inhibidor tirosina quinasa específico de AXL. Nuestros datos preclínicos muestran su eficacia en monoterapia disminuyendo la viabilidad celular in vitro tanto para AU565-R como HCC1954. Y, lo más importante, restaura la sensibilidad a trastuzumab en la línea celular con resistencia adquirida. Este efecto de resensibilización a trastuzumab, no se observó en la línea con resistencia primaria. Lo que nos llevó a hipotetizar que en este modelo la señalización de AXL no era driver en la resistencia a trastuzumab. Las HCC1954 poseen una mutación activadora en PIK3CA, la vía PI3K es una de las más relevantes en cáncer de mama con propiedades oncogénicas. Las mutaciones de PIK3CA es uno de los mecanismos de hiperactivación de la vía, esta mutación sería responsable de una activación constitutiva e independiente de la señalización upstream de receptores tirosina quinasa como HER2 y AXL. Por tanto, exploramos la inhibición farmacológica de la vía PI3K como estrategia para revertir la resistencia a trastuzumab en la línea con resistencia primaria y PIK3CA mutada. Utilizamos tres fármacos con mecanismos moleculares diferentes: un inhibidor de PI3K (BKM120), inhibidor de mTOR (everolimus) y un inhibidor dual de PI3K y mTOR (GDC-9080). Observamos como los tres fármacos en monoterpia eran eficaces y redujeron la viabilidad celular de HCC1954. El más efectivo de los inhibidores fue GDC-0980, probablemente debido a su inhibición en dos puntos de la vía que podría evitar la supresión de los mecanismos de feedback negativo. Estos datos ponen en relieve lo importante que es caracterizar la enfermedad metastásica e identificar potenciales dianas driver como objetivos terapéuticos. La importancia de este trabajo radica en la implicación traslacional de los hallazgos preclínicos. AXL podría ser un biomarcador predictivo de resistencia secundaria a trastuzumab. De este modo una potencial estrategia a evaluar sería un tratamiento con trastuzumab más inhibidor de AXL en aquellas pacientes con cáncer de mama HER2 positivo con progresión de enfermedad a trastuzumab y en las que el tumor asocia sobreexpresión de AXL (a nivel de proteína o RNA). El tratamiento de combinación permitiría bloquear la inducción de la transición epitelio-mesénquima y la vía HER2. Actualmente existen diferentes ensayos clínicos en fases tempranas que evalúan el papel de AXL como diana terapéutica con inhibidores orales de tirosina-quinasa o anticuerpos monoclonales. Además, las firmas génicas podrían ayudar a estratificar a las pacientes según su perfil molecular. Por otro lado, ya es una realidad en oncología como la determinación de las mutaciones de la vía PI3K y el desarrollo de inhibidores en ensayos clínicos nos ayuda a entender su valor predictivo y pronóstico. Los resultados de los ensayos clínicos que están en marcha nos ayudarán a comprender mejor el potencial terapéutico de los inhibidores de PI3K

Uncovering the molecular and cellular mechanisms of metastatic dormancy in luminal breast cancer

[eng] Breast cancer (BC) is the most common cancer in women and it represents the most common cause of cancer-­‐related death in women worldwide1. Advances in early detection, prevention, risk stratification, and therapeutic strategies as well as supportive care for patients have resulted in important improvements in mortality and reduction of cancer relapse. However, around 20% of the patients will present metastatic disease. Advanced breast cancer is still an incurable disease with a median overall survival (OS) of ∼3 years, while the 5-­‐year survival of only ∼25%2. Thus, the deeper understanding of cancer biology, inter-­‐ and intra-­‐tumor heterogeneity, and as well as, the biology behind the progression of tumor cells toward metastasis is urgent. To date, evidence suggests that both intrinsic properties of breast cancer cells and host organ microenvironment participate actively to this matter3. In general, detectable distant breast cancer metastases occur years, or even decades, after primary tumor diagnosis. Metastatic lesions are supposed to originate from disseminated tumor cells that underwent a period of dormancy4. Nevertheless, the molecular factors that promote the formation of detectable metastasis from disseminated tumor cells are largely unknown. Nowadays, the genomic revolution has transformed the landscape of cancer, leading possible an improvement in understanding of tumor biology. To try to explore BC capability in producing metastases, a molecular analysis consisting on transcriptome study was performed among a retrospective cohort of metastatic breast cancer patients. Paired tissue samples from primary and metastatic tumor were available to be analyzed.[spa] El cáncer de mama (CM) es el cáncer más común en las mujeres y representa la causa más común de muerte relacionada con el cáncer en las mujeres en todo el mundo. Los avances en la detección temprana, la prevención, la estratificación del riesgo y las estrategias terapéuticas, así como la atención de apoyo para los pacientes, han dado como resultado mejoras importantes en la mortalidad y la reducción de la recaída del cáncer. Sin embargo, alrededor del 20% de las pacientes presentarán enfermedad metastásica. El cáncer de mama avanzado sigue siendo una enfermedad incurable con una mediana de supervivencia general (SG) de ∼3 años, mientras que la supervivencia a 5 años de solo ∼25%. Por lo tanto, es urgente una comprensión más profunda de la biología del cáncer, la heterogeneidad inter e intratumoral y la biología detrás de la progresión de las células tumorales hacia la metástasis. Hasta la fecha, la evidencia sugiere que tanto las propiedades intrínsecas de las células como el microambiente del órgano huésped participan activamente en este asunto