Is there a correlation between HER2 gene amplification level and response to neoadjuvant treatment with trastuzumab and chemotherapy in HER2-positive breast cancer?

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

Endoscopic ultrasound-guided fine-needle aspiration cytology in the diagnosis of the gastrointestinal stromal tumor of the stomach.

BACKGROUND: This study aims to evaluate the usefulness of endoscopic ultrasound-guided fine-needle aspiration cytology (EUS-FNAC) in the diagnosis of the gastric gastrointestinal stromal tumor (GIST). METHODS: We retrospectively investigated the efficacy and accuracy of EUS-FNAC in the diagnosis of gastric GIST. Cytological smears and cytoblock sections including immunohistochemistry and mutational studies from patients diagnosed as gastric GISTs were retrieved. RESULTS: Thirty patients (mean age 68.8 years, range 32-88 years, Male:Female 1:1.7) were diagnosed by cytological and cytoblock study to have GIST. The size of tumors ranged from 1.6 to 25 cm (mean 6.0 cm). 7 (23%) cases were incidentally discovered. Location was: gastric body 13 (43.3%), fundus 8 (26.7%), antrum 7 (23.3%), cardia 2 (6.7%). The study of removed tumors was correlated with the cytological findings. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 75%, 100%, 100%, 46%, and 96%. There were no false-positive cases. The preoperative risk assessment of 27 cases with cytoblock were: none 3 (11.1%), very low 8 (29.6%), low 12 (44.4%), high 3 (11.1%), insufficient clinical data 1 (3.7%). The follow-up varied from 2 to 120 months (mean 46.7 months). Only 1 patient of the high-risk group died. The most frequent mutations found were those of c-KIT in exon 11. CONCLUSIONS: Pathological diagnosis was based on a combination of cytological, histopathological, and immunohistochemical features. EUS-FNAC is a reliable, accurate, and safe method for the diagnosis of GIST. The cytoblock allows tumor risk classification and mutational study of the cases.

Breast cancer subtype discrimination using standardized 4-IHC and digital image analysis.

Breast cancer is a heterogeneous disease. Surrogate classification of intrinsic subtypes of invasive carcinomas by combined immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 (4-IHC) has increased steadily since the 2011 St Gallen symposium, due to its rapid subtyping of tumors at a reasonable cost. An important step in improving 4-IHC reproducibility and reliability will be to provide reference values from the routine use of standardized 4-IHC followed by image analysis. The aims of the current study were (1) to analyze invasive breast carcinomas using standardized 4-IHC and quantitative image analysis and (2) to compare the results obtained in the classification of biological subtypes using current Ki67 and PR threshold values proposed by different authors to sub-classifying the luminal A-like and the luminal B-like (HER2-negative) subtypes. Five hundred twenty-one tumors were analyzed by standardized immunohistochemistry, with automatic image analysis, and HER2 FISH technique. Positivity for ER was found in 82.7% and for PR in 70.1% of cases. Using the Allred scoring system, hormone receptor results showed a bimodal distribution, particularly for ER. HER2 positivity was found in 15.7% of cases, and the mean Ki67 score was 32.3%. Using the most recently proposed surrogate definitions for the classification of luminal breast cancer subtypes, the percentages of different subtypes that we found were similar to those published with genomic platforms: 40.7% luminal A-like, 32.4% luminal B-like/HER2-negative, 9.8% luminal B-like/HER2-positive, 6.0% HER2-positive, and 11.1% triple negative. Standardized 4-IHC with automatic image analysis constitutes a low-cost method for surrogate definitions of biological subtypes of breast cancer that delivers accurate results in a day.

Fluorescence in situ hybridization analysis of the ALK gene in 2,045 non-small cell lung cancer patients from North-Western Spain (Galicia).

