Bone mineral density in breast cancer patients treated with adjuvant letrozole, tamoxifen, or sequences of letrozole and tamoxifen in the BIG 1-98 study (SAKK 21/07).

BACKGROUND: The risk of osteoporosis and fracture influences the selection of adjuvant endocrine therapy. We analyzed bone mineral density (BMD) in Swiss patients of the Breast International Group (BIG) 1-98 trial [treatment arms: A, tamoxifen (T) for 5 years; B, letrozole (L) for 5 years; C, 2 years of T followed by 3 years of L; D, 2 years of L followed by 3 years of T]. PATIENTS AND METHODS: Dual-energy X-ray absorptiometry (DXA) results were retrospectively collected. Patients without DXA served as control group. Repeated measures models using covariance structures allowing for different times between DXA were used to estimate changes in BMD. Prospectively defined covariates were considered as fixed effects in the multivariable models. RESULTS: Two hundred and sixty-one of 546 patients had one or more DXA with 577 lumbar and 550 hip measurements. Weight, height, prior hormone replacement therapy, and hysterectomy were positively correlated with BMD; the correlation was negative for letrozole arms (B/C/D versus A), known osteoporosis, time on trial, age, chemotherapy, and smoking. Treatment did not influence the occurrence of osteoporosis (T score < -2.5 standard deviation). CONCLUSIONS: All aromatase inhibitor regimens reduced BMD. The sequential schedules were as detrimental for bone density as L monotherapy.

Prognostic and predictive value of centrally reviewed Ki-67 labeling index in postmenopausal women with endocrine-responsive breast cancer: results from Breast International Group Trial 1-98 comparing adjuvant tamoxifen with letrozole.

PURPOSE: To evaluate the prognostic and predictive value of Ki-67 labeling index (LI) in a trial comparing letrozole (Let) with tamoxifen (Tam) as adjuvant therapy in postmenopausal women with early breast cancer. PATIENTS AND METHODS: Breast International Group (BIG) trial 1-98 randomly assigned 8,010 patients to four treatment arms comparing Let and Tam with sequences of each agent. Of 4,922 patients randomly assigned to receive 5 years of monotherapy with either agent, 2,685 had primary tumor material available for central pathology assessment of Ki-67 LI by immunohistochemistry and had tumors confirmed to express estrogen receptors after central review. The prognostic and predictive value of centrally measured Ki-67 LI on disease-free survival (DFS) were assessed among these patients using proportional hazards modeling, with Ki-67 LI values dichotomized at the median value of 11%. RESULTS: Higher values of Ki-67 LI were associated with adverse prognostic factors and with worse DFS (hazard ratio [HR; high:low] = 1.8; 95% CI, 1.4 to 2.3). The magnitude of the treatment benefit for Let versus Tam was greater among patients with high tumor Ki-67 LI (HR [Let:Tam] = 0.53; 95% CI, 0.39 to 0.72) than among patients with low tumor Ki-67 LI (HR [Let:Tam] = 0.81; 95% CI, 0.57 to 1.15; interaction P = .09). CONCLUSION: Ki-67 LI is confirmed as a prognostic factor in this study. High Ki-67 LI levels may identify a patient group that particularly benefits from initial Let adjuvant therapy.

A critical evaluation of secondary cancer risk models applied to Monte Carlo dose distributions of 2-dimensional, 3-dimensional conformal and hybrid intensity-modulated radiation therapy for breast cancer.

