Piero Tosi

MYC translocation-negative classical Burkitt lymphoma cases: an alternative pathogenetic mechanism involving miRNA deregulation

The molecular feature of Burkitt lymphoma (BL) is the translocation that places c‐Myc under the control of immunoglobulin gene regulatory elements. However, there is accumulating evidence that some cases may lack an identifiable MYC translocation. In addition, during the EUROFISH project, aiming at the standardization of FISH procedures in lymphoma diagnosis, we found that five cases out of 35 classic endemic BLs were negative for MYC translocations by using a split‐signal as well as a dual‐fusion probe. Here we investigated the expression pattern of miRNAs predicted to target c‐Myc, in BL cases, to clarify whether alternative pathogenetic mechanisms may be responsible for lymphomagenesis in cases lacking the MYC translocation. miRNAs are a class of small RNAs that are able to regulate gene expression at the post‐transcriptional level. Several studies have reported their involvement in cancer and their association with fragile sites in the genome. They have also been shown to control cell growth, differentiation, and apoptosis, suggesting that these molecules could act as tumour suppressors or oncogenes. Our results demonstrated a modulation of specific miRNAs. In particular, down‐regulation of hsa‐let‐7c was observed in BL cases, compared to normal controls. More interestingly, hsa‐mir‐34b was found to be down‐regulated only in BL cases that were negative for MYC translocation, suggesting that this event might be responsible for c‐Myc deregulation in such cases. This hypothesis was further confirmed by our in vitro experiments, which demonstrated that increasing doses of synthetic hsa‐mir‐34b were able to modulate c‐Myc expression. These results indicate for the first time that hsa‐mir‐34b may influence c‐Myc expression in Burkitt lymphoma as the more common aberrant control exercised by the immunoglobulin enhancer locus. Copyright

Origin of Microsatellite Instability in Gastric Cancer

Microsatellite instability (MSI) is observed in 13-44% of gastric carcinoma. The etiology of MSI in gastric carcinoma has not been clearly defined. To assess the role of mismatch repair in the development of MSI in gastric cancer, expression of hMSH2 and hMLH1 was explored. We examined 117 gastric carcinomas for MSI and observed instability at one or more loci in 19 (16%) of these tumors. Of the 19 tumors with MSI, nine exhibited low-rate MSI (MSI-L) with instability at <17% of loci, whereas the remaining 10 exhibited high-rate MSI (MSI-H) with instability at >33% of loci examined. Immunohistochemical staining for hMLH1 and hMSH2 was performed on eight of the tumors with MSI-H, five with MSI-L, and 15 tumors without MSI. All eight tumors with MSI-H showed loss of staining for either hMLH1 (n = 5) or hMSH2 (n = 3). In contrast, tumors with MSI-L or without MSI all showed normal hMSH2 and hMLH1 protein expression patterns. Moreover, all eight of the tumors with MSI-H also showed instability at BAT-26, whereas none of the MSI-L tumors or tumors without instability showed instability at BAT-26. These findings suggest that the majority of high-level MSI in gastric cancer is associated with defects of the mismatch repair pathway. Although larger studies are needed, BAT-26 appears to be a sensitive and specific marker for the MSI-H phenotype in gastric carcinoma.

Automated diagnosis of pigmented skin lesions.

Since advanced melanoma remains practically incurable, early detection is an important step toward a reduction in mortality. High expectations are entertained for a technique known as dermoscopy or epiluminescence light microscopy; however, evaluation of pigmented skin lesions by this method is often extremely complex and subjective. To obviate the problem of qualitative interpretation, methods based on mathematical analysis of pigmented skin lesions, such as digital dermoscopy analysis, have been developed. In the present study, we used a digital dermoscopy analyzer (DBDermo‐Mips system) to evaluate a series of 588 excised, clinically atypical, flat pigmented skin lesions (371 benign, 217 malignant). The analyzer evaluated 48 parameters grouped into 4 categories (geometries, colors, textures and islands of color), which were used to train an artificial neural network. To evaluate the diagnostic performance of the neural network and to check it during the training process, we used the error area over the receiver operating characteristic curve. The discriminating power of the digital dermoscopy analyzer plus artificial neural network was compared with histologic diagnosis. A feature selection procedure indicated that as few as 13 of the variables were sufficient to discriminate the 2 groups of lesions, and this also ensured high generalization power. The artificial neural network designed with these variables enabled a diagnostic accuracy of about 94%. In conclusion, the good diagnostic performance and high speed in reading and analyzing lesions (real time) of our method constitute an important step in the direction of automated diagnosis of pigmented skin lesions.

Human Papillomavirus Testing with the Hybrid Capture 2 Assay and PCR as Screening Tools

ABSTRACT The recognition of high-risk human papillomaviruses (HPVs) as etiological agents of cervical cancer has increased the demands to use testing for HPV for the detection of abnormal cervical smears and for cervical cancer screening. The present study compared the performance of the Hybrid Capture 2 (HC2) assay with that of PCR for the detection of significant cervical lesions in 1,511 women with different risks for HPV infections in three New Independent States of the former Soviet Union. The results showed that the level of agreement between the HC2 assay and PCR was substantial, with a kappa (Cohen) value of 0.669 (95% confidence interval [CI], 0.629 to 0.709). Of the 228 samples with discrepant results, 92 were positive by the HC2 assay but negative by PCR, whereas 136 samples were PCR positive but HC2 assay negative. The positive predictive values (PPVs) of the HC2 assay and PCR in detecting high-grade intraepithelial lesions (HSILs) were 4.5% (95% CI, 3.5 to 5.5%) and 3.6% (95% CI, 2.7 to 4.5%), respectively, and the negative predictive values (NPVs) were 99.6% (95% CI, 99.3 to 99.9%) and 99.3% (95% CI, 98.9 to 99.7%), respectively. The sensitivities of the HC2 assay and PCR for the detection of HSILs were 85.2 and 74.0%, respectively, and the specificities were 67.2 and 64.1%, respectively. In receiver operating characteristic (ROC) analysis, the performance of the HC2 assay for the detection of HSILs was excellent (P = 0.0001); the area under the ROC analysis curve was 0.858 (95% CI, 0.811 to 0.905), and the optimal balance between sensitivity (86.5%) and specificity (80%) was obtained with an HC2 assay cutoff level of 15.6 relative light units/positive control. Use of this cutoff would increase the specificity of the HC2 assay to 80.0% without compromising sensitivity. In conclusion, the results of PCR and the HC2 assay were concordant for 85% of samples, resulting in substantial reproducibility. Both tests had low PPVs, equal specificities, and equal (almost 100%) NPVs for the detection of HSILs; but the sensitivity of the HC2 assay was slightly better.

β-catenin and Gli1 are prognostic markers in glioblastoma

Glioblastomas (GBMs), the most common primary malignancies of the central nervous system, are highly aggressive and heterogeneous, and remain a dramatic therapeutic challenge. Markers mirroring the complex molecular profile of GBMs that are predictive of patient outcomes are needed to define novel multi-targeted treatment strategies. Resistance to current GBM therapies is partly due to a subpopulation of stem-like and other self-renewing cells (hereafter called glioma stem-like cancer cells, GSCC), which are therefore of key interest as therapeutic entry points. Wnt and Hedgehog are among the main pathways involved in GSCC renewal. β-catenin and Gli1 are markers of Wnt and Hedgehog activation respectively and both pathways are known to be altered in gliomas. To date, there are no investigations of Gli1 protein expression in GBM tissue, and recently a high expression of β-catenin has been found to have a poor prognostic impact in GBM patients in a study. We have therefore quantified the positivity for β-catenin, Gli1, as well as Ki-67, p53, and EGFR proteins on immunohistochemically-stained GBM sections from 106 patients in an investigation for potential predictive biomarkers. Correlation between these markers and survival was evaluated by pair-wise Pearson correlation coefficient and by bi-dimensional hierarchical clustering, followed by survival estimations using linear regression models and classification trees. We demonstrated that both β-catenin and, for the first time, Gli1 proteins are highly predictive markers of short survival, being found in 75 and 90% of the highly predictive trees, respectively, whereas Ki-67, p53 and EGFR were under 30% and thus, not considered as predictive. Our results indicate a role of β-catenin and Gli1 in GBM malignant behaviour, and suggest that inhibiting members of Wnt and Hedgehog pathways could be a valuable therapeutic strategy for GBM patients. See commentary: A magnifying glass on glioblastoma stem cell signaling pathways

Pathogenesis of posterior capsular opacification Part II: Histopathological and in vitro culture findings

ABSTRACT The most interesting sources of information about the pathogenesis of posterior capsular opacification seem to be histopathological studies and in vitro tissue cultures. Since our surgical technique is extracapsular cataract extraction, the explants we used for tissue culture consisted of the anterior capsule epithelial sheet without the equatorial germinative zone. We successfully overcame several problems by using the autologous plasma clot culture method. This medium, considered the optimal one for this type of culture, allowed us to study the heterogeneous behavior of the epithelial cells in culture. Using the plasma clot culture method, we were able to demonstrate in vitro fibroblastic transformation of the epithelial cells. Histopathological findings of particular cases of posterior capsule opacification and immunohistochemistry of the human lens are also reported.

Nuclear morphometry as an important prognostic factor in stage I renal cell carcinoma

Although 60% of Stage I renal carcinoma patients die from tumor within 5 years postoperatively, a considerable percentage survive that period. Nuclear grading can help to predict the outcome, but many of the patients are Grade 2, and the prognosis of this subclass is uncertain. Therefore, nuclear morphometry was carried out in 41 patients with Stage I renal cell carcinoma. Of these, 24 died within 5 years and 17 have survived that period. Using a mean nuclear area of 32 μm2 as the decision threshold, none of the 24 short‐term survivors are below that threshold and three of the long‐term survivors exceed that value (18% false‐positives) (99% confidence limit). Separate analysis with sets for learning and testing and Grade 2 patients gave similar results. The results show the essential prognostic value of morphometry in this set of patients with Stage I renal cell carcinoma.

An interlaboratory study to determine the presence of human papillomavirus DNA in esophageal carcinoma from China

Esophageal‐carcinoma samples originating from the high‐incidence area of China were tested in 2 different laboratories, each using a different degenerate PCR approach. Results confirmed the notion that none of the PCR approaches available for HPV‐DNA detection today, is optimal for detecting all known HPV types at equal sensitivity and specificity. In combining results obtained in both laboratories, HPV DNA was demonstrated in 20/117 (17.1%) esophageal‐carcinoma samples analyzed. HPV DNA was detected in 3/70 (4.3%) diagnostic biopsies, 7/23 (30.4%) surgical specimen and 10/24 (41.6%) cytological scrapings originating from the entire surface of the esophagus. Mucosotropic HPV types were present in 7/117 (6%) samples, only 3 being of the high‐risk types (HPV 16, 18, 33). Other mucosal types found were HPV 6, 11, 13, 53 and 54. Cutaneous HPV types were present in 14/117 (12.0%) samples. HPVs 20 and 38 were present in 3 (2.6%) of the total samples and, in each case, together with another HPV type within one lesion. Two putative new HPV types, DL347 and DL 369, were identified. Int. J. Cancer 81:225–228, 1999.

Neoplastic cells of Hodgkin's disease show differences in EBV expression between Kenya and Italy

The Epstein‐Barr Virus (EBV) has been implicated in the pathogenesis of Hodgkins disease (HD). However, the association of EBV with this disease varies greatly from series to series and from country to country. Epidemiological studies have shown differences in HD occurring in different parts of the world. In particular, it has been reported that HD in developing countries differs from HD in Western countries in terms of epidemiological, pathological and clinical characteristics. These discrepancies among populations suggest an interaction with environmental factors and a direct role of different etiological agents. At present, there are no data on the frequency of association of EBV with HD in equatorial Africa. In this study, a large series of HD cases have been collected at the University of Nairobi, Kenya, and at the Universities of Bologna and Siena, Italy. The cases have been reviewed and classified according to the REAL Classification and the presence of EBV has been assessed by in situ hybridization (ISH). A statistical difference in EBV expression was found between HD from Kenya and HD from Italy. EBV‐positive neoplastic cells were detected in 92% of Kenyan cases, whereas only 48% of Italian cases showed EBER1/2 positivity in the neoplastic cells. Our results suggest that, in Kenya, EBV plays a more direct role in the pathogenesis of HD, as it does for endemic Burkitt lymphoma.

HIV-associated malignant lymphomas in Kenya (Equatorial Africa)☆

The clinical and pathological features of acquired immune deficiency syndrome (AIDS)-related lymphomas, including their relationship with other viruses, such as Epstein-Barr virus (EBV) and human herpes virus-8 (HHV8), have been the subject of several studies from North America and Europe. No consistent data have been reported in Africa, where AIDS runs an epidemiological and clinical course different from that observed in Western countries. We retrospectively evaluated the presence of human immunodeficiency virus (HIV), HHV8, and EBV in 146 cases of malignant lymphomas collected in Kenya (Equatorial Africa), with the use of polymerase chain reaction (PCR) and in situ hybridization (ISH). The PCR technique confirmed HIV infection in 16 HIV-seropositive subjects (11%) and showed the presence of HIV sequences in five additional cases (3%) in which the occurrence of lymphoma was the only clinical manifestation. Our findings suggest that AIDS-related lymphomas are not pathogenetically homogenous, and different mechanisms may contribute to lymphomagenesis in these severely immunocompromised patients. In our series, no association of Hodgkins disease (HD) with HIV infection could be shown. Among non-HIV-related lymphomas, EBV was present in 94% of Burkitt lymphoma (BL) occurring in patients younger than 15 years of age, in 87% of HD independently of age, sex, and histological types, in 60% of anaplastic large cell lymphoma (ALCL), and to a lesser extent (13%) in large B-cell lymphoma (LBCL) cases. Only one tumor, a case of HD, showed HHV8 by PCR.