The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ganglio centinela positivo en estadios iniciales de cáncer de mama. 2018. Resumen de recomendaciones / Positive sentinel lymph node in early stages of breast cancer. 2018. Summary of recommendations

Sabemos que el estado de la axila sigue siendo un factor pronóstico determinante y fundamental en la elección del tratamiento adyuvante de nuestras pacientes. Varios ensayos clínicos han hecho esfuerzos para intentar demostrar que la biopsia del ganglio centinela es tan efectiva como la linfadenectomía en cuanto a la estadificación. Estos mismos estudios evidenciaron que, en la gran mayoría de los casos ­del 40% al 60%­, ese ganglio centinela es el único metastásico. Por lo tanto, luego se quiso demostrar la efectividad de esa biopsia de ganglio centinela, no solo en cuanto a la eficacia de la estadificación, sino en cuanto al control local, incluso en pacientes con hasta uno o dos ganglios comprometidos pero que recibieron el tratamiento local adecuado, esto es, la exéresis adecuada, el tratamiento radiante completo y el tratamiento adyuvante que correspondiera en esos casos

¿Es posible identificar factores pronósticos y predictivos en Cáncer de Mama Triple Negativo? / Is it possible to identify prognostic and predictive factors in Triple Negative breast cancer?

Introducción Debido a su negatividad para los receptores hormonales y de her2, los cánceres de mama Triple Negativos (cmtn) no tienen tratamiento específico y es la quimioterapia la única modalidad de tratamiento sistémico. Objetivos El objetivo de este trabajo es, en primer lugar, evaluar en los cmtn la factibilidad de utilizar a la infiltración linfocitaria y a la expresión de la proteína p53 y de los receptores de andrógenos como marcadores pronósticos (sobrevida global y período libre de enfermedad), y, en segundo lugar, determinar su utilidad como elementos predictivos de respuesta a la quimioterapia. Material y método Analizamos un grupo de pacientes con diagnóstico de cmtn tratadas en el Hospital Alemán de Buenos de Aires y en el Sanatorio Mater Dei desde diciembre de 2000 a diciembre de 2012. Resultados Se analizaron 21 pacientes con cmtn. En nuestra población, la quimioterapia se asoció con menor riesgo de recaída y mortalidad. La prevalencia en la expresión de la proteína p53 fue del 61,9% en la población general, del 72,7% en las recaídas y del 66,6% en las pacientes fallecidas. El 23,8% de las pacientes estudiadas presentó expresión de receptores androgénicos. La infiltración linfocitaria tumoral (tils) promedio fue del 20,5% (5% - 60%), siendo menor cuando se evalúa exclusivamente la población de pacientes que recayeron (17,7%) y aún menor cuando se evalúa la población de pacientes fallecidas (11,7 %). Discusión La quimioterapia tuvo un rr de sobrevida de 0,15 (ic: 0,06 - 0,78) con una p: 0,0021. La expresión de la proteína p53 tuvo un rr de 1,2 como factor de riesgo de mortalidad o progresión (ic: 0,23 - 9,07; p: 0,6). La expresión de receptores androgénicos tiene un rr de 1,6 como factor de riesgo de mortalidad o progresión (ic: 0,21 - 6,24; p: 0,41). La presencia de tils mayor al 20% es predictora de mortalidad y recurrencia con una p: 0,023. Conclusiones La determinación de tils junto con la evaluación en la expresión de la proteína p53 son herramientas útiles a la hora de definir la conveniencia de un tratamiento quimioterápico ya que ambas se asocian con aumento de la sobrevida total y de la sobrevida libre de enfermedad, aunque en nuestra población esta asociación no haya sido estadísticamente significativa para la p53 y sí para la presencia de tils

