Pellegrino Crafa

Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma

Background:Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma.Methods:The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours.Results:Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis.Conclusion:The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8 + cells in patients with hepatocellular carcinoma

BACKGROUND/AIMS Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

PTEN status in advanced colorectal cancer treated with cetuximab

Background:Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway.Methods:PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n=43) and on metastatic (n=24) sites in CRC patients treated with cetuximab.Results:The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; P<0.05), PFS (0.8 vs 8.2 months; P<0.001) and OS (2.9 vs 14.2 months; P<0.001).Conclusion:A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised.

A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR

Background Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP. Methodology/Principal Findings The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (80±5)% accuracy, (81±6)% sensitivity and (78±7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus ≥7, an important predictor of final outcome. Conclusions/Significance The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the “cancer field effect”, in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method posses the potential to enhance conventional diagnosis.

Estrogen Receptor α Is a Novel Marker Expressed by Follicular Dendritic Cells in Lymph Nodes and Tumor-Associated Lymphoid Infiltrates

During routine assessment of the hormonal phenotype of breast carcinomas, we detected expression of the estrogen receptor (ER) in the germinal centers of reactive lymphoid follicles surrounding malignant foci. To confirm and extend this finding, we compared ER-alpha, progesterone receptor (PR), and androgen receptor (AR) immunostaining in hyperplastic or metastatic lymph nodes obtained from patients with various pathology, disease location, gender and age. Irrespective of these parameters, we found that: 1) ER-alpha-positive cells were located prevalently in germinal centers, 2) the PR was weakly expressed by cells within and surrounding germinal centers, and 3) the androgen receptor was undetectable. Transcripts for ER-alpha and PR were also detected by reverse transcription-polymerase chain reaction on laser-microdissected lymph node germinal centers. Morphologically, the ER-positive cells resemble dendritic cells and by double immunostaining were found to express both CD21 and CD23, which is characteristic of follicular dendritic cells. Finally, we assessed the effects of Tamoxifen treatment by comparing the numbers of ER-positive follicular dendritic cells in lymph nodes obtained from breast cancer patients before and after treatment. The results show that Tamoxifen treatment generated larger germinal centers with more abundant ER(+)/CD21(+)/CD23(+) cells. Taken together, these results open new perspectives on the effects of sex steroids and their antagonists on the human response in cancer and inflammation.

Which breast carcinomas need HER-2/neu gene study after immunohistochemical analysis? Results of combined use of antibodies against different c-erbB2 protein domains

Aims:  Evaluation of HER2 gene amplification in breast cancers is a compelling, routine procedure. The aim of this work was to evaluate which breast carcinomas would really benefit from HER‐2/neu gene analysis.

Aggressive angiomyxoma of the spermatic cord. Two unusual cases occurring in childhood.

We report on two cases of aggressive angiomyxoma (AAM) of the spermatic cord occurring in two 13-year-old children. Clinically, the tumor simulated a mass of the spermatic cord. Histologically, it represented a poorly circumscribed, benign myxoid tumor, with a sparse population of stromal cells immunoreactive for vimentin and, focally, for smooth muscle actin. No immunostaining for desmin, S-100, p53, p21waf-1, c-Erb-B2 and estrogen-progesterone receptors was found. High proliferating cell nuclear antigen (PCNA) immunoexpression found in most of the tumor cells may explain the high risk of recurrence. AAM should be considered in the differential diagnosis of a spermatic cord mass occurring during infancy.

Biological variables in non-small cell lung cancer: comparison between immunocytochemical determination on fine needle aspirates from surgical specimens and immunohistochemical determination on tissue sections

A number of biological and predictive markers of non-small cell lung cancer (NSCLC) have been sought, but these have so far been mainly evaluated on surgically resected specimens. Given that fine needle aspiration biopsy (FNAB) is being increasingly used in the diagnosis of NSCLC, its application could be extended to the immunocytochemical detection of biological parameters at the time of diagnosis before surgery. In order to assess the reliability of estimating biological markers on fine needle aspirates (FNAs) from NSCLC, the aim of this study was to compare Ki67 growth fraction, p53 and bcl-2 protein expression as revealed by the immuncytochemical assessment of FNAs obtained from surgical samples with the immunohistochemical results obtained from the corresponding histological sections. FNAs were performed on surgical specimens obtained from 29 NSCLC patients. Ki67, p53 and bcl-2 were cytologically and histologically evaluable in respectively 25, 27 and 19 cases. Concordance between FNAs and corresponding paraffin sections was 84% for Ki67, 93% for p53 and 95% for bcl-2. All of the specimens whose biological parameters were studied by immunocytohistochemistry also underwent flow cytometric DNA analysis of FNAs taken from fresh surgical specimens. Of the 29 cases, 22 were aneuploid and seven diploid. The S-phase fraction (SPF) was evaluable in 62% of cases. Comparison of SPF results on FNAs with Ki67 values evaluated on the corresponding histologic and cytologic specimens, revealed a significant correlation only with histology. Good reproducibility was also found in relation to the immunocytochemical results obtained on FNAs from different areas of the same tumour, showing that tumour heterogeneity does not affect the method. The concordance between the immunocytochemical and immunohistochemical results suggests that FNAB may be a reliable procedure for the biological characterization of NSCLC. Given its limited invasiveness, FNAB could be used in vivo for the preoperative assessment of biological parameters in patients with operable or metastatic NSCLC.

Pleomorph poorly differentiated endocrine carcinoma of the rectum

We present a case of poorly differentiated endocrine carcinoma (PDEC) of the rectum identified immunohistochemically and characterized by a high degree of cellular pleomorphism, including bizarre giant cells. This case indicates that gastrointestinal PDECs are not restricted to small cell carcinomas. Among the multiple genes investigated, loss of heterozygosity (LOH) of the p53 locus without p53 immohistochemical accumulation, overexpression of c-kit and absent expression of p16 were seen.

Peculiar Findings in a Case of Bilateral Uveal Pigmented Lesions

Purpose: To describe a probable case of bilateral diffuse uveal melanocytic proliferation (BDUMP) with unusual manifestations and prognosis. Design: Case report. Methods: Clinical follow-up of the patient lasting 50 months with recurrent fundus examination using color photographs, angiography, ultrasound, and optical coherence tomography. Serological and radiological investigations were performed to assess possible extraocular alterations. Results: In both eyes patch-shaped pigmented alterations of the fundus were revealed. Fluorescein and indocyanine angiography evidenced corresponding areas of hyperfluorescent pinpoints and subtle serous detachment of the neurosensory retina, respectively. Ten months after the initial evaluation, flat pigmentary lesions appeared in the superior scleral surface of the right eye and underwent histological examination. After an initial decrease in visual acuity, the patient experienced a spontaneous recovery. He did not develop cataracts or any systemic malignancies. Conclusions: Although not all the criteria for the diagnosis were fulfilled, clinical findings were compatible with BDUMP. The presence of scleral pigmented lesions and the good visual prognosis may widen the spectrum of this rare disease.