The comparison of radiotherapy techniques regarding secondary cancer risk has yielded contradictory results possibly stemming from the many different approaches used to estimate risk. The purpose of this study was to make a comprehensive evaluation of different available risk models applied to detailed whole-body dose distributions computed by Monte Carlo for various breast radiotherapy techniques including conventional open tangents, 3D conformal wedged tangents and hybrid intensity modulated radiation therapy (IMRT). First, organ-specific linear risk models developed by the International Commission on Radiological Protection (ICRP) and the Biological Effects of Ionizing Radiation (BEIR) VII committee were applied to mean doses for remote organs only and all solid organs. Then, different general non-linear risk models were applied to the whole body dose distribution. Finally, organ-specific non-linear risk models for the lung and breast were used to assess the secondary cancer risk for these two specific organs. A total of 32 different calculated absolute risks resulted in a broad range of values (between 0.1% and 48.5%) underlying the large uncertainties in absolute risk calculation. The ratio of risk between two techniques has often been proposed as a more robust assessment of risk than the absolute risk. We found that the ratio of risk between two techniques could also vary substantially considering the different approaches to risk estimation. Sometimes the ratio of risk between two techniques would range between values smaller and larger than one, which then translates into inconsistent results on the potential higher risk of one technique compared to another. We found however that the hybrid IMRT technique resulted in a systematic reduction of risk compared to the other techniques investigated even though the magnitude of this reduction varied substantially with the different approaches investigated. Based on the epidemiological data available, a reasonable approach to risk estimation would be to use organ-specific non-linear risk models applied to the dose distributions of organs within or near the treatment fields (lungs and contralateral breast in the case of breast radiotherapy) as the majority of radiation-induced secondary cancers are found in the beam-bordering regions.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

Characterization of BMP signaling in breast development and tumorigenesis

Résumé L'influence des hormones reproductives sur le développement du cancer du sein a été établie au travers de nombreuse études épidémiologiques. Nous avons précédemment démontré que le gène Wnt-4 est un médiateur essentiel de la progestérone dans le développement lobulo-alvéolaire de l'épithélium mammaire. De plus, le rôle de la voie de signalisation Wnt dans la tumorigénèse de la glande mammaire mutine est largement établi. Pour comprendre sa fonction dans le cancer du sein, nous avons activée cette voie en surexprimant le gène Wnt-1 dans des cellules épithéliales primaires de sein, au moyen d'un rétrovirus. Ceci a conduit à la transformation oncogénique de ces cellules et à l'obtention d'un modèle de carcinogénèse du sein dénommé Wnt-1 HMEC. L'analyse de l'expression des gènes induits par la surexpression de Wnt-1 dans ces cellules, a permis d'identifier les gènes BMP4 et 7. Alors que des analyses de RT-PCR ont montré leur forte expression dans les cellules Wnt-1-HMECs, la présence d'une grande quantité de la protéine BMP7 a été constatée dans les tumeurs dérivées de ces cellules. L'importante phosphorylation des Smad 1, 5, S dans les Wnt-1 HMECs indique l'activation de la voie BMP, possiblement due à la stimulation ce celle-ci par BMP7. L'activation de la voie Wnt par la ß-Caténine, conduit à la transcription de BMP7, identifiant ainsi ce gène comme un gène cible de la voie canonique. La pertinence de nos observations a par ailleurs été confirmée par le fait que BMP7 est surexprimé dans les tumeurs de seins humains. Afin d'élucider la fonction de la voie BMP dans le sein, nous avons utilisé le modèle mutin. L'expression du gène BMP7 dans les souris transgéniques MMTV Wnt-1 s'est avérée élevée, démontrant qu'il est aussi un gène cible de la voie Wnt in-vivo. L'expression de l'ARN messager .codant pour la protéine BMP7 est induite lors du développement lobulo-alvéolaire, qui se fait sous l'influence de la progestérone et de Wnt-4. Ensemble, ces observations corroborent le fait qu'une stimulation avec de la progestérone suffit à induire la transcription du gène dans les 24h. Nos résultats coïncident d'autre part avec le fait que BMP7 est exprimé dans la couche myoépithéliale de l'épithélium où la voie Wnt est activée. L'analyse de souris reportrices de l'activité de la voie BMP, suggère une activation dans la couche luminale de l'épithélium durant tout le développement de la glande mammaire. Curieusement, cette même voie est active dans le mésenchyme lors de la mammogénèse embryonnaire. Finalement, nos analyses d'immunofluorescence démontrent la capacité de prolifération des cellules ayant activé BMP, ainsi que leur nette ségrégation d'avec les cellules exprimant le récepteur à la progestérone. Nos résultats démontrent que le gène BMP7 est un gène cible de la voie Wnt canonique dans le sein. Son expression dans la couche myoépitheliale est induite par Wnt-4, lui-même sécrété par les cellules luminales sensibles à la progestérone. La sécrétion de la protéine BMP7 conduit finalement à l'activation de la voie BMP dans les cellules négatives pour le récepteur à la progestérone. Abstract Epidemiological studies highlight the repetitive exposure to circulating progesterone as a major risk in the development of breast cancer. Work in our laboratory showed that Wnt-4 is an essential mediator of progesterone-driven side-branch formation, while Wnt signaling has long been established as strongly oncogenic in the mouse mammary gland. To address the role of Wnt in breast tumorigenesis we activated the pathway in primary human breast epithelial cells by means of refroviral Wnt-1 expression. This resulted in a Wnt1-induced breast carcinogenesis model, being referred to as Wnt-1-HMECs. Gene expression profiling revealed the Bone Morphogenetic Protein 4 and 7 (BMP4 and 7) a mong the most upregulated gene by ectopic Wnt-1 expression in primary HMECs. RT-PCR analysis confirmed elevated BMP4 and 7 mRNA levels in Wnt-1-infected HMECs, as well as strong BMP7 expression in the tumors derived from these cells. Smad 1, 5, 8 phosphorylation was high in Wnt-1HMECs whereas below detection limit in primary HMECs suggesting that the increased expression of BMP-7 results in activation of downstream signaling. Ectopic expressíon of a stabilized form of ßcatenin in primary HMECs resulted in increased transcription of BMP-7 suggesting that it is a target of canonical Wnt signaling. The clinical relevance of our observations was confirmed by the finding of BMP7 being upregulated in human breast tumor samples. To elucidate the role of BMP ligands in the breast in-vivo, we made use of the mouse model. Expression of the BMP7 gene was found to be increased in MMTV-Wnt-1 transgenic animals, suggesting that BMP7 may also be a Wnt 1 target gene in vivo. Expression of BMP7 was upregulated in mid-pregnancy which coincides with progesterone/Wnt induced side branching. BMP7 was induced within 24 hours by progesterone. Consistent with it being a target of canonical Wnt signaling, we demonstrated preferential expression of this ligand in the myoepithelial cells, the target cells of Wnt signals. In-vivo analysis of BMP signaling using a reporter mouse revealed the activation of the pathway in the luminal layer of the epithelium throughout postnatal development. Interestingly, during embryonic mammogenesis the pathway was found to be active in the mesenchyme. Immunofluorescence studies demonstrated that cells with BMP activity can proliferate. They also revealed a clear segregation between progesterone receptor positive cells and cells with active BMP signaling. Together our observations suggest that BMP-7 is a canonical Wnt signaling target both in HMECs and in the mouse mammary gland in-vivo. It is expressed in the myoepithelium possibly in response to Wnt-4, which is secreted by steroid receptor positive cells in response to progesterone. BMP-7 in turn may impinge on lumina) epithelial cells and activate BMP signaling in PR negative cells.