Identification of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements is a standard diagnostic test in patients with advanced non-small cell lung cancer (NSCLC). The current study describes the experience of ALK rearrangement detection of a referral center in the public health care system of Galicia in North-Western Spain. The fluorescence in situ hybridization (FISH) patterns of the ALK gene and the clinical and pathological features of these patients are reported. This study is also of interest for comparative purposes due to the relative geographical isolation of the area, which could have contributed to particular genetic features. A total of 2,045 tissue samples from NSCLC patients were collected between October 2010 and July 2015 and tested for ALK rearrangements by FISH. Examination of 1,686 paraffin-embedded tissue specimens and 395 cytological samples (306 cell block preparations and 53 cytological smears) was conducted, and any associations between the FISH results and clinicopathological features were assessed. The rate of successful evaluation was marginally higher in tissue samples than in cytological samples (92.9% vs. 84.1%); this difference was not significant. ALK rearrangements were identified in 82 patients(4%): 65 (79.3%) in tissue specimens, 15 (18.3%) in cell block samples and 2 (2.4%) in cytological smears. This genetic translocation appeared to be associated with a non-smoking history, younger age, female gender, stage IV and adenocarcinoma histological type. The findings demonstrate that ALK evaluation by FISH is feasible in tissue and cytological samples. The clinical and pathological features of the ALK-positive series of patients are similar to those previously reported in the literature.

Dual-colour CISH is a reliable alternative to FISH for assessment of topoisomerase 2-alpha amplification in breast carcinomas.

Anthracyclines are among the most powerful antineoplastic drugs available for breast cancer treatment. Although HER2 amplification has been postulated to predict anthracycline benefit, numerous reports have demonstrated that HER2/TOP2A co-amplification is the clinically useful predictive marker of response to anthracyclines. The standard technique to evaluate gene status for target therapy selection is fluorescence in situ hybridization (FISH), but this technique has some disadvantages. Dual-colour chromogenic in situ hybridization (CISH) is an extension of the FISH protocol that allows bright-field microscopy and thus represents a user-friendly alternative to FISH. In order to evaluate whether dual-colour CISH is a reliable alternative to FISH in determining TOP2A gene amplification and to determine the frequency with which TOP2A and HER2 were co-amplified, we analysed 100 invasive breast cancer specimens (70 consecutive and 30 HER2-amplified samples) using tissue microarrays. Thus, a 99 % agreement was found between TOP2A status determined by dual-colour CISH and FISH, as well as a high degree of correlation in TOP2A ratios using both techniques. TOP2A gene amplification was present in 8.6 % of the 70 consecutive samples studied, all of which were HER2-amplified. Co-amplification of TOP2A was observed in 46.5 % of the additional 30 HER2-amplified samples (no TOP2A amplification was seen in non-amplified HER2 samples). We conclude that dual-colour CISH represents an excellent alternative to FISH for determination of TOP2A gene status in invasive breast cancer. Our results showing TOP2A amplification only in HER2-amplified cases also add to the evidence that TOP2A determination should be restricted to those cases.

HER2 status determination using RNA-ISH--a rapid and simple technique showing high correlation with FISH and IHC in 141 cases of breast cancer.

AIMS: the assessment of the human epidermic growth factor receptor 2 (HER2) is currently performed in most laboratories using two techniques: Fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), and novel methodology is being investigated continuously in the assessment of HER2, such as SISH, CISH, DNA chips, ELISA or real time PCR to make assessment easier, faster or more accurate. RNA-ISH (RNA in Situ Hybridisation) is a new technique designed to detect mRNA expression levels, conducted by light microscope without the need for counting or grading systems in a total processing time of 4 hours. This study aims to determine if RNA-ISH is a viable and effective technique and a possible alternative to the currently used techniques by analysing and comparing genetic amplification (FISH) and protein levels (IHC) with mRNA over-expression (RNA-ISH) in 141 cases of breast cancer. RESULTS: This study demonstrated a 96.5% concordance between over-expression of HER2 as determined by RNA-ISH and gene amplification as determined by FISH. The relationship between RNA-ISH-evaluated and IHC-evaluated over-expression was equally well reflected with a 95.2% concordance. Importantly, a considerable reduction in processing and evaluation time was achieved of only 4 hours. CONCLUSIONS: We conclude that the probe developed for RNA-ISH represents a viable, effective possible alternative to FISH and IHC for analysing HER2 status in primary breast tumours.

Fluorescent in situ hybridization heating pretreatment: the key is temperature control.

Fluorescent in situ hybridization (FISH) is a very useful tool for diagnostic and prognostic purposes in pathology. However, many laboratories still experience troubles when applying FISH to paraffin material. To overcome these difficulties, different pretreatments which include enzymatic digestion have been described. Usually, previous to digestion, a heating step is performed. The aim of this study was to compare the efficiency of the heating step with different buffers and different heating methods. We conclude that the main factor in the heating pretreatment is the temperature control, irrespective of the buffer used. Best results are obtained with any buffer by heating the slides to 99°C for 15 min followed by 10 min at room temperature.

