[Influence of hormonal factors on triple-negative breast cancer prognosis]. / Influence des facteurs hormonaux sur le pronostic des cancers du sein triple négatifs.

OBJECTIVES: Triples negative breast cancer defined by the absence of expression of the hormone receptors and HER2 protein, are considered as aggressive tumours with bad outcome in comparison to the hormone sensitive tumours. The aim of the study was to evaluate the link between hormone factors and prognostic factors of triple-negative tumours. METHODS: All patients managed for a triple-negative breast cancer between January, 2009 and December, 2013 were included. For every patient, collected data were the clinical, histological, adjuvant or neoadjuvant treatments, as well as survival data. RESULTS AND CONCLUSION: During the study period, 1682 patients were operated for a breast cancer, among which 1444 presented at least an invasive tumour. One hundred and fifty-five women (10.7%) had a negative triple tumour. The average age of diagnosis was 56.4years, is significantly younger than for patients with other types of tumours, P=0.0001. For women with a triple-negative tumour, the parity was the only hormonal factor identified as an independent factor for axillary lymph node involvement (OR=1.53; 95% CI [1.10-2.25] P=0.02) and previous hormone replacement therapy as an independent factor of locoregional recurrence (OR=0.13 [0.005-0.64] P=0.001). We did not find any hormonal factor predictive of distant metastasis. We did not find any difference in overall survival according to the parity (P=0.72), the Body mass index (P=0.62) or the use of HRT (P=0.49). CONCLUSION: Hormone factors seem to have a prognostic implication for triple-negative despite the absence of hormone receptors expression.

[Prognosis impact of breast cancer adjuvant radiotherapy delay]. / Impact pronostique du délai d'induction de la radiothérapie adjuvante dans le cancer du sein.

OBJECTIVES: To evaluate delay to access to adjuvant radiotherapy for women with breast cancer and to study impact on prognosis. METHODS: A restrospective descriptive study in the teaching hospital of Tours between 1st January 2007 and 31th December 2013. All women managed for an invasive breast cancer during this period were included with exclusion of women with indication of chemotherapy (neoadjuvant/adjuvant). Delay between surgery and radiotherapy were recorded. Overall survival and recurrence free survival were used to evaluate the impact of delays on prognosis. RESULTS: Of the 1855 women with an invasive breast cancer, 904 (48.7%) had an adjuvant radiotherapy without chemotherapy. In the whole population, a delay surgery-radiotherapy>90 days was found as an independent factor negatively impacting recurrence free survival (HR=2.12 [1.03-4.36] p=0.04). In the group of patient with a breast conservative surgery, a delay surgery-radiotherapy>65 days was found as an independent factor negatively impacting recurrence free survival with HR=2.29 [1.16-4.54], p=0.02. A delay surgery-radiotherapy>70 days was found as an independent factor negatively impacting Overall survival and HR=3.41 [1.005-11.62], p=0.04. CONCLUSION: Delay to access to adjuvant radiotherapy is an independent factor impacting patient's survival, especially in the case of breast conservative therapy.

[Presentation and outcome of breast cancer under 40 years - A French monocentric study]. / Cancer du sein chez les patientes de moins de 40 ans : présentation et caractéristiques évolutives.

OBJECTIVE: The aim of our study was to evaluate the impact of young age on breast cancer presentation and women's prognosis. METHODS: We performed a descriptive retrospective study in the university teaching hospital of Tours from January 2007 to December 2013. All women managed for an invasive breast cancer were included. The population was divided in 2 groups according to age: ≤40 years and>40 years. We studied differences in histological, management and outcome characteristics. RESULTS: Two thousand and eighty three women with an invasive breast cancer were included. A hundred and fifty five in the group of women with an age ≤40 years and 1928 in the group of women with an age>40 years. Histological characteristics of breast cancer in younger women were worse than in their older counterparts (with more aggressive features: grade 3, negative hormone receptors, positive Her 2 status, triple negative molecular sub-type). Overall survival was lower in young women than in women age>40 years (P=0.05),as was recurrence free survival (P<0.001), locoregional recurrence free survival (P=0.02) and distant metastasis free survival(P<0.001). Age≤40 years was an independent factor predictive of poor recurrence free survival. CONCLUSION: In our study we found an impact of age≤40 years on invasive breast cancer presentation and prognosis.

Clinicopathologic predictors of lymph node metastasis in breast cancer patients according to molecular subtype.

