José Santini

Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls.

Detection of circulating tumor cells (CTCs) morphologically may be a promising new approach in clinical oncology. We tested the reliability of a cytomorphologic approach to identify CTCs: 808 blood samples from patients with benign and malignant diseases and healthy volunteers were examined using the isolation by size of epithelial tumor cell (ISET) method. Cells having nonhematologic features (so-called circulating nonhematologic cells [CNHCs]) were classified into 3 categories: CNHCs with malignant features, CNHCs with uncertain malignant features, and CNHCs with benign features. CNHCs were found in 11.1% and 48.9% of patients with nonmalignant and malignant pathologies, respectively (P < .001). CNHCs with malignant features were observed in 5.3% and in 43.1% of patients with nonmalignant and malignant pathologies, respectively. Cytopathologic identification of CTCs using the ISET method represents a promising field for cytopathologists. The possibility of false-positive diagnosis stresses the need for using ancillary methods to improve this approach.

MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

Clinical Impact of the Detection of BRAF Mutations in Thyroid Pathology: Potential Usefulness as Diagnostic, Prognostic and Theragnostic Applications

A BRAF somatic mutation at residue 600 of the BRAF protein (BRAFV600E) is highly prevalent in papillary thyroid carcinomas (PTC). This mutation occurs in approximately 44% (from 29% to 83%) of PTC depending on the different studies. BRAFV600E is almost always found in PTC with a papillary or a mixed follicular/papillary architecture, being rarer in other subtypes of PTC. The discovery of the BRAFV600E mutation in tissue and fine-needle aspiration (FNA) is diagnostic for PTC and has been frequently associated with worse clinical prognosis. However, some studies failed to reveal this prognostic association. Transcriptional and post-transcriptional modulation of PTC with a BRAF mutation has been evaluated in some recent studies. Current therapeutic approaches targeting BRAF are being tested in clinical trials, particularly in more aggressive PTC. In this review, we will first discuss the diagnostic value of a BRAF mutation for PTC diagnosis. The prognostic role of a BRAFV600E mutation is then outlined and discussed in the context of other well-accepted clinicopathological prognostic parameters for PTC (age, gender, pTNM stage, histological subtype). Finally, the currently and potentially used treatments targeting BRAF in patients with PTC are presented.

Diagnostic value of immunohistochemistry for the detection of the BRAF V600E mutation in papillary thyroid carcinoma: Comparative analysis with three DNA-based assays

BACKGROUND The aim of this study was to compare the detection of BRAF(V600E) by immunohistochemistry (IHC) using a mutation-specific antibody with molecular biology methods for evaluation of papillary thyroid carcinoma (PTC) patients. PATIENTS AND METHODS This study concerned 198 consecutive conventional PTC patients, of which the majority were women (133/198; 67%), with a mean age of 56 years (range 19-79 years). BRAF mutation analysis was performed using DNA-based (direct sequencing, pyrosequencing, and SNaPshot) and IHC (VE1 antibody) methods. The sensitivity and specificity of IHC for BRAF(V600E) was compared with the molecular biology data. RESULTS A BRAF mutational result was obtained in 194 cases. A BRAF(V600E) mutation was detected in 153/194 (79%) cases of PTC when using at least one molecular method, and in 151/194 (78%) cases with IHC. No false positive results were noted using IHC to detect the BRAF(V600E) mutation. Besides this mutation, other rare BRAF mutations (BRAF(V600K) and BRAF(K601E)), used as negative controls, were consistently negative with IHC. The sensitivity and specificity of IHC for the detection of this mutation were 98.7% and 100% respectively. The IHC test demonstrated excellent performance at a level equivalent to two DNA-based counterparts (pyrosequencing and SNaPshot). Failure to achieve a result was more frequent with the direct sequencing method than with the three other methods. CONCLUSION IHC using the VE1 antibody is a specific and sensitive method for the detection of the BRAF(V600E) mutation in PTC. IHC may be an alternative to molecular biology approaches for the routine detection of this mutation in PTC patients.