BRAF mutation in solid cell nest hyperplasia associated with papillary thyroid carcinoma. A precursor lesion?

We describe a case of solid cell nest hyperplasia associated with papillary thyroid carcinoma in a 48-year-old man with goiter. The entire gland was examined; in 1 section, the cells of 1 solid cell nest were in close contact with a follicular variant of papillary microcarcinoma. A second follicular variant of papillary microcarcinoma, 1 follicular adenoma, hyperplastic nodules, and some lymphoid aggregates were also found. Scattered p63-positive cells were found in the second papillary microcarcinoma. After microdissection, the same BRAF(V600E) mutation was found both in a pool of 5 solid cell nests and in the adjacent papillary microcarcinoma. BRAF(V600E) mutation and the previously unreported BRAF(G593D) mutation along with p.G606G silent change were found in the second papillary microcarcinoma, but no mutations were detected in the follicular adenoma or in the 2 other pools of solid cell nests screened for BRAF gene mutations. These findings support a histogenetic link between the main cells of solid cell nests and papillary thyroid carcinoma, and suggest solid cell nest hyperplasia as a precursor lesion of papillary thyroid carcinoma.

Extension of coronary artery disease is associated with increased IL-6 and decreased adiponectin gene expression in epicardial adipose tissue.

UNLABELLED: Epicardial adipose tissue (EAT) expresses lower levels of adiponectin in patients with CAD and higher levels of inflammatory mediators such as IL-6 and leptin than subcutaneous adipose tissue. This showed one important role of EAT in coronary artery disease. However, the relationship of EAT adiponectin and IL-6 levels to the extension of coronary artery disease has not hitherto been determined. We sought to determine whether the levels of adiponectin and interleukin-6 (IL-6) mRNA in epicardial adipose tissue are associated with the extension of coronary artery disease (CAD). METHODS: Angiographic and hormones expression were evaluated from epicardial and subcutaneous adipose tissue. 92 patients (58 CAD, 34 non-CAD) who underwent cardiac surgery. Adiponectin and IL-6 mRNA levels were measured by real time RT-PCR in epicardial and subcutaneous adipose tissue (SAT) following angiographic evaluation of their coronary arteries. RESULTS: We found that epicardial adipose tissue of CAD expressed lower levels of adiponectin mRNA and higher levels of IL-6 mRNA than that of non-CAD patients. As the number of injured arteries rose, adiponectin mRNA levels decreased (r=-0.402, p<0.001) and IL-6 mRNA increased (r=0.514, p<0.001) in epicardial adipose tissue. CONCLUSIONS: The extension of CAD is significantly associated with the expression of adiponectin and IL-6 mRNA in EAT. These findings suggest that low adiponectin and high IL-6 expression by EAT may contribute to CAD extension.

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.

Immunohistochemical expression of HIF-1alpha in response to early myocardial ischemia.

This study aims to evaluate the effects of ischemia on the myocardial fibers and the expression of the transcriptional factor for angiogenesis hypoxia-inducible factor-1 alpha (HIF-1alpha) in human heart specimens. We have prospectively analyzed the HIF-1alpha expression in human ischemic hearts with the ABC-inmunohistochemistry technique and amplification by biotinylated tyramide. The relationship between the expression of HIF-1alpha and the temporal evolution of ischemia has also been evaluated. As pathomorphological diagnosis of early myocardial ischemia has many problems in human autopsy material with less than 4 to 6 h after clinical onset, we suggest that HIF-1alpha is helpful in the early acute myocardial infarction diagnosis, so it stains necrotic areas within the first 2 h. The amplification procedure provides a higher intensity of the final staining without losing specificity. It is concluded that in normal cardiac fibers, basal expression of HIF-1alpha is not appreciable, but it steadily increases after ischemia. With regard to the practical applicability in forensic field, our observations suggest that positive immunohistochemical expression of HIF-1alpha on heart samples may be used as a reliable indicator of myocardial damage in cases without cardiac lesion evidence, using conventional microscopy. This method is especially useful and may provide definitive proof of myocardial ischemia in unexpected deaths without previous symptoms, or in forensic cases with a short period of clinical manifestations. In addition, it may have been involved in possible future cardiovascular therapies.