PURPOSE: We present a large institutional study to determine factors predictive of axillary lymph node (LN) metastasis in breast cancer according to molecular subtype. METHODS: We conducted a retrospective analysis of our prospectively maintained breast cancer database study using data from of women managed from January 2009 through December 2013. Clinicopathologic characteristics were correlated with lymph node status and outcome according to breast cancer molecular subtyping. RESULTS: LN metastases were detected in 464 (32.1%) of 1444 women with breast cancer. By multivariate analysis, independent factors predictive of LN involvement were: for the luminal A subtype (n=776): tumour size: OR=1.05 [95% CI: 1.03-1.07] P<0.0001; lymphovascular invasion: OR=3.06 [95% CI: 1.80-5.20] P<0.0001 and tumour grade: OR=1.65 [95% CI: 1.07-2.58] P=0.026. For luminal B subtype (n=441): age: OR=0.97 [95% CI: 0.95-0.99] P=0.004; tumour size: OR=1.03 [95% CI: 1.01-1.05] P=0.002; lymphovascular invasion: OR=3.21 [95% CI: 1.92-5.44] P<0.0001; inflammatory breast cancer: OR=12.36 [95% CI: 2.18-243.3] P=0.019. For the HER2 subtype (n=72): lymphovascular invasion: OR=7.87 [95% CI: 2.10-35.2] P=0.003. For the triple negative subtype (n=155): parity: OR=1.53 [95% CI: 1.10-2.25] P=0.02; tumour size: OR=1.03 [95% CI: 1.01-1.05] P=0.002 and lymphovascular invasion: OR=7.13 [95% CI: 2.46-22.8] P=0.00048. CONCLUSION: This retrospective study provides valuable insight into LN involvement of patients with primary breast cancer according to molecular subtyping.

[Predictive factors of conservative breast surgery after neoadjuvant chemotherapy for breast cancer]. / Facteurs prédictifs de traitement conservateur après chimiothérapie néo-adjuvante dans le cancer du sein.

OBJECTIVES: The aim of our study was to evaluate the existence of predictive factors of conservative breast surgery after neoadjuvant chemotherapy (NAC) for breast cancer. METHODS: We included all women with invasive breast cancer who received NAC and underwent breast surgery between January 2007 and December 2013 in our institution. Univariable and multivariable analyses were performed to determine the association between clinical and histological factors and conservative breast surgery. RESULTS: During the study period, 229 women were included of whom 73 had breast conservative surgery (32%). At univariable analysis, significant predictive factors were age (OR 0.97 [CI 95% 0.95-0.99], P=0.02), radiological size (OR 0.97 [CI 95% 0.96-0.99], P<0.001), multifocality (OR 0.53 [CI 95% 0.27-1.05], P=0.06), breast inflammation (OR 0.15 [CI 95% 0.07-0.32], P<0.001) and the type of hormone receptors (P=0.12). In multivariable analysis, all these factors but age were significant factors and thus considered as independent predictive factors. CONCLUSION: This work permitted to identify independent predictive factors of breast conservative surgery after NAC for breast cancer that will be included in a risk scoring system that we aim to evaluate prospectively.

[Impact of pathological complete response to neoadjuvant chemotherapy in invasive breast cancer according to molecular subtype]. / Impact de la réponse histologique complète à la chimiothérapie néo-adjuvante pour cancer du sein selon le sous-type moléculaire.

OBJECTIVES: The aim of this study was to evaluate the impact of pathological complete response (pCR) on overall survival (OS) and recurrence-free survival (RFS) according to molecular subtypes in women treated for an invasive breast cancer after neoadjuvant chemotherapy (NAC). METHODS: All women (n=225) managed with a neoadjuvant chemotherapy for an invasive breast cancer in our institution between January 2007 and December 2013 were included. The characteristics of patients with pCR (pCR-1), breast pCR and axillary pCR were compared to those without pCR (pCR-0) according to the molecular subtypes: luminal A (n=62), luminal B (n=77), Her-2 (n=31) and triple negative (n=55). RESULTS: NAC concerned 225 patients of whom 36 (16%) had pCR. Achievement of pCR led to significantly better overall survival in women with Her-2 tumors (35% versus 100%, P=0.035) and also to significantly better locoregional survival in women treated for triple negative tumors (P=0.026). Predictive factors of pCR were a high pathologic grade: OR=2.39, IC 95% (1.19-4.83), P=0.008; Her-2 molecular subtype (P=0.008); positive estrogenic hormonal receptors (P=0.006), a positive Her-2 receptor: OR=2.58, IC 95% (1.20-5.54), P=0.01. CONCLUSION: Achievement of pCR is an intermediate marker of survival in women managed with NAC for breast cancer.

The SK3/K(Ca)2.3 potassium channel is a new cellular target for edelfosine.