New prognostic factors and calculators for outcome prediction in patients with recurrent glioblastoma: A pooled analysis of EORTC Brain Tumour Group phase I and II clinical trials.

BACKGROUND: Prognostic models have been developed to predict survival of patients with newly diagnosed glioblastoma (GBM). To improve predictions, models should be updated with information at the recurrence. We performed a pooled analysis of European Organization for Research and Treatment of Cancer (EORTC) trials on recurrent glioblastoma to validate existing clinical prognostic factors, identify new markers, and derive new predictions for overall survival (OS) and progression free survival (PFS).¦METHODS: Data from 300 patients with recurrent GBM recruited in eight phase I or II trials conducted by the EORTC Brain Tumour Group were used to evaluate patient's age, sex, World Health Organisation (WHO) performance status (PS), presence of neurological deficits, disease history, use of steroids or anti-epileptics and disease characteristics to predict PFS and OS. Prognostic calculators were developed in patients initially treated by chemoradiation with temozolomide.¦RESULTS: Poor PS and more than one target lesion had a significant negative prognostic impact for both PFS and OS. Patients with large tumours measured by the maximum diameter of the largest lesion (⩾42mm) and treated with steroids at baseline had shorter OS. Tumours with predominant frontal location had better survival. Age and sex did not show independent prognostic values for PFS or OS.¦CONCLUSIONS: This analysis confirms performance status but not age as a major prognostic factor for PFS and OS in recurrent GBM. Patients with multiple and large lesions have an increased risk of death. With these data prognostic calculators with confidence intervals for both medians and fixed time probabilities of survival were derived.

