Sandra Rosini

Mutational analysis of the HER2 gene in lung tumors from Caucasian patients : Mutations are mainly present in adenocarcinomas with bronchioloalveolar features

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR‐single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776–779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776–779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K‐ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K‐ras mutations in 110 (27.3%) cases. EGFR, HER2 and K‐ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Effective Assessment of egfr Mutation Status in Bronchoalveolar Lavage and Pleural Fluids by Next-Generation Sequencing

Purpose: The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells. Experimental Design: We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS. Results: In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS. Conclusions: The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies. Clin Cancer Res; 19(3); 691–8. ©2012 AACR.

Immune cells in periapical granuloma: Morphological and immunohistochemical characterization*

Samples of periapical granulomas obtained from 12 patients were examined using light and electron microscopes and monoclonal antibodies. Monocytes/macrophages, lymphocytes, and plasma cells were nearly always the most abundant cell populations. Ultrastructural analysis showed close contacts between macrophages and cells of the lymphoid lineage, with the lymphoid cells frequently demonstrating blastic features. Immunohistochemical staining with the anti-interleukin 2 receptor antibody showed that the concentration of labeled cells was quite low. The vast majority were lymphocytes, though some mast cells were also labeled. Mast cells were chiefly located in perivascular areas and interleukin 2 receptor-positive mast cells were frequently associated with lymphoid cells. mast cells could be part of a negative feedback mechanism in the immune response. By releasing histamine, they would block the immune response and by absorbing interleukin 2 they would remove it as an immune system stimulant.

The expression of LEC/CCL16, a powerful inflammatory chemokine, is upregulated in ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disease of unknown aetiology and pathogenesis. The presence in the colonic mucosa of reactive cells expressing proinflammatory cytokines and chemokines is associated with high levels of IL-10, an anti-inflammatory cytokine. Our aim was to investigate the role of IL-10 and the β chemokine LEC/CCL16 selectively up-regulated by IL-10 in inflammatory cell recruitment and cytokine and chemokine production during UC. We studied histologically, immunohistochemically and ultrastructurally colonic biopsies from 20 active UC patients and 10 control specimens taken far from any macroscopically detectable lesion in age and sex-matched patients with noninflammatory bowel disease. In active UC, immature dendritic cells (DCs) in the LP are associated with IL-10 in the T cell rich area. Furthermore, most of the LP-infiltrating macrophages strongly expressed LEC/CCL16, a chemokine upregulated by IL-10. To evaluate if LEC/CCL16 plays a role in the inflammatory reaction present in UC, we performed morphological studies in mice injected s.c. with syngeneic tumor cells engineered to produce LEC/CCL16. We found that the LEC protein locally released by LEC-gene-transfected tumor cells is a potent proinflammatory chemokine that induces the recruitment of a reactive infiltrate, and an angiogenic process mirroring that in human UC. In conclusion our data indicate that: 1) LEC is endowed with a powerful inflammatory activity and 2) upregulated in active UC, when IL-10 expression is elevated in a T cell rich area, 3) this upregulation can be seen as a pro-inflammatory pathway triggered by IL-10 in UC.

The Lack of Epithelial Interleukin-7 and BAFF/BLyS Gene Expression in Prostate Cancer as a Possible Mechanism of Tumor Escape from Immunosurveillance

Purpose: The human prostate is endowed with intraepithelial and stromal lymphocytes, which may develop lymphoid follicles (LF) and allow a local immune response. We sought to investigate whether interleukin (IL)-7 and BAFF/BLyS, two fundamental survival factors for T and B cells, are expressed in the normal and neoplastic prostate and affect intraprostatic lymphocyte homeostasis. Experimental Design: We have used real-time reverse transcription-PCR of microdissected prostatic glands and confocal microscopy to detect cytokine production, combined with immunohistochemistry to characterize intraprostatic lymphocytes. Results: Prostatic epithelia constitutively produce IL-7 and, to a lesser extent, BAFF/BLyS. Indeed, we show that IL-7 receptor α is expressed by intraepithelial T lymphocytes and parafollicular T cells, whereas BAFF-R is found on periglandular B lymphocytes and mantle zone B cells of LFs. Prostate-homing B and T lymphocytes are scarcely proliferating, whereas most of them express the antiapoptotic protein bcl-2 and reveal a low apoptotic index in the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay. The transition from normal to neoplastic glands in prostate cancer (PCa) is marked by a dramatic decline of IL-7 and BAFF/BLyS production. Accordingly, PCa is characterized by a significant reduction of intraepithelial lymphocytes and loss of LFs. B-cell and T-cell expression of bcl-2 decrease, whereas the apoptotic events increase. The remaining PCa-infiltrating lymphocytes are mostly CD8+ T cells that lack terminal differentiation and barely penetrate neoplastic glands. Conclusions: These results suggest that epithelial IL-7 and BAFF/BLyS production support intraprostatic lymphocyte survival. Its loss in PCa is associated with a severe depletion of prostate-associated lymphocytes and points to a novel tumor escape mechanism.

Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Building Sustainable Capacity for Disease Diagnosis in Sub-Saharan Africa: Case Studies of Cooperation in Diagnostic Pathology

One of the most important outcomes of globalization’s complex and debated processes is the possibility of promoting and upgrading services in geographically remote areas. Globalization yields the easiest means of transportation and communication, which makes networking a relatively simple process. Moreover, innovative sound technologies, such as tele-consultation, provide these networks with a solid base for continuation and further buildup. These possibilities apply to the field of Medicine in general, and Pathology in particular. These new opportunities are very promising in regard to the endeavor of building sustainable capacities for disease diagnosis in sub-Saharan Africa.

Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study

Human Papillomavirus (HPV) E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001–2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/μL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC) cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A “one sample for all” approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

GISTs: From the History to the Tailored Therapy

Gastrointestinal stromal tumours (GISTs) represent the most common non-epithelial mesenchymal tumours of the gastrointestinal tract. The role of the pathologist in the differential diagnosis of GISTs, as well as the correct understanding of these neoplasia by detailed clinicopathologic, biological and genetic studies, are becoming increasingly important in optimizing the management of these tumours and to develop new therapies for the treatment of advanced diseases.

Overview on Molecular Markers to Implement Cervical Cancer Prevention: Challenges and Perspectives

A decline in the incidence and mortality of cervical cancer has been observed in most Western countries since the first third of the past century. This may be mainly attributed to the introduction of Pap test, that is considered one of the milestones in medicine. Nevertheless, worldwide burden of cervicocarcinoma is still enormous. This fact is well known among the scientific community, and has led to an extensive search for optional screening tools and predictive markers, currently under testing in different countries.