Introduction Because of its negativity for hormonal and her2 receptors, Triple Negative tumors do not benefit from anti hormonal treatments or with anti her2. They have no specific treatment and chemotherapy being the only form of systemic treatment. Objectives The objective of this study is, first, to evaluate in Triple Negative tumors feasibility of using lymphocyte infiltration, the expression of p53 protein and androgen receptors as prognostic markers (overall survival and disease-free), and, second, evaluate their usefulness as predictors of response to chemotherapy. Materials and method We intend to analyze those patients diagnosed with tnbc treated at the German Hospital of Buenos Aires and at the Mater Dei Sanatorium from December 2000 to December 2012. Results Twenty-one patients with tnbc were analyzed. In our population, chemotherapy was associated with a lower risk of relapse and mortality. The prevalence in the p53 mutation was 61.9% in the general population, 72.7% in relapses and 66.6% in deceased patients. Tumor lymphocytic infiltration (tils) on average was 20.5% (5% - 60%), being lower when only the population of patients who relapsed were evaluated (17.7%), and even lower when evaluating the population of deceased patients (11.7%). Discussion Chemotherapy had a survival rr of 0.15 (ci: 0.06 - 0.78) with a p: 0.0021. The p53 protein mutation had a rr of 1.2 as a risk factor for mortality or progression (ci: 0.23 - 9.07; p: 0.6). The expression of androgen receptors has a rr of 1.6 as a risk factor for mortality or progression (ci: 0.21 - 6.24; p: 0.41). The presence of tils greater than 20% is a predictor of mortality and recurrence with a p: 0.023. Conclusions The determination of tils together with determination of the p53 protein mutation are useful tools in determining the suitability of a chemotherapeutic treatment since both are associated with increased total survival and disease-free survival, although in our population this association was not statistically significant for p53.

Invasive micropapillary carcinoma of the breast overexpresses MUC4 and is associated with poor outcome to adjuvant trastuzumab in HER2-positive breast cancer.

BACKGROUND: Invasive micropapillary carcinoma of the breast (IMPC) is a histological tumor variant that occurs with low frequency characterized by an inside-out formation of tumor clusters with a pseudopapillary arrangement. IMPC is an aggressive tumor with poor clinical outcome. In addition, this histological subtype usually expresses human epidermal growth factor receptor 2 (HER2) which also correlates with a more aggressive tumor. In this work we studied the clinical significance of IMPC in HER2-positive breast cancer patients treated with adjuvant trastuzumab. We also analyzed mucin 4 (MUC4) expression as a novel biomarker to identify IMPC. METHODS: We retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry. RESULTS: IMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient's age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1-6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma. CONCLUSION: In HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

PURPOSE: Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. EXPERIMENTAL DESIGN: Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. RESULTS: TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). CONCLUSIONS: We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. ©2016 AACR.

A phase II dose-escalation trial of perioperative desmopressin (1-desamino-8-d-arginine vasopressin) in breast cancer patients.

Desmopressin (dDAVP) is a well-known peptide analog of the antidiuretic hormone vasopressin, used to prevent excessive bleeding during surgical procedures. dDAVP increases hemostatic mediators, such as the von Willebrand factor (vWF), recently considered a key element in resistance to metastasis. Studies in mouse models and veterinary trials in dogs with locally-advanced mammary tumors demonstrated that high doses of perioperative dDAVP inhibited lymph node and early blood-borne metastasis and significantly prolonged survival. We conducted a phase II dose-escalation trial in patients with breast cancer, administering a lyophilized formulation of dDAVP by intravenous infusion in saline, 30-60 min before and 24 h after surgical resection. Primary endpoints were safety and tolerability, as well as selection of the best dose for cancer surgery. Secondary endpoints included surgical bleeding, plasma levels of vWF, and circulating tumor cells (CTCs) as measured by quantitative PCR of cytokeratin-19 transcripts. Only 2 of a total of 20 patients experienced reversible adverse events, including hyponatremia (grade 4) and hypersensitivity reaction (grade 2). Reactions were adequately managed by slowing the infusion rate. A reduced intraoperative bleeding was noted with increasing doses of dDAVP. Treatment was associated with higher vWF plasma levels and a postoperative drop in CTC counts. At the highest dose level evaluated (2 µg/kg) dDAVP appeared safe when administered in two slow infusions of 1 µg/kg, before and after surgery. Clinical trials to establish the effectiveness of adjunctive perioperative dDAVP therapy are warranted. This trial is registered on www.clinicaltrials.gov (NCT01606072).

