Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. PATIENTS AND METHODS: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. RESULTS: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. SIGNIFICANCE: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.

Magnetic resonance imaging of breast cancer: does the time interval between biopsy and MRI influence MRI-pathology discordance in lesion sizing?

Background Breast magnetic resonance imaging (MRI) is more accurate than ultrasound and mammography in estimating local extension of both invasive breast cancer and ductal carcinoma in situ (DCIS) and it is part of a breast cancer patient's preoperative management. Purpose To verify if time interval between breast biopsy and preoperative MRI, lesion margins, and biopsy technique can influence tumor sizing on MRI. Material and Methods By a database search, we retrospectively identified all women with a newly diagnosed, biopsy-proven, primary breast cancer who underwent MRI before surgery. The time interval between biopsy and MRI, the type of biopsy procedure, and various pathological features of tumors were collected. We defined the concordance between MRI and pathology measurements as a difference of <5 mm in lesion sizing. Results One hundred and sixty-six women (mean age, 51.4 ± 10.4 years) were included. The time interval between biopsy and MRI showed only a weak correlation with the absolute MRI-pathology difference (r = 0.236). Stratifying the whole cohort of patients using a cutoff value of 30 days, we found that the MRI-pathology discordance was significantly higher in patients with a biopsy-MRI time interval >30 days ( P < 0.05). By means of multivariate analysis, we found that DCIS subtype and the presence of poorly defined margins on MRI are the only two factors independently and strongly associated with MRI-pathology discordance in lesion sizing. Conclusion Size, histology, and margins of tumors may affect the accuracy of MRI measurements. The type of biopsy procedure and the time interval between biopsy and preoperative MRI are not independently associated to MRI-pathology discordance.

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.

Microscopic esophagitis in gastro-esophageal reflux disease: individual lesions, biopsy sampling, and clinical correlations.

Patients with non-erosive reflux disease may show microscopic damage. This study is aimed to describe distribution, sensitivity, and specificity of histological lesions (i.e., basal cell hyperplasia-BH, papillae elongation-PE, dilatation of intercellular spaces-DIS, intraepithelial eosinophils-IE, neutrophils, and erosions) and sampling criteria. Four groups were identified on the basis of symptoms, endoscopy, and pH monitoring: (1) erosive esophagitis (n = 48), (2) non-erosive esophagitis with abnormal pH (n = 59), (3) non-erosive esophagitis with normal pH (n = 12), and (4) controls (n = 20). Biopsies were taken at the Z-line and 2 and 4 cm above it. BH, PE, DIS, IE, neutrophils, and erosions were assessed. A global severity score was calculated on the basis of the above parameters and allowed the distinction of patients from controls with 80% sensitivity and 85% specificity. Lesions were more severe at Z-line than proximally and more expressed in erosive than in non-erosive disease, although more than 70% of latter patients still showed histological damage. Esophageal biopsy seems very attractive in non-erosive disease where it may contribute to diagnosis and play a role in the comparative evaluation of different therapies.

Influence of Tumor Subtype, Radiological Sign and Prognostic Factors on Tumor Size Discrepancies Between Digital Breast Tomosynthesis and Final Histology.

Background Influence of tumor subtype, radiological sign and prognostic factors on tumor size discrepancies between DBT and final histology has not been completely investigated so far. Purpose To study the influence of tumor subtype, radiological sign and prognostic factors on tumor size discrepancies between digital breast tomosynthesis and final histology. Material and methods This is a retrospective study conducted between January 2015 and December 2016. After IRB approval, 130 consecutive patients with breast cancer diagnosed with digital breast tomosynthesis (DBT) were evaluated. A discrepancy between DBT and final histology was considered present if the difference was above the cut-off of 5 mm. Tumor subtype, radiological sign and prognostic factors were evaluated in patients with discrepancies. Descriptive statistic and non-parametric tests were used. Results A total of 105 cases of cancer, in 96 patients, all female, were included. Mean age was 61 years (range: 35-82 yrs). In 19 (18.1%) cases, discrepancies were found: 13 (68.4%) were underestimated by DBT. For tumor subtype, 10 (52.6%) were infiltrating lobular carcinomas (ILC) (p < 0.01). Fourteen (73.7%) discordant cases were architectural distortions (p < 0.01). Prognostic factors did not affect tumor size discrepancies. Conclusion ILC or an architectural distortion represents the majority of cases of tumor size discrepancies between DBT and final histology.

