Morphological evaluation of tumor-infiltrating lymphocytes (TILs) to investigate invasive breast cancer immunogenicity, reveal lymphocytic networks and help relapse prediction:a retrospective study

Tumor-infiltrating lymphocytes (TILs) in breast cancer are a key representative of the tumor immune microenvironment and have been shown to provide prognostic and predictive biomarkers. The extent of lymphocytic infiltration in tumor tissues can be assessed by evaluating hematoxylin and eosin (H&E)-stained tumor sections. We investigated tissue microarrays of 31 invasive breast cancer patients, looking at quantity and topological distribution of CD3+, CD8+, CD20+, Ki67+, FoxP3+ TILs and CD3+/FoxP3+, CD8+/FoxP3+ cell ratios. We separately evaluated TILs at the invasive edge and at the center of the tumor, to find any clinical implications of tumor heterogeneity. No statistically significant difference was found in quantity and distribution of both TIL subsets and TIL ratios, by comparing patients who suffered from a local or distant recurrence of the tumor (relapse group: 13 patients) with patients not showing cancer relapse (non-relapse group: 18 patients). In the whole sample, we observed three main statistically significant positive correlations: (1) between CD3+ and CD8+ T-cells; (2) between FoxP3+ and Ki67+ lymphocyte infiltration; (3) between CD3+/FoxP3+ cell ratio (C3FR) and CD8+/FoxP3+ cell ratio (C8FR). Tumor heterogeneity and stronger positive TIL associations were found in the non-relapse group, where both CD3–CD8 and FoxP3-Ki67 inter-correlations were found to be significant at the center of the tumor, while the correlation between C3FR and C8FR was significant at the invasive edge. No correlations between TIL subsets were detected in the relapse group. Our findings suggest the existence of stronger inter-subtype lymphocytic networks in invasive breast cancer not showing recurrence. Further evaluations of clinical and topological correlations between and within TIL subsets are needed, in addition to the assessment of TIL quantification and distribution, in order to follow up on whether morphological evaluation of TILs might reveal the underlying lymphocytic functional connectivity and help relapse prediction

Neutrophil gelatinase-associated lipocalin (NGAL) predicts response to neoadjuvant chemotherapy and clinical outcome in primary human breast cancer

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC

Cannabinoid receptor CB2 drives HER2 pro-oncogenic signaling in breast cancer

Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different models of cancer. However, the biological role of these receptors in tumor physio-pathology is still unknown. We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen and Freiburg between 1997 and 2010. CB2 mRNA expression was also analyzed in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the HER2 rat ortholog (neu) and lacks CB2, and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by co-localization, coimmunoprecipitation and proximity ligation assays. We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis. We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade, and that an increased CB2 expression activates the HER2 prooncogenic signaling machinery at the level of the tyrosine kinase c-SRC. Finally, HER2 and CB2 form heteromers in cancer cells. Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and suggest that CB2 may be a biomarker with prognostic value in these tumors

Activation of the orphan receptor GPR55 by lysophosphatidylinositol promotes metastasis in triple-negative breast cancer

The orphan G protein-coupled receptor GPR55 has been directly or indirectly related to basic alterations that drive malignant growth: uncontrolled cancer cell proliferation, sustained angiogenesis, and cancer cell adhesion and migration. However, little is known about the involvement of this receptor in metastasis. Here, we show that elevated GPR55 expression in human tumors is associated with the aggressive basal/triple-negative breast cancer population, higher probability to develop metastases, and therefore poor patient prognosis. Activation of GPR55 by its proposed endogenous ligand lysophosphatidylinositol confers pro-invasive features on breast cancer cells both in vitro and in vivo. Specifically, this effect is elicited by coupling to Gq/11 heterotrimeric proteins and the subsequent activation, through ERK, of the transcription factor ETV4/PEA3. Together, these data show that GPR55 promotes breast cancer metastasis, and supports the notion that this orphan receptor may constitute a new therapeutic target and potential biomarker in the highly aggressive triple-negative subtypeThis work was supported by grants from Spanish Ministry of Economy and Competitiveness [PI11/00295 to CS, PI14/01101 to CS and EP-G, SAF2013-46183-R to MQ, and SAF2014-54705-R to MV-M, supported with European Regional Development (FEDER) funds] and Madrid Regional Government (S2010/BMD-2308 to MG, and 2010/BMD-2359 to MQ). EPG was a recipient of a Postdoctoral Research Contract from Fundación Científica Asociación Española Contra el Cáncer and a Federation of the Societies of Biochemistry and Molecular Biology (FEBS) Short-term Fellowship. SB-B and SC-L are recipients of a Formación de Profesorado Universitario (FPU) fellowship and a Ramón y Cajal research contract, respectively, from the Spanish Ministry of Economy and Competitivenes