Targeting Stat3 induces senescence in tumor cells and elicits prophylactic and therapeutic immune responses against breast cancer growth mediated by NK cells and CD4+ T cells.

Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4(+) T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.

Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer.

BACKGROUND: The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference. METHODS: Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS. RESULTS: The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors. CONCLUSIONS: We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.

Alterations in c-Src/HER1 and estrogen receptor α signaling pathways in mammary gland and tumors of hexachlorobenzene-treated rats.

Hexachlorobenzene (HCB) is an organochlorine pesticide that acts as an endocrine disruptor in humans and rodents. The development of breast cancer strongly depends on endocrine conditions modulated by environmental factors. We have demonstrated that HCB is a tumor co-carcinogen in rats and an inducer of proliferation in MCF-7 cells, in an estrogen receptor α (ERα)-dependent manner, and of migration in MDA-MB-231 breast cancer cell line. In the present study, we examined HCB effect on c-Src/human epidermal growth factor receptor (HER1) and ERα signaling pathways in mammary glands and in N-nitroso-N-methylurea (NMU)-induced mammary tumors in rats. Furthermore, we evaluated histopathological changes and serum hormone levels. Rats were separated into four groups: control, HCB (100 mg/kg b.w.), NMU (50 mg/kg b.w.) and NMU-HCB. Our data show that HCB increases c-Src and HER1 activation, c-Src/HER1 association, and Y699-STAT5b and ERK1/2 phosphorylation in mammary glands. HCB also enhances Y537-ERα phosphorylation and ERα/c-Src physical interaction. In tumors, HCB also induces c-Src and HER1 activation, c-Src/HER1 association, as well as T308-Akt and Y699-STAT5b phosphorylation. In addition, the pesticide increases ERα protein content and decreases p-Y537-ERα levels and ERα/c-Src association in tumors. HCB increases serum 17-beta estradiol and prolactin contents and decreases progesterone, FSH and LH levels in rats without tumors, while the opposite effect was observed in rats with tumors. Taken together, our results indicate that HCB induces an estrogenic effect in mammary gland, increasing c-Src/HER1 and ERα signaling pathways. HCB stimulates c-Src/HER1 pathway, but decreases ERα activity in tumors, appearing to shift them towards a higher malignancy phenotype.

Modulation of endothelial cell migration and angiogenesis: a novel function for the "tandem-repeat" lectin galectin-8.

Angiogenesis, the growth of new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. Glycan-mediated recognition has been proposed to play an instrumental role in mediating cell-cell and cell-matrix interactions. Galectins (Gal), a family of glycan-binding proteins with affinity for ß-galactosides and a conserved sequence motif, can decipher glycan-containing information and mediate cell-cell communication. Galectin-8 (Gal-8), a member of this family, is a bivalent "tandem-repeat"-type galectin, which possesses 2 CRDs connected by a linker peptide. Here, we show that Gal-8 is endowed with proangiogeneic properties. Functional assays revealed a critical role for this lectin in the regulation of capillary-tube formation and EC migration. Moreover, Matrigel, either supplemented with Gal-8 or vascular endothelial growth factor (VEGF), injected in mice resulted in induction of in vivo angiogenesis. Remarkably, Gal-8 was expressed both in the cytoplasm and nucleus in ECs of normal and tumor vessels. Furthermore, CD166 [activated leukocyte cell adhesion molecule (ALCAM)] was identified as a specific Gal-8-binding partner in normal vascular ECs. Collectively, these data provide the first evidence demonstrating an essential role for Gal-8 in the regulation of angiogenesis with critical implications in tumor biology.