Axillary ultrasound and Fine-Needle Aspiration Cytology in the preoperative staging of axillary node metastasis in breast cancer patients.

OBJECTIVE: A prospective observational clinical study was undertaken to assess the accuracy of preoperative Axillary Ultrasound (AUS) plus Fine-Needle Aspiration Cytology (FNAC) as well as and its clinical utility, that is the capacity of the information supplied by the test to guide the clinical decision-making. MATERIALS AND METHODS: from January 2013 to August 2015, 400 female patients with pT1-3 cN0 operable breast cancer underwent AUS with FNAC at the Breast Unit of the "IRCCS San Martino-IST" in Genoa (Italy). RESULTS: 127 out of 400 patients (31.7%) had axillary lymph node metastases; in 69 out of 127 node-positive patients (54.3%) AUS detected at least one abnormal lymph node, and in 56 out of 127 patients (44.1%) the abnormal sonographic pattern of the lymph node was coupled with a positive FNAC finding. No false-positive finding by both AUS-alone or combined AUS/FNAC was observed. AUS-alone had sensitivity of 54.3% (69/127), specificity of 100% (273/273), PPV of 100% (69/69), NPV of 82.5% (273/331), and accuracy of 85.5% (342/400). Combined AUS/FNAC had sensitivity of 44.1% (56/127), specificity of 100% (273/273), PPV of 100% (56/56), NPV of 79.4% (273/344), and accuracy of 82.2% (329/400). CONCLUSIONS: AUS-alone or combined AUS/FNAC had a high accuracy rate coupled with a more than satisfactory efficiency due to their low costs and easy access for the preoperative staging of the axilla. Notably, AUS-alone might be suggested for the preoperative staging of patients with early stage breast cancer because FNAC did not increased the specificity but reduced the sensitivity of the technique. Patients with negative findings might undergo either SLNB or close observation while waiting for the definitive results of ongoing SOUND randomized clinical trial.

Clinical decision-making in atypical and suspicious categories in fine-needle aspiration cytology of the breast.

BACKGROUND: Fine-needle aspiration cytology (FNAC) is a simple and reliable technique to assess breast lesions, although a definitive differential diagnosis (benignity vs. cancer) is achieved approximately in 60-70% of cases because an inadequate (C1), atypical (C3) or suspicious (C4) category is otherwise reported. PATIENTS AND METHODS: A retrospective analysis of 763 cases with C3 or C4 reports was performed to define their positive predictive value (PPV), as well as the practical implications of clinical and imaging findings as for clinical decision-making. FNACs were collected from January 2003 to September 2012 at the Breast Unit of IRCCS "A.O.U. San Martino-IST" Genoa, with each being received later to definitive histology. The PPV for cancer of C3/C4 categories were computed to measure the accuracy of FNAC; moreover, the PPV was also stratified according to clinical, mammography and sonography data alone or by their combination. RESULTS: The PPV of C3 and C4 was 21.1% (80/380) and 84.1 % (322/383), respectively. Within each C3/C4 category, a significant direct correlation (p<0.001) between the suspicion index of clinical, mammography and sonography data and cancer detection rate was always observed. The PPV of C3/C4 stratified by the combination of clinical and imaging findings showed satisfactory values in the C3 category only when there was an agreement between clinical and imaging findings, whereas the PPV of the C4 category was always remarkably high (ranging from 92.3% to 100%). CONCLUSION: the diagnostic work-up in C4 reports or in patients with a C3 report but with an high suspicion index at clinical or imaging examination should be preferably implemented by means of a core biopsy to optimize the therapeutic planning; given a C3 report with dubious clinical and/or imaging findings, an excisional biopsy (or in alternative vacuum-assisted breast biopsy with complete removal of the nodule) should be preferably performed in order to reach a definitive histological dia gnosis with no further delay.