Cancer du sein: radiothérapie et signalisation oestrogénique [Breast cancer: radiotherapy and estrogen signaling].

Hormone receptors are expressed in more than 75% of breast cancer. Therefore, two prescription modalities of endocrine therapy could be proposed: either sequential or concomitant to breast cancer irradiation. If combined to radiotherapy, is endocrine therapy a radiosensitizer? Does endocrine therapy enhance the risk factor of radio-induced toxicity? Here, we will distinguish the interaction of ionizing radiation combined with therapies targeting oestrogen receptor (REα) from the interaction of ionizing radiation with oestrogen. This review aims at making clear all these items.

Robust vaccine-elicited cellular immune responses in breast milk following systemic Simian Immunodeficiency Virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Primary breast lymphoma: patient profile, outcome and prognostic factors. A multicentre Rare Cancer Network study.

BACKGROUND: To asses the clinical profile, treatment outcome and prognostic factors in primary breast lymphoma (PBL). METHODS: Between 1970 and 2000, 84 consecutive patients with PBL were treated in 20 institutions of the Rare Cancer Network. Forty-six patients had Ann Arbor stage IE, 33 stage IIE, 1 stage IIIE, 2 stage IVE and 2 an unknown stage. Twenty-one underwent a mastectomy, 39 conservative surgery and 23 biopsy; 51 received radiotherapy (RT) with (n = 37) or without (n = 14) chemotherapy. Median RT dose was 40 Gy (range 12-55 Gy). RESULTS: Ten (12%) patients progressed locally and 43 (55%) had a systemic relapse. Central nervous system (CNS) was the site of relapse in 12 (14%) cases. The 5-yr overall survival, lymphoma-specific survival, disease-free survival and local control rates were 53%, 59%, 41% and 87% respectively. In the univariate analyses, favorable prognostic factors were early stage, conservative surgery, RT administration and combined modality treatment. Multivariate analysis showed that early stage and the use of RT were favorable prognostic factors. CONCLUSION: The outcome of PBL is fair. Local control is excellent with RT or combined modality treatment but systemic relapses, including that in the CNS, occurs frequently.

Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H.

Background : Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10. Objectives: To analyse molecular pathomechanisms of hyperproliferation and hyperkeratosis, we investigated the defects in mechanosensation and mechanotransduction in keratinocytes carrying the K10R156H mutation. Methods: Differentiated primary human keratinocytes infected with lentiviral vectors carrying wild-type K10 (K10wt) or mutated K10R156H were subjected to 20% isoaxial stretch. Cellular fragility and mechanosensation were studied by analysis of mitogen-activated protein kinase activation and cytokine release. Results: Cultured keratinocytes expressing K10R156H showed keratin aggregate formation at the cell periphery, whereas the filament network in K10wt cells was normal. Under stretching conditions K10R156H keratinocytes exhibited about a twofold higher level of filament collapse compared with steady state. In stretched K10R156H cells, higher p38 activation, higher release of tumour necrosis factor-alpha and RANTES but reduced interleukin-1 beta secretion compared with K10wt cells was observed. Conclusions: These results demonstrate that the R156H mutation in K10 destabilizes the keratin intermediate filament network and affects stress signalling and inflammatory responses to mechanical stretch in differentiated cultured keratinocytes.

Recent advances in tumour antigen-specific therapy: in vivo veritas.

Modern cancer therapies should strive not only to eliminate malignant tissues but also to preserve healthy tissues and the patient's quality of life. Antigen-specific immunotherapy approaches are promising for either aspect since they are designed to only act against tissues expressing 1 or more specified tumour antigens. In order to develop successful vaccine and adoptive transfer protocols, longitudinal monitoring of cancer patients taking part in clinical trials is mandatory. Here, in vivo expansion of antigen-specific cells, as well as their ex vivo functional status represent important parameters to be analysed. To obtain results that most closely reflect the cells' in vivo status, functional assays must be carried out with as little in vitro culturing as possible. The present minireview discusses recent advances in these domains.