Programa nacional de diagnóstico de la sobreexposición de HER2 en pacientes con cáncer de mama / National program for the diagnosis of HER2 overexpression in breast cancer patients

Introducción: Por medio de la realización del testeo de la sobreexpresión y/o amplificación de HER2 los médicos pueden seleccionar adecuadamente a las pacientes que se beneficiarán de la terapia anti HER2. En Argentina, antes de 2003 sólo pocos patólogos realizaban la prueba de HER2 utilizando métodos no estandarizados de inmunohistoquímica (IHQ) con resultados dudosos y no reproducibles y además existiendo algunas partes del país sin posibilidades de diagnóstico. Objetivo: Generar un Programa Nacional de Detección de la sobreexpresión de HER2 que permita el acceso al diagnóstico en todo el país, mediante una técnica estandarizada y validada. Integrar un equipo de patólogos entrenados en IHC, que pudieran satisfacer las demandas realizando en forma rápida y fidedigna la detección de la sobreexpresión de HER2. Materiales y método: En agosto de 2003 se formó una red de trece patólogos; dentro del grupo se designó un coordinador, responsable del entrenamiento y la evaluación de los centros participantes, y dos consultores técnicos a cargo del control de calidad (interno y externo) y la estandarización de la técnica. Desde febrero de 2004 hasta diciembre de 2010 se realizaron las determinaciones de la sobreexpresión de HER2 mediante la técnica de IHC, con un anticuerpo policlonal anti HER2 (Dako), recuperación antigénica en microondas, sistema de detección EnVision (Dako) y revelado con diaminobenzidina. Para interpretar los resultados se usó el score de 0 a 3+, considerándose positivos tumores que muestren intensidad moderada (2+) o intensa (3+) en toda la membrana celular, en más del 30% de las células evaluadas. Resultados: Se realizaron 34.640 casos (Figura 5). Conclusiones: La formación del Programa Nacional de HER2 permitió el acceso al test de HER2 en todo el país con una técnica estandarizada y reproducible en todos los centros participantes. La prevalencia de HER2 en nuestra muestra fue del 13,2% (4.573) y similar a las publicadas en otras poblaciones.

TNF alpha acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-kappa B-dependent pathways.

Tumor necrosis factor alpha (TNF alpha) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF alpha, the participation of TNF alpha receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNFalpha induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappa B (NF-kappa B) transcriptional activation. A TNF alpha-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-kappa B transcriptional activation and cell proliferation, just like wild-type TNF alpha, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF alpha signaling and biological effect. Moreover, in vivo TNF alpha administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-kappa B activity, Bay 11-7082, resulted in regression of TNF alpha-promoted tumor. Bay 11-7082 blocked TNF alpha capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-xLin vivo and in vitro. Our results reveal evidence for TNF alpha as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF alpha antagonists and NF-kappa B pharmacological inhibitors in established breast cancer treatment.

Progestins induce transcriptional activation of signal transducer and activator of transcription 3 (Stat3) via a Jak- and Src-dependent mechanism in breast cancer cells.

Interactions between steroid hormone receptors and signal transducer and activator of transcription (Stat)-mediated signaling pathways have already been described. In the present study, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in BALB/c mice and in the human breast cancer cell line T47D. We found that C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 and that MPA treatment of C4HD cells up-regulated Stat3 protein expression. In addition, MPA induced rapid, nongenomic Stat3, Jak1, and Jak2 tyrosine phosphorylation in C4HD and T47D cells. MPA treatment of C4HD cells also resulted in rapid c-Src tyrosine phosphorylation. These effects were completely abolished by the progestin antagonist RU486. Abrogation of Jak1 and Jak2 activity by transient transfection of C4HD cells with dominant negative (DN) Jak1 or DN Jak2 vectors, or inhibition of Src activity by preincubation of cells with the Src family kinase inhibitor PP2, blocked the capacity of MPA to induce Stat3 phosphorylation. Treatment of C4HD cells with MPA induced Stat3 binding to DNA. In addition, MPA promoted strong Stat3 transcriptional activation in C4HD and T47D cells that was inhibited by RU486 and by blockage of Jak1, Jak2, and Src activities. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F mutant had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Finally, we addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells' ability to form tumors in syngeneic mice. Our results have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation, which is in turn an obligatory requirement for progestin stimulation of both in vitro and in vivo breast cancer growth.

