François Guillemin

Comparison of models to predict nonsentinel lymph node status in breast cancer patients with metastatic sentinel lymph nodes: a prospective multicenter study.

PURPOSE Several models have been developed to predict nonsentinel lymph node (non-SN) status in patients with breast cancer with sentinel lymph node (SN) metastasis. The purpose of our investigation was to compare available models in a prospective, multicenter study. PATIENTS AND METHODS In a cohort of 561 positive-SN patients who underwent axillary lymph node dissection, we evaluated the areas under the receiver operating characteristic curves (AUCs), calibration, rates of false negatives (FN), and number of patients in the group at low risk for non-SN calculated from nine models. We also evaluated these parameters in the subgroup of patients with micrometastasis or isolated tumor cells (ITC) in the SN. RESULTS At least one non-SN was metastatic in 147 patients (26.2%). Only two of nine models had an AUC greater than 0.75. Three models were well calibrated. Two models yielded an FN rate less than 5%. Three models were able to assign more than a third of patients in the low-risk group. Overall, the Memorial Sloan-Kettering Cancer Center nomogram and Tenon score outperform other methods for all patients, including the subgroup of patients with only SN micrometastases or ITC. CONCLUSION Our study suggests that all models do not perform equally, especially for the subgroup of patients with only micrometastasis or ITC in the SN. We point out available evaluation methods to assess their performance and provide guidance for clinical practice.

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan localisation in cultured tumour cells.

Intracellular photosensitiser localisation significantly influences the mechanism of response to photodynamic therapy (PDT), since the primary sites of damage are closely related to the specific sensitiser distribution. Foscan® subcellular localisation in the MCF-7 human adenocarcinoma cell line has been studied by means of confocal microscopy and microspectrofluorometry. The fluorescence topographic profiles recorded after cells costained with Foscan® and organelle-specific fluorescent probes revealed that Foscan® presents low localisation in lysosomes and a weak accumulation in mitochondria. Alternatively, the Foscan® fluorescence topographic profile turned out to colocalise perfectly with that obtained for the endoplasmic reticulum (ER) and the Golgi apparatus. The patterns of fluorescence derived from confocal microscopy studies were consistent with predominant localisation of Foscan® in these organelles. Furthermore, evaluation of enzymatic activity of selected organelles immediately after laser light irradiation (650 nm) indicated the Golgi apparatus and ER as the primary damaged sites resulting from Foscan®-mediated PDT in the MCF-7 cell line. To our knowledge, this is the first study to demonstrate unambiguously that the ER and the Golgi apparatus are preferential sites of Foscan® accumulation.

Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication

The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.

Relationship between subcellular localisation of Foscan and caspase activation in photosensitised MCF-7 cells.

The present study investigates the relationship between the subcellular localisation of Foscan® and intrinsic apoptotic pathway post Foscan®-based photodynamic therapy (PDT). With this purpose, mammary carcinoma MCF-7 cells were incubated with Foscan® for 3 or 24 h and then subjected to equitoxic light doses. Fluorescence microscopy revealed very good Foscan® co-localization to endoplasmic reticulum (ER) and Golgi apparatus after 3 h incubation with MCF-7 cells. Progressive increase in incubation time shows leakage of Foscan® from Golgi apparatus. Twenty-four hours incubation yielded a fluence-dependent enhanced induction of the ER-resident glucose-regulated protein 78 (Bip/GRP78), along with a weak mitochondrial damage, thus underscoring the ER as the main site of photodamage after prolonged incubation. Analysis of events implicated in apoptotic pathway after 24 h incubation demonstrated photodamage to Bcl-2 protein in total cellular extract, but not in the mitochondrial fraction. We further determined an increase in caspases-7 and -6 activation, which was strongly related to the expression of GRP78. The above findings demonstrate that Foscan® localisation in ER improves the photoactivation of the caspase-7 apoptotic pathway, which is poorly related to mitochondrial damage.

Validation of the Tenon breast cancer score for predicting non-sentinel lymph node status in breast cancer patients with sentinel lymph node metastasis: a prospective multicenter study

Background Axillary lymph node dissection (ALND) is the standard treatment for patients with sentinel node (SN) metastasis, but most of these patients have negative non-sentinel nodes (non-SN). We have developed a scoring system (the Tenon score) to help identify a subgroup of patients who have a low risk of having non-SN metastases and who may thus forgo ALND. Here we validated the Tenon score in an independent cohort of SN-positive patients. Patients and methods We tested the accuracy of the Tenon score for predicting non-SN status in a prospective multicenter study of 226 SN-positive breast cancer patients. We calculated the false-negative rate, sensitivity, specificity, and positive (PPV) and negative predictive values (NPV). Receiver operating characteristics (ROC) curves were constructed and the areas under the curve (AUC) were calculated as a measure of discriminatory capacity. Results At least one non-SN was positive in 63 patients (27.9%). One hundred and twenty (53.1%) of the 226 patients had a Tenon score of 3.5 or less. Among these 120 patients, five had at least one positive non-SN. With a score cut-off of 3.5, the negative predictive value was 95.8% and the false-negative rate was 4.2%. Overall, the Tenon score accurately predicted non-SN status, with an AUC of 0.82 (95% confidence interval, 0.77–0.88). Conclusion In this multicenter study of an independent patient population, the Tenon score was accurate and reproducible for predicting non-SN status in breast cancer patients. The simplicity and reliability of the variables on which the Tenon score is based may be an advantage over other scoring systems.