Assessment of Morphology, Antigenicity, and Nucleic Acid Integrity for Diagnostic Thyroid Pathology Using Formalin Substitute Fixatives

BACKGROUND With the advent of the formaldehyde standard law in France, and because of the impact of new methods for diagnosis and prognosis in pathology, formalin replacement in surgical pathology laboratories is currently being discussed in France. However, a set of criteria must be assessed before introducing a formalin substitute fixative. The objective of this study was to compare formalin substitute fixation with formalin fixation and cryoconservation of tissues from several benign and malignant thyroid pathologies with respect to morphology, antigenicity, and nucleic acid (RNA, DNA, microRNA) integrity. METHODS Calibrated specimens (200 mg, 1 cm(2) each) from four conventional papillary thyroid carcinomas, four follicular variant of papillary thyroid carcinomas, three minimally invasive follicular carcinomas, four thyroid adenomas, five thyroid nodular hyperplasias, and five normal thyroid tissues were fixed for 6, 12, or 24 hours, in different fixatives (formalin, Glyo-Fixx, FineFIX, ExcellPlus, RCL2) at room temperature or at 4 degrees C. Tissues were stained (hematoxylin-eosin, periodic acid Schiff, trichromic Masson, and Sweet-Gordon staining) and their antigenicity determined by immunohistochemistry (performed with HBME-1, galectin-3, CK19, vimentin, CD31, and KL1 antibodies). Evaluation by four pathologists was made blinded. The quantity and quality of DNA, RNA, and two representative microRNA extracted from deparaffinized sections of paraffin embedded specimen were compared with that of cryosections. RESULTS The staining and morphology were not altered by the use of different fixatives. However, formalin, FineFIX, and RCL2 gave the best results for immunohistochemistry. Moreover, FineFIX and RCL2 gave the highest amount of nucleic acids and of the best quality. CONCLUSIONS All the formalin substitute fixatives used in this study provided good histomorphologic quality for the different stained thyroid tissues, but individually, some fixatives performed better for immunohistochemical and molecular biological procedures for different thyroid pathologies.

Thyroid tumours of uncertain malignant potential: frequency and diagnostic reproducibility

The term thyroid tumours of uncertain malignant potential (TT-UMP) has been proposed for a subgroup of follicular-patterned thyroid tumours for which benignancy or malignancy cannot be assessed with certainty. The frequency, diagnostic reproducibility, immunohistochemistry and molecular genetic profiling of such tumours have been poorly explored. We, therefore, investigated (1) the frequency of TT-UMP diagnosed in a single institution (Nice, France: 2004–2008), (2) the observer variation among four pathologists, (3) whether immunohistochemical and molecular genetic profiling of TT-UMP provide additional information concerning such lesions. A series of 31 diagnosed TT-UMP (2.9%) out of 1,078 consecutive thyroidectomies were analysed. It comprised 15 follicular thyroid tumours of UMP (FT-UMP) and 16 well-differentiated tumours of UMP (WDT-UMP). Observer concordance was 70% for all TT-UMP. More than 50% of FT-UMP expressed galectin-3 and CK19, whereas more than 50% of WDT-UMP expressed HBME-1. Five cases of TT-UMP showed N-RAS mutations, while one showed H-RAS mutation and another PAX8/PPARγ rearrangement. In conclusion, the frequency of TT-UMP is low in our institution. Diagnostic reproducibility is within the same range as other published data on follicular-patterned thyroid tumours. The ancillary methods have a low impact on aiding diagnosis of such lesions.

CONCOMITANT B.I.D. RADIOTHERAPY AND CHEMOTHERAPY WITH CISPLATIN AND 5-FLUOROURACIL IN UNRESECTABLE SQUAMOUS-CELL CARCINOMA OF THE PHARYNX: CLINICAL AND PHARMACOLOGICAL DATA OF A FRENCH MULTICENTER PHASE II STUDY