Inhibition of in vivo breast cancer growth by antisense oligodeoxynucleotides to type I insulin-like growth factor receptor mRNA involves inactivation of ErbBs, PI-3K/Akt and p42/p44 MAPK signaling pathways but not modulation of progesterone receptor activity.

The present study addresses the effect of targeting type I insulin-like growth factor receptor (IGF-IR) with antisense strategies in in vivo growth of breast cancer cells. Our research was carried out on C4HD tumors from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice. We employed two different experimental strategies. With the first one we demonstrated that direct intratumor injection of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. In the second experimental strategy, we assessed the effect of intravenous (i.v.) injection of AS [S]ODN on C4HD tumor growth. This systemic treatment also resulted in significant reduction in tumor growth. The antitumor effect of IGF-IR AS[S]ODNs in both experimental protocols was due to a specific antisense mechanism, since growth inhibition was dose-dependent and no abrogation of tumor proliferation was observed in mice treated with phosphorothioate sense ODNs (S[S]ODNs). In addition, IGF-IR expression was inhibited in tumors from mice receiving AS[S]ODNs, as compared to tumors from control groups. We then investigated signal transduction pathways modulated in vivo by AS[S]ODNs treatment. Tumors from AS[S]ODN-treated mice of both intratumoral and intravenous protocols showed a significant decrease in the degree of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation. Activation of two of the main IGF-IR signaling pathways, phosphatidylinositol 3-kinase (PI-3K)/Akt and p42/p44 mitogen-activated protein kinases (MAPK) was abolished in tumors growing in AS[S]ODN-treated animals. Moreover, ErbB-2 tyrosine phosphorylation was blocked by in vivo administration of AS[S]ODNs. On the other hand, we found no regulation of either progesterone receptor expression or activity by in vivo AS[S]ODNs administration. Our results for the first time demonstrated that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODNs.

Reproducibilidad del grado nuclear en el cáncer de mama: una experiencia multicéntrica / Reproducibility of nuclear grade in breast neoplasm: a multicenter experience

El grado nuclear es considerado uno de los elementos de valor pronóstico en el carcinoma de mama. Resulta de interés verificar cuál es el grado de reproducibilidad de los patólogos generales al consignar el grado nuclear considerando que la mayor parte de los estudios anatomopatológicos de los carcinomas de mama los realizan patólogos "no expertos". Para ello se tomó una serie de 15 carcinomas de mama de los cuales 10 fueron seleccionados al azar y 5 por su particular dificultad para clasificar el grado nuclear. Diez patólogos generales trabajando en forma separada examinaron los casos y les asignaron el grado nuclear. Se hizo una primera asignación de acuerdo al criterio que cada observador utilizaba habitualmente para sus informes y una segunda siguiendo a una guía escrita. e estudiaron los datos por medio del análisis de la varianza. Los resultados mostraron que tanto para los casos seleccionados al azar como para la serie total no hubo diferencias significativas entre los observadores. La guía escrita, sorprendentemente, en lugar de disminuir las diferencias las aumentó. Un análisis del comportamiento individual de los observadores permitió detectar que dos de ellos tenían una gran variación entre ambas asignaciones, lo que se consideró como una influencia determinante para los resultados de todo el grupo. Cuando se analizó la diferencia interobservador para discriminar los tumores de alto grado (G3) del resto, se observó una buena reproducibilidad en todo el grupo participante. Estos resultados nos permiten concluir que, en esta serie examinada por patólogos generales, se obtuvo una aceptable reproducibilidad, en especial cuando se trató de identificar a los tumores con mayor riesgo.

