Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm

Efecto de la Doxorubicina sobre MKP-1 en el Cáncer de Mama Humano y Asociación entre la Expresión de MKP-1 y la Evolución de las Pacientes

Objetivos: La fosfatasa 1 de las protein-quinasas activadas por mitógenos (Mitogen-activated protein kinase (MAPK) phosphatase-1, MKP-1) regula por defosforilación la actividad de las vías de señalización de las protein-quinasas activadas por mitógenos, la quinasa regulada por señales extracelulares (extracellular signal-regulated kinase, ERK), la quinasa terminal c-Jun (c-Jun NH2-terminal kinase, JNK) y p38. MKP-1 también media en los mecanismos de quimioresistencia en cáncer de mama y se inhibe por la actividad de la doxorrubucina en modelos celulares de cáncer de mama. Este trabajo aquí presentado tiene como objetivo caracterizar los efectos de la doxorrubicina sobre MKP-1 y sobre las MAPK en cáncer de mama e investigar su relevancia como factor pronóstico en esta enfermedad. Experimental Design: Los efectos de la doxorubicin en MKP-1, la forma fosforilada (p) de ERK1/2 (p-ERK1/2), de p-JNK y de p-p38 se estudiaron en un panel de líneas celulares de cáncer de mama humano por técnicas de Western blot y en tumores humanos cultivados ex vivo por inmunohistoquímica. De forma adicional, MKP-1 se estudió en una serie de tejidos mamarios desde parénquima normal hasta carcinoma infiltrante de mama, y en una cohorte amplia de pacientes de cáncer de mama con seguimiento clínico. Results: MKP-1 se expresaba en niveles bajos en el tejido mamario normal y en la hiperplasia ductal, detectándose sobreexpresión en el carcinoma ductal in situ. MKP-1 también se sobreexpresaba en casi el 50% de los tumores mamarios. De forma similar en las líneas de cáncer de mama y en los tumores cultivados ex vivo, de forma general la doxorrubicina redujo la expresión de MKP-1 e incrementó los niveles de p-ERK1/2 y JNK. Aquellos casos, sin embargo, que sobreexpresaban MKP-1, la doxorrubicina no afectó la expresión de MKP-1 ni de las formas fosforiladas de las MAPK. Por último, la sobreexpresión de MKP-1 se reveló como un factor de mal pronóstico en los análisis uni- y multivariados para la supervivencia libre de enfermedad en las pacientes con cáncer de mama estudiadas. Conclusions: MKP-1 se sobreexpresa en la transformación maligna de la mama y se asocia a una peor supervivencia en las pacientes con cáncer de mama. Además, MKP-1 es inhibida por la doxorrubicina en esta enfermedad.Purpose: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) regulates by dephosphorylation the activity of the major intracellular signaling pathways mediated by mitogen-activated protein kinase [extracellular signalregulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38]. MKP-1 mediates breast cancer chemoresistance and is repressible by doxorubicin in breast cancer cells. This work is aimed to characterize doxorubicin effects on MKP- 1 and phospho-MAPKs in human breast cancers and to study the clinical relevance of MKP-1 expression in this tumor. Experimental Design: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a broad panel of human breast cancer cells by Western blotting and in human breast cancer were assayed ex vivo by immunohistochemistry. MKP-1 expression was also assayed in a spectrum of normal to malignant breast lesions and in a large series of breast cancer patients with clinical follow-up. Results: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in approximately 50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin downregulated MKP-1, and upregulated activation of ERK1/2 and JNK, in most of studied cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or activation levels of MAPKs. With regard to patient outcome, MKP-1 overexpression was a significant adverse prognostic factor for relapse both by univariate and multivariate analysis. Conclusions: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in human breast cancer

Efecto de la doxorubicina sobre MKP-1 en el cáncer de mama humano y asociación entre la expresión de MKP-1 y la evolución de las pacientes