PURPOSE The aim of this phase II study conducted on unresectable squamous cell carcinoma (USCC) of the oro- and hypopharynx was to associate twice-a-day (b.i.d.) continuous nonaccelerated radiotherapy with concomitant cisplatin (CP)-5-fluorouracil (5-FU) chemotherapy, both given at full dose. Feasibility, efficacy, survival, and pharmacokinetic-pharmacodynamic relationships were analyzed. METHODS AND MATERIALS Fifty-four consecutive patients with strictly USCC of oro- and/or hypopharynx received continuous b.i.d. radiotherapy (RT) (2 daily fractions of 1.2 Gy, 5 days a week, with a 6-h minimal interval between fractions). Total RT dose was 80.4 Gy on the oropharynx and 75.6 Gy on the hypopharynx. Three chemotherapy (CT) courses of CP-5-FU were given during RT at 21-day intervals (third not delivered after the end of RT). CP dose was 100 mg/m2 (day 1) and 5-FU was given as 5-day continuous infusion (day 2-day 6: 750 mg/m2/day cycle 1, 750 mg total dose/day cycle 2 and 3). Pharmacokinetics was performed for 5-FU (105 h follow-up) and CP (single sample at 16 h). Special attention was paid to supportive care. RESULTS Good feasibility of RT was observed (85.2% of patients with total dose > 75 Gy). Five patients received 1 CT cycle, 34: 2 cycles, and 15: 3 cycles. The most frequent and severe acute toxicities were mucositis with grade 3-4 occurring in 28% at cycle 1 and 86% at cycle 2, as well as neutropenia (43% at cycle 2). Locoregional control at 6 months was observed in 66.7% of patients. No late toxicity above grade 2 RTOG was noticed. CP dose and 5-FU AUC(0-105h) were significantly linked to grade 3-4 neutropenia (cycle 2). Cumulative total platinum (Pt) concentration and Karnofsky index were the only independent predictors of locoregional control at 6 months. Finally, total RT dose and total Pt concentration were the only independent predictors of specific survival. CONCLUSION This protocol showed good locoregional response with an acceptable toxicity profile. Pharmacokinetic survey is probably an effective approach to further reduce toxicity and improve efficacy. A multicentric randomized phase III study, now underway, should confirm these encouraging results.

Complications of the myocutaneous platysma flap in intraoral reconstruction.

Pedicled myocutaneous flaps remain important tools in head and neck reconstruction. Evaluation of their complications are necessary to judge their merits.

The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection

Aims: The aim of this study was to investigate the usefulness of polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection (LCM). Methods: The PCR DNA amplification method was used to detect M. tuberculosis in granulomas microdissected from one section stained by haematoxylin and eosin (H&E) from a formalin‐fixed paraffin‐embedded specimen. The results were compared to those obtained from PCR performed from 10 whole paraffin sections of 5 µm each, and with the histology, culture and the patients clinical findings. Results: Forty‐nine formalin‐fixed and paraffin‐embedded samples from 49 patients with a histological suspicion of a mycobacterial infection were investigated. Using culture as the reference method, the sensitivity for the detection of M. tuberculosis was 92% and the specificity was 100% using PCR from microdissected granulomas and were similar to those obtained by using PCR from 10 whole sections. Conclusions: The PCR method of examination of microdissected granulomas from deparaffinised sections is a sensitive, specific and rapid method for the detection of M. tuberculosis in formalin‐fixed and paraffin‐embedded samples. The method is as sensitive as that using PCR on 10 whole tissue sections, thus making it suitable for small biopsies. However, although these methods reduce the delay in diagnosis, culture remains the gold standard for identification of mycobacteria in tissue. Culture also allows for the testing of antibiotic sensitivity of any isolated species, in this way determining appropriate treatment.

DENTALMAPS: Automatic Dental Delineation for Radiotherapy Planning in Head and Neck Cancer

PURPOSE To propose an automatic atlas-based segmentation framework of the dental structures, called Dentalmaps, and to assess its accuracy and relevance to guide dental care in the context of intensity-modulated radiotherapy. METHODS AND MATERIALS A multi-atlas-based segmentation, less sensitive to artifacts than previously published head-and-neck segmentation methods, was used. The manual segmentations of a 21-patient database were first deformed onto the query using nonlinear registrations with the training images and then fused to estimate the consensus segmentation of the query. RESULTS The framework was evaluated with a leave-one-out protocol. The maximum doses estimated using manual contours were considered as ground truth and compared with the maximum doses estimated using automatic contours. The dose estimation error was within 2-Gy accuracy in 75% of cases (with a median of 0.9 Gy), whereas it was within 2-Gy accuracy in 30% of cases only with the visual estimation method without any contour, which is the routine practice procedure. CONCLUSIONS Dose estimates using this framework were more accurate than visual estimates without dental contour. Dentalmaps represents a useful documentation and communication tool between radiation oncologists and dentists in routine practice. Prospective multicenter assessment is underway on patients extrinsic to the database.