Reproducibilidad del grado nuclear en el cáncer de mama: una experiencia multicéntrica / Reproducibility of nuclear grade in breast neoplasm: a multicenter experience

El grado nuclear es considerado uno de los elementos de valor pronóstico en el carcinoma de mama. Resulta de interés verificar cuál es el grado de reproducibilidad de los patólogos generales al consignar el grado nuclear considerando que la mayor parte de los estudios anatomopatológicos de los carcinomas de mama los realizan patólogos "no expertos". Para ello se tomó una serie de 15 carcinomas de mama de los cuales 10 fueron seleccionados al azar y 5 por su particular dificultad para clasificar el grado nuclear. Diez patólogos generales trabajando en forma separada examinaron los casos y les asignaron el grado nuclear. Se hizo una primera asignación de acuerdo al criterio que cada observador utilizaba habitualmente para sus informes y una segunda siguiendo a una guía escrita. e estudiaron los datos por medio del análisis de la varianza. Los resultados mostraron que tanto para los casos seleccionados al azar como para la serie total no hubo diferencias significativas entre los observadores. La guía escrita, sorprendentemente, en lugar de disminuir las diferencias las aumentó. Un análisis del comportamiento individual de los observadores permitió detectar que dos de ellos tenían una gran variación entre ambas asignaciones, lo que se consideró como una influencia determinante para los resultados de todo el grupo. Cuando se analizó la diferencia interobservador para discriminar los tumores de alto grado (G3) del resto, se observó una buena reproducibilidad en todo el grupo participante. Estos resultados nos permiten concluir que, en esta serie examinada por patólogos generales, se obtuvo una aceptable reproducibilidad, en especial cuando se trató de identificar a los tumores con mayor riesgo. (AU)

Metástasis cardíaca: su diagnóstico antemorten por hallazgos electrocardiográficos inusuales / Heart metastasis: its antemortem diagnosis by unusual electrocardiographic findings

Un hombre de 60 años desarrolló una imagen persistente de infarto agudo de miocardio, un hemibloqueo posterior izquierdo, un bloqueo sinoatrial de 2§ grado 312 y un bloqueo A-V de 1er grado, seguido de disociación A-V, en el curso de un carcinoma pavimentoso broncopulmonar. Se formuló el diagnóstico de metástasis cardíaca y se observó una significativa mejoría de la conducción A-V luego de la administración de corticoides. En la necropsia se comprobaron metástasis en la cara posterior, septum interventricular y aurícula derecha. No ha sido referida una asociación semejante de cambios electrocardiográficos por metástasis cardíacas, en la literatura disponible

Metástasis cardíaca: su diagnóstico antemorten por hallazgos electrocardiográficos inusuales / Heart metastasis: its antemortem diagnosis by unusual electrocardiographic findings

Un hombre de 60 años desarrolló una imagen persistente de infarto agudo de miocardio, un hemibloqueo posterior izquierdo, un bloqueo sinoatrial de 2º grado 312 y un bloqueo A-V de 1er grado, seguido de disociación A-V, en el curso de un carcinoma pavimentoso broncopulmonar. Se formuló el diagnóstico de metástasis cardíaca y se observó una significativa mejoría de la conducción A-V luego de la administración de corticoides. En la necropsia se comprobaron metástasis en la cara posterior, septum interventricular y aurícula derecha. No ha sido referida una asociación semejante de cambios electrocardiográficos por metástasis cardíacas, en la literatura disponible (AU)

Manual operativo de anatomía patológica del cáncer de mama / Operational manual of pathological anatomy of breast cancer

El informe anatomopatológico es una pieza clave para la definición del tratamiento y el pronóstico de las pacientes con cáncer de mama. La naturaleza multidisciplinaria del tratamiento de los tumores mamarios, hace indispensable la estandarización del reporte de patología. El cáncer de mama es una enfermedad muy prevalente y muchos profesionales se ven involucrados en su tratamiento. Por ello es esencial homogeneizar el lenguaje, de manera tal de que el informe pueda ser leído e interpretado por todos los miembros del equipo responsable del cuidado de la mujer. La aparición de nuevas técnicas moleculares, lejos de reducir el valor de la patología tradicional, aumenta la necesidad de un procesamiento de tejidos adecuado, de modo de garantizar un material adecuadamente tratado para realizar determinaciones complementarias. Queremos agradecer la invalorable colaboración de la Sociedad Argentina de Patología, la Sociedad Argentina de Citología y de nuestros Consultores Académicos sin cuya generosidad y solidez científica esta guía no podría haberse realizado.