Predictive factors of non-sentinel lymph node involvement in patients with invasive breast cancer and sentinel node micrometastases.

Patient-related, tumor-related, and sentinel node (SN)-related factors have been identified with the aim of predicting non-SN status in patients with SN micrometastases. According to our previous experience, primary tumor size (p=0.005) and the presence of lymphovascular invasion (LVI) (p=0.000) significantly predicted non-SN status in patients with SN micrometastasis; moreover, non-SN metastases were never detected in patients with pT1a-1b, G1, and no LVI. A prospective assessment was undertaken in a validation set of 126 patients to confirm these findings. Univariate analysis indicated that primary tumor size (p=0.05), Scarff-Bloom-Richardson (SBR) grade (p=0.008), LVI (p=0.001), and the number of mitoses/mm(2) (p=0.01) were significant predictors of non-SN status. By logistic regression analysis, tumor size (p=0.03), LVI (p=0.001), grade (p=0.003) and the number of mitoses/mm(2) (p=0.01) were the only variables remaining in the model. Three subsets of patients were identified: i) 18.3% of patients (pT1, G1, and no LVI) had tumor-negative non-SN (no risk group); ii) 37.3% of patients (number of mitoses/mm(2) <10, SBR grade II-III) had a rate of tumor-positive non-SN <15% (intermediate risk); iii) 44.4% of patients had a mean rate of non-SN involvement of 46% (high risk). By these parameters, more than 50% of patients could be selectively spared unnecessary axillary lymph node dissection without staging or therapeutic benefit, especially in patients with well-differentiated pT1 tumors without LVI.

Flat epithelial atypia: comparison between 9-gauge and 11-gauge devices.

BACKGROUND: This study aimed to establish if women with a diagnosis of flat epithelial atypia (FEA) without residual microcalcifications at stereotactic vacuum-assisted breast biopsy (VABB) could be managed with mammographic follow-up (FU) instead of surgery and to compare 9-gauge and 11-gauge devices. PATIENTS AND METHODS: From October 2003 to January 2011, 2382 VABB procedures were performed (1373 with 11-gauge and 1009 with 9-gauge). We found 121 cases of pure FEA that were surgically treated: 57 with a 9-gauge device (group 1) and 64 with an 11-gauge device (group 2). The underestimation rate (UR) of malignancy for patients without and those with residual microcalcifications for each VABB device was calculated. Differences between groups were analyzed with the Fischer exact test. RESULTS: The overall UR of FEA was 4% (2 of 57) with the 9-gauge device and 8% (5 of 64) with the 11-gauge device. With a 9-gauge device, the UR for patients without residual microcalcifications was 0% (0 of 46), and the UR for patients with residual microcalcifications was 18% (2 of 11). With an 11-gauge device, the UR for patients without residual microcalcifications was 0% (0 of 39), the UR for patients with residual microcalcifications at post-biopsy mammograms was 16% (5 of 25). With a 9-gauge device, 80% (46 of 57) of patients did not have residual microcalcifications after VABB. With an 11-gauge device, 60% (39 of 64) of patients had no residual microcalcifications after VABB. Differences between the 9-gauge and 11-gauge devices were statistically significant (P < .05). CONCLUSION: Women with FEA without residual microcalcifications after VABB can be managed conservatively. Nine-gauge VABB is associated with a lower percentage of residual microcalcifications compared with an 11-gauge device, but it is safe to follow patients with FEA if all calcifications are removed with the core biopsy.

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.