BACKGROUND AND PURPOSE: The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function. EXPERIMENTAL APPROACH: Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and ¹²5I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca²(+) -activated K(+) channel, SK3/K(Ca)2.3. KEY RESULTS: Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/K(Ca)2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit ¹²5I-Apamin binding to this SK(Ca) channel; rather, it reduced the calcium sensitivity of SK3/K(Ca)2.3 channel and dramatically decreased intracellular Ca²(+) concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SK(Ca) channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/K(Ca)2.3 protein in a SK3/K(Ca)2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine. CONCLUSIONS AND IMPLICATIONS: Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/K(Ca)2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.

Apc mutation induces resistance of colonic cells to lipoperoxide-triggered apoptosis induced by faecal water from haem-fed rats.

Recent epidemiological studies suggest that high meat intake is associated with promotion of colon cancer linked with haem-iron intake. We previously reported that dietary haem, in the form of either haemoglobin or meat, promotes precancerous lesions in the colon of rats given a low-calcium diet. The mechanism of promotion by haem is not known, but is associated with increased lipid peroxidation in faecal water and strong cytotoxic activity of faecal water on a cancerous mouse colonic epithelial cell line. To better understand the involvement of faecal water components of haem-fed rats in colon-cancer promotion, we explored the effect of faecal water on normal [adenomatous polyposis coli (Apc)+/+] or premalignant cells (Apc-/+). Further, we tested if this effect was correlated to lipoperoxidation and 4-hydroxynonenal (HNE). We show here for the first time that heterozygote Apc mutation represents a strong selective advantage, via resistance to apoptosis induction (caspase 3 pathway), for colonic cells exposed to a haem-iron-induced lipoperoxidation. The fact that HNE treatment of the cells provoked the same effects as the faecal water of rats fed the haem-rich diet suggests that this compound triggers apoptosis in those cells. We propose that this mechanism could be involved in the promotion of colon carcinogenesis by haem in vivo.

Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.

S-phase fraction and DNA ploidy in 633 T1T2 breast cancers: a standardized flow cytometric study.

The lack of a standardized methodology for quantifying DNA ploidy and S-phase fraction (SPF) by flow cytometry is hindering routine use of these markers in breast cancer management. In a retrospective clinical multicenter study, we validated a standardized flow cytometry protocol. We tested 633 frozen T(1)T(2), N(0)N(1), M(0) breast tumors obtained in four institutions. Cell preparation was standardized, and precise rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. DNA aneuploidy was observed in 61.0% of cases. No significant difference was observed among centers. Aneuploidy and high SPF were associated with large tumor size, node involvement, high histological grade, and hormone receptor negativity. In the overall population (median follow-up, 69 months), patients with medium and high SPF values had shorter disease-free survival (DFS) than those with low SPF values (P < 0.0001). Ploidy had no significant influence. By Cox analysis, SPF, pN, and estrogen receptor status were independent predictors of DFS (P = 0.0002, P = 0.001, and P = 0.05). In node-negative patients, SPF was the only predictor of DFS (P = 0.01), whereas in node-positive patients, the risk of relapse increased with both high SPF (P = 0.003) and estrogen receptor negativity (P = 0.004). Low SPF values distinguished grade II tumors with a particularly good outcome. Our results strongly support the use of SPF in multicenter studies and clinical trials and suggest that node-negative patients with slowly proliferating tumors do not require systemic adjuvant therapy.

DNA and cell cycle analysis as prognostic indicators in breast tumors revisited.

Both DNA ploidy and S-phase ploidy are promising prognostic factors for node-negative breast cancer patients. Based largely on the analysis of one large study, much of the reported problems with these factors have been caused by some unappreciated complexities in categorizing DNA ploidy into low- and high-risk groups and the lack of some necessary adjustments to eliminate unwanted correlations between DNA S-phase and ploidy. When both DNA ploidy and S-phase are compensated properly, they become independent prognostic markers, forming a powerful prognostic model.

Effect of an alpha-linolenic acid-rich diet on rat mammary tumor growth depends on the dietary oxidative status.

To investigate whether the oxidative status of an 18:3(n-3) polyunsaturated fatty acid (PUFA)-enriched diet could modulate the growth of chemically induced rat mammary tumors, three independent experiments were performed. Experiments I and II examined the variation of tumor growth by addition of antioxidant (vitamin E) or a prooxidant system (sodium ascorbate/2-methyl-1,4-naphthoquinone) to a 15% linseed oil diet rich in 18:3(n-3). Experiment III addressed the role of PUFA in the tumor growth modulation by vitamin E. For this purpose, we compared the effect of vitamin E in 15% fat diets containing a high level of 18:3(n-3) (linseed oil, high-PUFA diet) or devoid of 18:3(n-3) (hydrogenated palm/sunflower oil, low-PUFA diet). In Experiments I-III, tumor growth increased in the presence of vitamin E compared with control (without vitamin E). Furthermore, it decreased when prooxidant was added. In contrast, no difference was observed when the diet was low in PUFA, suggesting that sensitivity of PUFA to peroxidation may interfere with tumor growth. This observation was supported by growth kinetic parameter analysis, which indicated that tumor growth resulted from variations in cell loss but not from changes in cell proliferation. These data show that, in vivo, PUFA effects on tumor growth are highly dependent on diet oxidative status.