Recent Improvements in the Use of Synthetic Peptides for a Selective Photodynamic Therapy

Photodynamic therapy (PDT) is a relatively new cytotoxic treatment, predominantly used in anti-cancer approaches, that depends on the retention of photosensitizers in tumor and their activation after light exposure. Photosensitizers are photoactive compounds such as porphyrins and chlorins that upon photoactivation, effect strongly localized oxidative damage within the target cells. The ability to confine activation of the photosensitizer by restricting illumination to the tumor allows for a certain degree of selectivity. Nevertheless, the targeted delivery of photosensitizers to defined cells is a major problem in PDT of cancer, and one area of importance is photosensitizer targeting. Alterations or increased levels in receptor expression of specific cellular type occur in the diseased tissues. Therefore, photosensitizers can be covalently attached to molecules such as peptides, leading to a receptor-mediated targeting strategy. These active-targeting approaches may be particularly useful for anti-vascular PDT. Moreover, it has been shown that the photocytotoxicity of photodynamic drugs could be enhanced by delivering high amounts of a photosensitizer into subcellular organelles such as the nucleus where nucleic acids represent target molecules sensitive to photodamage. The recent progresses in the use of active-targeting strategy with synthetic peptides and the interest of using an active-targeting strategy in PDT, which could allow efficient cellular internalization of photosensitizers, are described in this review.

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI‐DNA complexes

p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild‐type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy.

Primary Photodamage Sites and Mitochondrial Events after Foscan® Photosensitization of MCF-7 Human Breast Cancer Cells¶

To determine the initial photodamage sites of Foscan®‐mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF‐7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5′‐diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan®‐mediated PDT in MCF‐7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan® photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan®‐based PDT were very different from that of overall cell death. By 24 h post‐PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan®‐mediated MCF‐7 cell death by late and indirect mechanism.

Foscan® (mTHPC) photosensitized macrophage activation : enhancement of phagocytosis, nitric oxide release and tumour necrosis factor-α-mediated cytolytic activity

SummaryPhotodynamic activation of macrophage-like cells contributes to an effective outcome of photodynamic therapy (PDT) treatment. The possibility for an enhancement of macrophage activity by photosensitization with meta-tetra(hydroxyphenyl)chlorin (mTHPC) (1 μg ml–1) was studied in U937, monocyte cell line differentiated into macrophages (U937Φ cells). Phagocytic activity of U937Φ cells was evaluated by flow-cytometry monitoring of ingestion of fluorescein-labelled Escherichia coli particles. Increase in irradiation fluence up to 3.45 mJ cm–2 (corresponding irradiation time 15 s) resulted in significant increase in fluorescence signal (145%), while at higher light fluences the signal lowered down to the untreated control values. A light energy-dependent production of tumour necrosis factor-alpha (TNF-α) by photosensitized macrophages was further demonstrated using the L929 assay. The maximum TNF-α mediated cytolysis was observed at 28 mJ cm–2 and was 1.7-fold greater than that in control. In addition, we demonstrated a fluence-dependent increase in nitric oxide (NO) production by mTHPC-photosensitized macrophages. NO release increased gradually and reached a plateau after irradiation fluence of 6.9 mJ cm–2. Cytotoxicity measurements indicated that the observed manifestations of mTHPC-photosensitized macrophage activation took place under the sublethal light doses. The relevance of the present findings to clinical mTHPC-PDT is discussed.

Results of Preoperative Lymphoscintigraphy for Breast Cancer Are Predictive of Identification of Axillary Sentinel Lymph Nodes

The aim of this study was to identify the variables associated with successful peroperative sentinel lymph node (SLN) localization. We studied 201 patients with T1, T2, N0 invasive breast cancer who underwent a SLN procedure from 1999 to 2003. Of these 201 patients, 55 underwent peritumoral and 146 underwent periareolar radioisotope injection before the blue dye injection. All patients were operated on by breast conservative surgery and axillary dissection after SLN biopsy. Age, weight, menopausal status, previous biopsy, localization of the tumor, results of lymphoscintigraphy, site of radiotracer injection, tumor size, tumor grade, experience of surgeons, and the number of invaded axillary nodes were analyzed to determine whether they had any significant correlation with successful identification of SLN. Variables found to have a statistically significant influence on the SLN identification rate and on preoperative lymphoscintigraphy identification were introduced into a univariate and multivariate logistic regression model. In multivariate analysis, successful lymphoscintigraphy (P < 0.0001) and the absence of metastatic axillary nodes (P < 0.005) were associated with successful identification of SLNs. The peritumoral injection of radiotracer (P < 0.001), patient age > 60 years (P < 0.003), and localization of the tumor in the upper outer quadrant (P < 0.004) were associated with failure of lymphoscintigraphic visualization of SLN. The technique of SLN detection thus appears to be better for patients with low risk of invaded axillary lymph nodes.