Quantitative Real Time PCR assessment of hormonal receptors and HER2 status on fine-needle aspiration pre-operatory specimens from a prospectively accrued cohort of women with suspect breast malignant lesions.

OBJECTIVES: Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptor status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic, semi-quantitative nature, relying in part on subjective interpretation. METHODS: In the present study, we tested a molecular approach to define ER, PR, and HER2 status in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used quantitative Real Time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene copy number (GCN). RESULTS: ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover, PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its transcript, correctly stratifying HER2+ and HER2- patients. Finally, there was a strongly significant agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained on pathological tissue specimens. CONCLUSIONS: The present results support a comprehensive approach to determine ER, PR, and HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for therapeutic strategies like neoadjuvant treatment.

Atypical ductal hyperplasia diagnosed at 11-gauge vacuum-assisted breast biopsy performed on suspicious clustered microcalcifications: could patients without residual microcalcifications be managed conservatively?

OBJECTIVE: The purpose of our study was to establish whether it might be safe for women with a diagnosis of atypical ductal hyperplasia (ADH) at stereotactically guided vacuum-assisted breast biopsy without any residual microcalcification after the procedure to undergo mammographic follow-up instead of surgical biopsy. MATERIALS AND METHODS: From October 2003 to January 2009, 1173 consecutive 11-gauge vacuum-assisted breast biopsy procedures were performed. ADH was found in the specimens of 114 patients who underwent vacuum-assisted breast biopsy for a single cluster of suspicious microcalcifications smaller than 15 mm; 49 had residual microcalcifications, and 65 had microcalcifications completely removed by the procedure. Of 49 patients with residual microcalcifications, 41 underwent surgical biopsy. Of 65 patients without residual microcalcifications, 26 underwent surgical biopsy, 35 were not surgically treated and were managed conservatively with mammographic follow-up, and 4 had follow-up of less than 24 months. RESULTS: In 41 patients with residual microcalcifications who underwent surgical biopsy, 8 malignant lesions were found at surgery. The underestimation rate was 20% (8/41). In 26 patients without residual microcalcifications who underwent surgical biopsy, no malignant lesions were found. One malignant lesion was found in the 35 patients managed conservatively at follow-up. The underestimation rate in patients without residual microcalcifications using surgical biopsy or mammographic follow-up as the reference standard was 1.6% (1/61). CONCLUSION: Patients without residual microcalcifications after vacuum-assisted breast biopsy could possibly be managed in a conservative way with mammographic follow-up.

Reliability and reproducibility of a RNA preamplification method for low-density array analysis from formalin-fixed paraffin-embedded breast cancer samples.

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.

Predictive and prognostic significance of neuron-specific enolase (NSE) in non-small cell lung cancer.

BACKGROUND: The predictive and prognostic role of neuron-specific enolase (NSE) in non-small cell lung cancer (NSCLC) is still under debate. PATIENTS AND METHODS: To study these aspects, serum NSE was prospectively measured at baseline of first-line chemotherapy treatment and tested for correlation with clinical outcome in 129 advanced NSCLC patients. RESULTS: An objective response was achieved in 27 out of 65 (41.5%) patients with NSE < 8.6 ng/ml and in 38 out of 64 (59.4%) patients with NSE > or = 8.6 ng/ml (p = 0.05). Logistic analysis confirmed the positive association between objective response and NSE values > or = 8.6 ng/ml (odds ratio = 1.69; 95% confidence interval: 1.09-2.63; p = 0.02). Overall median survival was 10.8 months. A statistically significant prognostic effect on survival was found for performance status, stage and response to treatment, but not for baseline NSE value. CONCLUSION: Based on these data, baseline circulating tumor NSE levels appear to have a weak predictive role, but not a prognostic significance in patients with advanced NSCLC submitted to standard chemotherapy.

Buccal mucosa cells as in vivo model to evaluate gefitinib activity in patients with advanced non small cell lung cancer.

PURPOSE: To evaluate the role of pretreatment and posttreatment expression in buccal mucosa cells of signal transduction proteins activated by epidermal growth factor receptor, including phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated mitogen-activated protein kinase (p-MAPK), and phosphorylated AKT (p-AKT), in predicting gefitinib activity in advanced non-small cell lung cancer patients. Expression of the same proteins was also assessed on corresponding tissue samples for comparison. Moreover, EGFR gene mutations and copy number were analyzed. EXPERIMENTAL DESIGN: Protein expression was evaluated by standard immunocytochemistry in buccal smears, obtained by scraping immediately before and after 2 weeks of gefitinib treatment, and in the available archival tumor specimens. EGFR gene mutations were evaluated by direct sequencing and gene copy number was determined by fluorescence in situ hybridization. Data were correlated with gefitinib toxicity and objective response. RESULTS: Fifty-eight patients with pretreated advanced non-small cell lung cancer were enrolled and nine of these patients (15%) showed an objective response to gefitinib (including two complete responses). Toxicity (P = 0.025) and baseline p-AKT expression in buccal mucosa cells (P = 0.061) showed a potential predictive role. On the contrary, the probability of achieving an objective response was not affected by pretreatment expression of EGFR, p-EGFR, and p-MAPK, either in buccal mucosa or in tumor tissue. Responders showed a nonstatistically significant trend toward a more pronounced reduction in the expression of p-EGFR, p-MAPK, and p-AKT after gefitinib treatment. Among responders, five of six (83%) tumors showed EGFR gene mutation, whereas none of the tumors from patients with stable or progressive disease did (P < 0.001). CONCLUSIONS: Epithelial cells obtained from buccal mucosa may be used to assess the pharmacodynamic effect of EGFR-targeted agents, and pretreatment p-AKT expression may be a possible predictive biomarker of in vivo gefitinib activity.

Mediastinal chondrosarcoma.

The authors report a rare case of primary chondrosarcoma of the anterior mediastinum showing unusual pathological and clinical features, namely 1) the lack of any anatomical relationship between the tumor and cartilage-containing organs, and 2) an indolent behavior with long-term survival. In spite of early disease recurrence and repeated surgery, the patient is in good health five years after primary surgery. The reported case suggests that 1) primary chondrosarcomas of the anterior mediastinum may have a better prognosis than previously recognized, 2) the disease can remain confined within the chest for as long as five years, and 3) repeated surgery may contribute to long-term survival.