[Standardization and quality control in the evaluation of proliferation parameters in T1T2, N0N1, M0 breast cancer: multicentric retrospective study II. DNA-ploidy and S-phase fraction]. / Standardisation et mise en place d'une assurance qualité dans l'évaluaton des paramètres de prolifération de 1,003 cancers du sein T1T2, N0N1, M0: étude multicentrique II. ADN-ploïdie et phase de sythèse d'ADN.

As part of a clinical research project, proliferative parameters were studied in primary breast cancer: standardization and technical validation of thymidine kinase (TK), thymidylate synthase (TS) and protein tyrosine kinase (PTK) are described. A total of 633 frozen tumor specimens, available in four institutions, was analyzed in three flow cytometry laboratories for DNA content and percentage of S-phase cells (%S) measurement. 1) The standardization step consisted in developing a common protocol for sample preparation; then, common cell suspensions were analyzed in order to perform an inter-laboratory control. Objective guidelines were elaborated to interpret DNA histograms in breast carcinoma. 2) DNA-aneuploidy was observed in 61% of cases of the retrospective series. Compared with DNA-aneuploid tumors, mean %S was significantly lower in case of DNA-diploidy (respectively: 6.4% and 2.2%, p < 0.001). When compared between the four institutions, %S distributions did not differ significantly. 3) %S is strongly correlated with TK, TS and PTK and high percentages were also observed in high grade tumors or tumor without hormone receptors. These results show that a standardization in using flow cytometers and DNA software allows multicenter studies.

Suppression of the promoter effect of polyunsaturated fatty acids by the absence of dietary vitamin E in experimental mammary carcinoma.

Because lipoperoxides seem able to modulate tumor growth, we have examined the concomitant effects of dietary antioxidant (vitamin E) and of a high amount of polyunsaturated fatty acids (PUFA) in a model of chemically-induced mammary carcinogenesis. Two groups of rats received a high fat diet with or without added vitamin E and mammary tumors were initiated in both groups by an injection of carcinogen. Tumor growth was followed in both groups. We found that tumor incidence and growth were decreased in rats with no added vitamin E in diet, suggesting a protective role of oxidized PUFA at later stages of carcinogenesis.

Detection and typing of human papillomaviruses by in situ hybridization with biotinylated oligonucleotide mixtures.

The value of biotinylated oligonucleotide probes for screening and typing by in situ hybridization of the most frequent genital human papillomavirus infections (HPVs 6, 11, 16, 18, 31, and 33) was assessed. Optimal hybridization conditions were defined on a panel of paraffin-embedded tissue sections previously characterized with HPV full genome probes. Mixtures of oligonucleotides rather than single oligonucleotides were used to improve sensitivity and specificity. All HPV-positive specimens were detected by the screening mixture with a sensitivity and specificity similar to that of full genome probes. Typing mixtures were highly specific for each HPV type. This study confirms the potential of oligonucleotide probes for detecting and typing HPV infections.

[Intraepithelial lesions of the cervix uteri and human papillomaviruses: comparative study of histological data and in situ hybridization. Apropos of a series of 77 cervical biopsies]. / Lésions intraépithéliales du col utérin et papillomavirus humains: étude comparative des donnéss de l'histologie et de l'hybridation in situ. A propos d'une série de soixante-dix-sept biospsies cervicales.

Seventy seven biopsy samples of cervical mucosa were tested for the presence of human papillomavirus (HPV) by immunohistochemistry and in situ hybridization. From the 38 samples identified as condyloma or cervical intraepithelial neoplasia (CIN), 31 were positive after in situ hybridization and 14 after immunochemical analysis. HPV 6 was found exclusively in condyloma acuminata (2 samples) whereas the HPV 16 probe essentially hybridized with high grade intraepithelial lesions (CIN II, CIN III). Low grade intraepithelial lesions (flat condyloma, CIN I) demonstrated a larger diversity of HPV types (HPV 16, 18, 31, 33). A close correlation was demonstrated between the histologic features of lesions and their HPV 6 or HPV 31 content but not for other HPV types. HPV 31 containing lesions showed a peculiar architecture with numerous, elongated papillae resulting in a spiked appearance.