Objetivos: La fosfatasa 1 de las protein-quinasas activadas por mitógenos (Mitogen-activated protein kinase (MAPK) phosphatase-1, MKP-1) regula por defosforilación la actividad de las vías de señalización de las protein-quinasas activadas por mitógenos, la quinasa regulada por señales extracelulares (extracellular signal-regulated kinase, ERK), la quinasa terminal c-Jun (c-Jun NH2-terminal kinase, JNK) y p38. MKP-1 también media en los mecanismos de quimioresistencia en cáncer de mama y se inhibe por la actividad de la doxorrubucina en modelos celulares de cáncer de mama. Este trabajo aquí presentado tiene como objetivo caracterizar los efectos de la doxorrubicina sobre MKP-1 y sobre las MAPK en cáncer de mama e investigar su relevancia como factor pronóstico en esta enfermedad. Experimental Design: Los efectos de la doxorubicin en MKP-1, la forma fosforilada (p) de ERK1/2 (p-ERK1/2), de p-JNK y de p-p38 se estudiaron en un panel de líneas celulares de cáncer de mama humano por técnicas de Western blot y en tumores humanos cultivados ex vivo por inmunohistoquímica. De forma adicional, MKP-1 se estudió en una serie de tejidos mamarios desde parénquima normal hasta carcinoma infiltrante de mama, y en una cohorte amplia de pacientes de cáncer de mama con seguimiento clínico. Results: MKP-1 se expresaba en niveles bajos en el tejido mamario normal y en la hiperplasia ductal, detectándose sobreexpresión en el carcinoma ductal in situ. MKP-1 también se sobreexpresaba en casi el 50% de los tumores mamarios. De forma similar en las líneas de cáncer de mama y en los tumores cultivados ex vivo, de forma general la doxorrubicina redujo la expresión de MKP-1 e incrementó los niveles de p-ERK1/2 y JNK. Aquellos casos, sin embargo, que sobreexpresaban MKP-1, la doxorrubicina no afectó la expresión de MKP-1 ni de las formas fosforiladas de las MAPK. Por último, la sobreexpresión de MKP-1 se reveló como un factor de mal pronóstico en los análisis uni- y multivariados para la supervivencia libre de enfermedad en las pacientes con cáncer de mama estudiadas. Conclusions: MKP-1 se sobreexpresa en la transformación maligna de la mama y se asocia a una peor supervivencia en las pacientes con cáncer de mama. Además, MKP-1 es inhibida por la doxorrubicina en esta enfermedad.Purpose: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) regulates by dephosphorylation the activity of the major intracellular signaling pathways mediated by mitogen-activated protein kinase [extracellular signalregulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38]. MKP-1 mediates breast cancer chemoresistance and is repressible by doxorubicin in breast cancer cells. This work is aimed to characterize doxorubicin effects on MKP- 1 and phospho-MAPKs in human breast cancers and to study the clinical relevance of MKP-1 expression in this tumor. Experimental Design: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a broad panel of human breast cancer cells by Western blotting and in human breast cancer were assayed ex vivo by immunohistochemistry. MKP-1 expression was also assayed in a spectrum of normal to malignant breast lesions and in a large series of breast cancer patients with clinical follow-up. Results: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in approximately 50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin downregulated MKP-1, and upregulated activation of ERK1/2 and JNK, in most of studied cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or activation levels of MAPKs. With regard to patient outcome, MKP-1 overexpression was a significant adverse prognostic factor for relapse both by univariate and multivariate analysis. Conclusions: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in human breast cancer

Multidisciplinary consensus on optimising the detection of NTRK gene alterations in tumours

The recent identification of rearrangements of neurotrophic tyrosine receptor kinase (NTRK) genes and the development of specific fusion protein inhibitors, such as larotrectinib and entrectinib, have revolutionised the diagnostic and clinical management of patients presenting with tumours with these alterations. Tumours that harbour NTRK fusions are found in both adults and children; and they are either rare tumours with common NTRK fusions that may be diagnostic, or more prevalent tumours with rare NTRK fusions. To assess currently available evidence on this matter, three key Spanish medical societies (the Spanish Society of Medical Oncology (SEOM), the Spanish Society of Pathological Anatomy (SEAP), and the Spanish Society of Paediatric Haematology and Oncology (SEHOP) have brought together a group of experts to develop a consensus document that includes guidelines on the diagnostic, clinical, and therapeutic aspects of NTRK-fusion tumours. This document also discusses the challenges related to the routine detection of these genetic alterations in a mostly public Health Care System.SEOM, SEAP and SEHOP have received financial support for this project in the form of unrestricted collaboration in the logistics of expert meeting from Bayer and Roche

Liquid biopsy in oncology: A consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology

[ES] Los pacientes con cáncer y alteraciones oncogénicas potencialmente tratables están aumentando de forma considerable. El diagnóstico de estas alteraciones permite frecuentemente realizar un tratamiento personalizado inicial o a la progresión, así como conocer información predictiva sobre la eficacia de la inmunoterapia. Sin embargo, en un 25% de los casos, la biopsia de tejido inicial no es informativa o no es posible realizar el perfil genómico tumoral a la progresión por la dificultad para obtener nuevas biopsias de tejido tumoral. Por ello, son necesarias alternativas diagnósticas eficaces para la estratificación molecular, que permitan una valoración dinámica del perfil genómico tumoral, como la biopsia líquida, reflejando además la heterogeneidad genómica en el seno del mismo paciente. Actualmente, existen distintas técnicas diagnósticas de biopsia líquida, cada una con distintos grados de precisión y rendimiento. El objetivo de este consenso de la Sociedad Española de Anatomía Patológica (SEAP) y la Sociedad Española de Oncología Médica (SEOM) es evaluar la viabilidad y efectividad de las distintas técnicas de biopsia líquida en el paciente con cáncer en la práctica clínica diaria. Los expertos de este consenso concluyen que la biopsia líquida es una alternativa aceptable a la biopsia de tejido para el estudio de biomarcadores. Sin embargo, es importante estandarizar los procedimientos preanalíticos y analíticos, garantizar su reproducibilidad, así como generar informes clínicos estructurados y comprensibles. La implementación de comités multidisciplinares de evaluación de las alteraciones moleculares es fundamental para mejorar estos procesos y favorecer las decisiones terapéuticas más adecuadas para cada paciente con cáncer en función de perfil genómico.[EN] The proportion of cancer patients with tumours that harbour a potentially targetable genomic alteration is increasing considerably. The diagnosis of these genomic alterations can lead to tailoring of treatment, at the onset of disease or during progression, as well as providing additional, predictive information on the efficacy of immunotherapy. However, in up to 25% of cases, the initial tissue biopsy is inadequate for precision oncology and, in many cases, tumour genomic profiling at progression is not possible due to technical limitations of obtaining new tumour tissue specimens. Efficient diagnostic alternatives are therefore required for molecular stratification, such as liquid biopsy. This technique enables the evaluation of the tumour genomic profile dynamically and as well as capturing intra-patient genomic heterogeneity. To date, there are several diagnostic techniques available for use in liquid biopsy, each with different precision and performance levels. The objective of this consensus statement of the Spanish Society of Pathology (SEAP) and the Spanish Society of Medical Oncology (SEOM) is to evaluate the viability and effectiveness of the different methodological approaches of liquid biopsy in cancer patients, and the potential application of this method to current clinical practice. The experts contributing to this consensus statement agree that, according to current evidence, liquid biopsy is an acceptable alternative to tumour tissue biopsy for the study of biomarkers in various clinical settings. It is therefore important to standardise pre-analytical and analytical procedures to ensure reproducibility and to generate structured and accessible clinical reports. It is essential to appoint multidisciplinary tumour molecular committees to oversee these processes and to enable the most suitable therapeutic decisions for each patient according to the genomic profile

Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies

Pembrolizumab Plus Gemcitabine in the Subset of Triple-Negative Advanced Breast Cancer Patients in the GEICAM/2015-04 (PANGEA-Breast) Study

The PANGEA-Breast trial evaluated a new chemo-immunotherapeutic combination that would synergistically induce long-term clinical benefit in HER2-negative advanced breast cancer patients. Treatment consisted of 21-day cycles of 200 mg of pembrolizumab (day 1) plus gemcitabine (days 1 and 8). The primary objective was the objective response rate (ORR). The tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumor, and the myeloid-derived suppressor cells (MDSCs) level in peripheral blood, were analyzed to explore associations with treatment efficacy. Considering a two-stage Simon's design, the study recruitment was stopped after its first stage as statistical assumptions were not met. A subset of 21 triple-negative breast cancer (TNBC) patients was enrolled. Their median age was 49 years; 15 patients had visceral involvement, and 16 had ≤3 metastatic locations. Treatment discontinuation due to progressive disease (PD) was reported in 16 patients. ORR was 15% (95% CI 3.2-37.9). Four patients were on treatment >6 months before PD. Grade ≥3 treatment-related adverse events were observed in 8 patients, where neutropenia was the most common. No association was found between TILs density, PD-L1 expression or MDSCs levels and treatment efficacy. ORR in TNBC patients also did not meet the assumptions, but 20% were on treatment >6 months.Research grant from Merck’s (MSD in Europe) Investigator Initiated Studies Program6.575 Q1 JCR 20211.349 Q1 SJR 2021No data IDR 2021UE

Combination of KIR2DS4 and FcγRIIa polymorphisms predicts the response to cetuximab in KRAS mutant metastatic colorectal cancer

Cetuximab is a standard-of-care treatment for RAS wild-type metastatic colorectal cancer (mCRC) but not for those harbor a KRAS mutation since MAPK pathway is constitutively activated. Nevertheless, cetuximab also exerts its effect by its immunomodulatory activity despite the presence of RAS mutation. The aim of this study was to determine the impact of polymorphism FcγRIIIa V158F and killer immunoglobulin-like receptor (KIR) genes on the outcome of mCRC patients with KRAS mutations treated with cetuximab. This multicenter Phase II clinical trial included 70 mCRC patients with KRAS mutated. We found KIR2DS4 gene was significantly associated with OS (HR 2.27; 95% CI, 1.08-4.77; P = 0.03). In non-functional receptor homozygotes the median OS was 2.6 months longer than in carriers of one copy of full receptor. Multivariate analysis confirmed KIR2DS4 as a favorable prognostic marker for OS (HR 6.71) in mCRC patients with KRAS mutation treated with cetuximab. These data support the potential therapeutic of cetuximab in KRAS mutated mCRC carrying non-functional receptor KIR2DS4 since these patients significantly prolong their OS even after heavily treatment. KIR2DS4 typing could be used as predictive marker for identifying RAS mutated patients that could benefit from combination approaches of anti-EGFR monoclonal antibodies and other immunotherapies to overcome the resistance mediated by mutation in RAS

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm