Mauro Boiocchi

Overexpression of folate binding protein in ovarian cancers

The high affinity folate binding protein (FBP) is overexpressed in ovarian cancers. However, its role in the pathogenesis and biological behaviour of these neoplasms is not clearly understood. Using the monoclonal antibody (MAb) MOv 18 and cytofluorimetric analysis, we investigated FBP expression in frozen neoplastic tissues from 136 patients diagnosed with epithelial ovarian cancer. FBP values were compared with clinico‐pathological characteristics (age, stage, histologic grade, histologic type, DNA ploidy, percentage of S‐phase cells, and previous chemotherapeutic treatment). Some amount of FBP overexpression was observed in 122 of the 136 tumours examined. The overall mean value of FBP fluorescence index (FBP FI) was 5.6 (median 2.7; min 0.8 − max, 78.9). By univariate analysis, FBP FI was overexpressed to a higher degree in ovarian neoplasms with high histologic grade, advanced stage, serous histology, aneuploid status, and high percentage of cells in S‐phase. Of the total number (136) of cases, 106 had all the parameters assessed and were thus selected for stepwise selection procedure. The only significant independent variable was the percentage of S‐phase cells, which accounted for about 31% of variance of FBP FI. Our results indicate that FBP is associated with parameters of biological aggressiveness in ovarian cancers. Int. J. Cancer 74:193‐198, 1997.

High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability

Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI- cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI- CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 +/- 6.2 versus 4.6 +/- 4.1, P < 0.001), CD8 (13 +/- 6.4 versus 3.7 +/- 3.8, P < 0.001), and TIA-1 (11.2 +/- 6.5 versus 1.9 +/- 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI- tumors, as revealed by the expression of granzyme B (5.3 +/- 4.5 versus 0.6 +/- 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 +/- 2.1 versus 2.6 +/- 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients.

Microsatellite Instability and High Content of Activated Cytotoxic Lymphocytes Identify Colon Cancer Patients with a Favorable Prognosis

Colorectal cancers with high-frequency microsatellite instability show peculiar clinicopathological features and a favorable clinical outcome. We investigated whether the improved prognosis for these cancers is related to the content of activated cytotoxic intraepithelial T lymphocytes. Microsatellite instability and the amount of activated cytotoxic lymphocytes were analyzed according to clinicopathological features, survival, and disease recurrence in 109 right-sided colon carcinomas from 245 consecutive patients with stage II/III colon cancer that underwent radical surgery. High-frequency microsatellite instability was found in 43% of stage II/III proximal colon cancers and was associated with significantly higher numbers of activated cytotoxic lymphocytes. High-frequency microsatellite instability, as well as the content of intratumoral-activated cytotoxic T lymphocytes correlated with improved overall and disease-free survival, particularly in patients with stage III tumors. Multivariate analysis revealed that patients with both features had a risk of death and relapse markedly lower than that associated with microsatellite status or intratumoral cytotoxic lymphocytes separately. The presence of local cytotoxic immune responses is probably the major determinant of the good clinical course of patients with microsatellite unstable colon cancer. Furthermore, high-frequency microsatellite instability coupled with a high content of intratumoral cytotoxic lymphocytes may identify a subset of colon cancer patients with a favorable clinical outcome, particularly in stage III disease.

Coordinated expression and amplification of the MDM2, CDK4, and HMGI‐C genes in atypical lipomatous tumours

Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13–15. The 12q13–15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI‐C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13–15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2‐negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI‐C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI‐C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13–15 rearrangements and HMGI‐C activation and at the other end are ALTs with ring chromosomes, 12q13–15 amplification with overrepresentation of the HMGI‐C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI‐C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy. Copyright

Regression of Ocular Adnexal Lymphoma After Chlamydia Psittaci–Eradicating Antibiotic Therapy

PURPOSE Some infectious agents contributing to lymphomagenesis have been considered targets for new therapeutic strategies. Chlamydia psittaci DNA has been detected in 80% of ocular adnexal lymphomas. The present pilot study was carried out to assess whether C psittaci-eradicating antibiotic therapy is associated with tumor regression in ocular adnexal lymphomas. PATIENTS AND METHODS Nine patients with C psittaci-positive marginal-zone B-cell lymphoma of the ocular adnexa at diagnosis or relapse were treated with doxycycline 100 mg, bid orally, for 3 weeks. The presence of C psittaci DNA in peripheral-blood mononuclear cells (PBMCs) was also assessed before and after treatment in seven patients. Objective lymphoma regression was assessed 1, 3, and 6 months after therapy conclusion and every 6 months during follow-up. RESULTS All patients completed antibiotic therapy with excellent tolerability. At 1 month from doxycycline assumption, chlamydial DNA was no longer detectable in PBMCs of all four positive patients. Objective response was complete in two patients, partial response (> 50%) was observed in two patients, and minimal response (< 50%) was observed in three patients. Duration of response in the seven responders was 12+, 29+, 31+, 8+, 7+, 2+, and 1+ months, respectively. CONCLUSION C psittaci-eradicating antibiotic therapy with doxycycline is followed by objective response in patients with ocular adnexal lymphoma, even after multiple relapses of the disease. A confirmatory, large, phase II trial is warranted to confirm whether this fast, cheap, and well-tolerated therapy could replace other more aggressive strategies as first-line treatment against ocular adnexal lymphomas.

Expression of folate binding protein as a prognostic factor for response to platinum‐containing chemotherapy and survival in human ovarian cancer

Overexpression of the folate binding protein (FBP) is a common feature in epithelial ovarian cancer, but its prognostic significance is not clearly understood. We investigated whether FBP in epithelial ovarian cancer specimens is a predictor of response to chemotherapy and survival. Between 1990 and 1995, 99 patients with epithelial ovarian cancer underwent primary surgery and were treated with chemotherapeutic regimens including platinum derivatives. First‐line chemotherapy was performed in 58 patients with residual disease and in 41 patients without residual disease after primary laparotomy. FBP expression level was determined in frozen specimens by cyto‐fluorimetric assay using the MOv 18 monoclonal antibody (MAb). Association of FBP fluorescence index (FI) with clinical characteristics, response to chemotherapy, and survival was studied by univariate and multivariate analysis.

B-cell non-Hodgkin's lymphoma and hepatitis C virus infection: a systematic review.

A high prevalence of hepatitis C virus (HCV) infection in patients with B‐cell non‐Hodgkins lymphoma (B‐NHL) has been reported in some, but not all, studies, and the association showed a strong regional variation. We conducted a systematic review of the prevalence of HCV infection in case series of B‐NHL and, when an appropriate control group was available, of the odds ratio of B‐NHL associated with HCV infection. A high HCV prevalence in B‐NHL was found in southern and eastern Europe, Japan and the southern United States, but not in central and northern Europe, Canada, northern United States, or a few Asian countries. Possible sources of heterogeneity and bias are discussed. The odds ratio of B‐NHL for HCV infection was relatively weak, ranging from 2 to 4 in most studies. Thus, even if the observed association were causal, the percentage of cases of B‐NHL attributable to HCV infection would be relatively low (10%) also in countries with a high prevalence of HCV infection in the general population, and extremely low in other countries. This may explain apparent inconsistencies between studies. Potential mechanisms of action are also discussed.

Effect of methylenetetrahydrofolate reductase 677C-->T polymorphism on toxicity and homocysteine plasma level after chronic methotrexate treatment of ovarian cancer patients.

MTHFR is a critical enzyme that regulates the metabolism of folate and methionine, both of which are important factors in DNA methylation and synthesis. Subjects with the 677C→T variant have impaired remethylation of Hcy to methionine that could determine hyperhomocysteinemia. Remethylation of Hcy into methionine and DNA methylation are also affected by MTX treatment. Thus, a combined effect between MTX and reduced activity of the MTHFR 677C→T polymorphism could occur, leading to toxicity. In a clinical trial, 43 ovarian cancer patients were treated with low doses of MTX. During MTX therapy, 12 patients (27.9%) developed G3/4 WHO toxicity. In these 12 patients, we observed 6 G3/4 thrombocytopenias, 1 G3 neutropenia, 1 G3 anemia, 9 G3 mucositis cases and 1 G4 mucositis case. A significant association was observed between toxicity and TT MTHFR 677 genotype (p < 0.0001). G3/4 toxicity occurred in 10 of 13 (77%), 1 of 17 (6%) and 1 of 13 (8%) patients with the TT, CT and CC MTHFR genotypes, respectively. According to the logistic regression model, patients with the TT genotype had a relative risk of 42.0 (95% CI 4.2–418.6) of developing G3/4 toxicity compared to patients with the CC and CT genotypes. Patients with the TT genotype had Hcy plasma levels after MTX therapy significantly (p = 0.0001) higher than basal levels (mean ± SD = 16.71 ± 4.72 vs. 12.48 ± 3.57 μmol/l); moreover, they also had higher Hcy plasma levels after MTX than patients with other MTHFR 677 genotypes (CC mean ± SD = 9.87 ± 3.61 μmol/l and CT mean ± SD = 11.48 ± 3.13 μmol/l). Finally, significant associations were observed between G3/4 WHO toxicity and higher Hcy plasma levels after MTX treatment (p = 0.0004). In conclusion, our data suggest that the TT MTHFR 677 genotype is associated with marked MTX‐induced hyperhomocysteinemia and could represent a pharmacogenetic marker for toxicity after chronic treatment with low doses of MTX.

Human immunodeficiency virus-associated systemic lymphomas may be subdivided into two main groups according to Epstein-Barr viral latent gene expression.

PURPOSE We report a pathologic characterization of human immunodeficiency virus (HIV)-associated systemic lymphomas, including the association of Epstein-Barr virus (EBV) in different categories. PATIENTS AND METHODS Eighty-seven HIV-associated non-Hodgkins lymphoma (NHL) were classified according to classic NHL classification and a recent description of morphologic variants of high-grade B-cell NHL. Seventy-one cases were immunophenotypically-genotypically characterized, whereas, in 49 representative cases, the association of EBV was assessed by nonisotopic in situ hybridization (ISH) and the immunohistochemical demonstration of latent membrane protein-1 (LMP-1). In addition, 14 Hodgkins disease (HD) cases, occurring in patients with HIV infection, were investigated for the frequency of LMP-1 expression. RESULTS Most lymphomas were of B-cell derivation and showed a blastic cell morphology, with (1) small noncleaved cells (SNCCs; 36 cases), (2) large noncleaved cells (10 cases), and (3) immunoblasts, usually polymorphic (12 cases). Moreover, 12 cases were classified as anaplastic large-cell (ALC) Ki-1-positive (Ki-1+) lymphoma. Combined ISH studies (for viral DNA and EBV RNA [EBER]) and immunohistologic demonstration of LMP-1 suggested that there were differences in viral latent gene expression between ALC Ki-1+ or immunoblastic lymphomas (usually EBV+, LMP-1+), and EBV-infected cells of SNCC lymphomas, which did not show LMP-1 expression. A high proportion (10 of 14) of LMP-1+ HD cases was found. CONCLUSION Differences in EBV association and LMP-1 expression were found between a major group of HIV-associated systemic NHL with blastic cell morphology, including SNCC lymphoma and its variants, and anaplastic cell lymphomas. A proportion of immunoblastic (polymorphic) lymphomas was different in viral latent gene expression from other blastic cell systemic lymphomas. It is concluded that only a group of these lymphomas (most ALC Ki-1+ and HD cases, along with a nonnegligible fraction of immunoblastic lymphomas) seems to be linked etiopathologically to EBV.

Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma.

OBJECTIVE Type II mixed cryoglobulinemia (type II MC) is often characterized by features of indolent B cell lymphoma (IBCL) found on pathologic examination of bone marrow, whereas the clinical evidence does not indicate a neoplastic disorder. To better address the issue of indolent malignant versus nonmalignant bone marrow lymphoproliferation underlying type II MC, molecular analyses of B cell clonality were performed in the present study, in conjunction with clinical and pathologic characterization. METHODS Polymerase chain reaction DNA amplification of immunoglobulin heavy chain genes was performed in bone marrow biopsy specimens obtained from 15 selected patients with type II MC, all infected with hepatitis C virus. Five of them had also developed overt B cell lymphoma (OBCL) during followup. Bone marrow features were consistent with IBCL in 9 of the 15 patients (group 1) and with reactive lymphoplasmacytosis in 6 of the 15 (group 2). RESULTS An oligoclonal B cell expansion was detected in 6 of 9 baseline bone marrow lesions from group 1 patients (biclonal or monoclonal expansion in the remaining 3 cases), and in 6 of 6 from group 2 patients. OBCL was always monoclonal. Selected lesions were analyzed by clonospecific hybridization and by cloning and sequence analysis in patients who had developed OBCL at followup. In 4 of 5 cases, OBCL did not originate from the dominant B cell clones that were overexpanded in the putative neoplastic baseline bone marrow lesions. OBCL clones showed significant homology with rheumatoid factor database sequences. CONCLUSION Based on the present results, as well as on evidence from previous studies of liver lesions, oligoclonal non-neoplastic B cell proliferation in the course of chronic infection-related inflammation appears to be the key feature of type II MC. Of note, molecular evidence from target tissues supports the clinical findings both at the time of type II MC diagnosis and in cases of OBCL complication. Bone marrow pathologic findings resembling those of IBCL should thus be considered in the light of clinical and molecular evidence.