Die Rolle des MDM2-SNP309 beim sporadischen Nierenzellkarzinom

Das mouse double minutes 2-Protein (MDM2) ist der zelluläre Hauptregulator des Tumorsuppressors p53, eines Transkriptionsaktivators von DNA-Reparatur- und Apoptosegenen, dessen Funktion von intakten Zellen nicht benötigt wird. MDM2 führt p53 über Ubiquitinierung dem proteasomalen Abbau zu. Im Falle einer DNA-Schädigung wird die Bildung des MDM2/p53-Komplexes durch Phosphorylierung von p53 unterbunden. Auf diese Weise wird p53 freigesetzt und steht der Zelle sofort zur Einleitung von Reparatur- bzw. Apoptoseprozessen zur Verfügung. Von MDM2 ist ein single nucleotide polymorphism (SNP) in der Promoterregion von Intron 1 bekannt, der zu einer erhöhten Affinität des Transkriptionsaktivators SP1 führt. Bindet SP1 an den Promotor, resultiert eine erhöhte MDM2-Expression, der p53-Signalweg wird herunterreguliert. Es konnte nachgewiesen werden, dass dieser SNP an Position 309 (Austausch der Base T durch G) mit vermehrter Tumorbildung bei erblichen, aber auch sporadischen Krebserkrankungen einhergeht. Darüber hinaus konnte für einige Tumoren ein früheres Erkrankungsalter bei Patienten mit dem SNP gezeigt werden. Bislang liegen noch keine Daten zum sporadischen Nierenzellkarzinom (NZK) bei Europäern vor. Im Rahmen dieser Arbeit wurde daher die Verteilung der Genotypen des MDM2-SNP309 innerhalb einer Gruppe von 257 überwiegend europäischen Patienten mit Nierenzellkarzinom ermittelt (101 hellzellige, 32 papilläre und 102 chromophobe NZK) und mit der einer Kontrollgruppe ohne Tumorerkrankung (111 Proben) verglichen. Die DNA wurde aus den formalinfixierten und in Paraffin eingebetteten Normalgewebeproben extrahiert. Die Amplifizierung des Restriktions-Fragment-Längen-Polymorphimus (RFLP) erfolgte mittels PCR unter Verwendung fluoreszenz-markierter Oligonukleotide. Für den sich anschließenden Verdau wurde das Restriktionsenzym MspA1 verwendet. Danach erfolgte die Kapillarelektrophorese. Der Vergleich der Verteilung der Genotypen beider Gruppen erfolgte anhand des Exakten Tests nach Fisher. Zusätzlich wurde die Korrelation des Polymorphismus mit NZK-Subtypen, histopathologischen Kriterien, Alter, Geschlecht und Überleben untersucht. Die Analyse der Überlebensdaten wurde mit dem Log Rank Test durchgeführt. Die Verteilung der Genotypen von Patienten mit Nierenzellkarzinom unterschied sich nicht wesentlich von der der Kontrollgruppe. Es war ebenfalls keine Korrelation zwischen dem Genotyp und dem NZK-Subtyp, Erkrankungsalter, Geschlecht, Überleben oder den histopathologischen Parametern erkenntlich. Auch bezüglich der Allelverteilung ergab sich keine Korrelation. So kommt diese Arbeit zu dem Schluss, dass das Vorkommen des SNP an der Position 309 des MDM2-Gens keinen Einfluss auf ein erhöhtes Erkrankungsrisiko für das Nierenzellkarzinom hat. Es liegt ebenfalls kein Zusammenhang zwischen dem SNP und einem besonders niedrigen Erkrankungsalter, dem Tumorstadium oder -subtyp, dem Geschlecht und dem Überleben bei europäischen Nierenzellkarzinom-Patienten vor

Reduced ovarian reserve in young early breast cancer patients: preliminary data from a prospective cohort trial

Abstract Background The numerous side effects of chemotherapy in patients with breast cancer are well known. However, the precise effects of chemotherapy on ovarian function in premenopausal women are poorly investigated. The patients are at risk of developing sexual hormone deficiency and impaired fertility. This prospective cohort study addresses predictive parameters of ovarian reserve after chemotherapy. Methods Fifty-one premenopausal women (28–46 years) with primary breast cancer were included in the trial. All of them received anthracycline-based chemotherapy (n = 18), or combinations with taxanes (n = 30), or anthracycline-free chemotherapy (n = 3). Changes in hormone levels (LH, FSH, E2 and Anti-Müllerian hormone (AMH)), antral follicle count (AFC), and amenorrhea were determined before (V1), and 6, 12 and 24 months after the initiation of chemotherapy (V2-V4). Quality of life parameters were evaluated. The additional impact of parity, BMI, and smoking on ovarian reserve was also assessed. Results AFC and AMH fell very markedly after chemotherapy and did not return to pre-treatment levels until V4. A significant positive correlation was noted in AFC before and 1 year after chemotherapy. AMH levels at V2-V4 were significantly correlated with those registered at V1. AFC and AMH were negatively correlated with age. Continued smoking had a significant detrimental effect on AFC after 24 months. LH and FSH levels increased between V1 and V2 and fell at V3 and V4, but stayed above pre-chemotherapy values. Two years after the start of chemotherapy 31/51 patients were amenorrhoic while 17 resumed their menstrual cycle; this was not influenced by the type of chemotherapy or age. Non-smokers were 13 times more likely to resume their menstruation than smokers. Quality of life (QL) was significantly lower 6 months after the initiation of chemotherapy. QL at one and 2 years after chemotherapy did not differ significantly from pre-chemotherapy scores. Conclusions Our study contributes to a better understanding and prediction of ovarian reserve in young early breast cancer patients undergoing chemotherapy. The data suggest that personal counseling in regard of the preservation of fertility should be offered especially to patients of a higher age, with low AMH levels or low follicle counts. Patients should be advised to stop smoking in order to enhance the likelihood of preserving their fertility