Riccardo Dolcetti

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.

Hodgkin's disease and human immunodeficiency virus infection: clinicopathologic and virologic features of 114 patients from the Italian Cooperative Group on AIDS and Tumors.

PURPOSE To describe virologic, clinicopathologic, and therapeutic features of a large series of Italian patients with Hodgkins disease (HD) and human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS From November 1986 to March 1994, 114 cases were observed. The relationship between Epstein-Barr virus (EBV) and HD was determined by an in situ hybridization technique, immunostaining for EBV-encoded latent membrane protein-1 (LMP-1) expression, and Southern blotting. Twenty-six patients were included in a prospective study evaluating the combination of chemotherapy (CT) with zidovudine. RESULTS Combined approach on EBV study revealed that 14 (78%) of 18 patients were EBV-associated. An almost equivalent distribution of EBV subtypes was observed in EBV-carrying cases, indicating that in the HIV setting, type 2 EBV also may be pathogenetically involved in HD development. In comparing these 114 patients with our single-institutional series of 104 HIV-negative patients with HD, we observed at presentation a younger median age (29 v 38 years); a prevalence of males (90% v 56%); and a higher percentage of stage IV disease (52% v 15%), presence of B symptoms (77% v 35%), and extranodal disease (63% v 29%). The complete remission (CR) rate (58%) and median survival (13 months) of patients treated prospectively were similar to that of patients treated with standard CT regimens. The statistically significant favorable prognostic factors for survival being the following: achievement of CR, CD4+ count greater than 250/microL, and no prior diagnosis of AIDS at onset of HD. CONCLUSION Our virologic findings indicate that HIV-related HD is more closely associated with EBV than HD in the general population. The peculiar clinicopathologic findings, the role of some prognostic factors, and the possibility of cure of HIV-related HD have been demonstrated.

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.

Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma

Functional characterization of signaling pathways that critically control mantle cell lymphoma (MCL) cell growth and survival is relevant to designing new therapies for this lymphoma. We herein demonstrate that the constitutive activation of Akt correlates with the expression of the phosphorylated, inactive form of PTEN. Phosphatidyl-inositol-3 kinase (PI3-K)/Akt or mammalian target of rapamycin (mTOR) inhibition decreased the growth of both primary MCL cultures and established cell lines and antagonizes the growth-promoting activity of CD40 triggering and IL-4. These effects are mediated by nuclear accumulation of the p27(Kip1) inhibitor induced by down-regulation of the p45(Skp2) and Cks1 proteins, which target p27(Kip1) for degradation. Moreover, Akt inhibition down-regulated cyclin D1 by promoting its proteasome-dependent degradation driven by GSK-3. Intriguingly, mTOR inhibition affected cyclin D1 proteolysis only in MCL cells in which GSK-3 is under the direct control of mTOR, suggesting that different MCL subsets could be differently responsive to mTOR inhibition. Finally, PI3-K/Akt inhibitors, but not rapamycin, induced variable levels of caspase-dependent apoptosis and reduced telomerase activity. These results indicate that Akt and mTOR activation have distinct functional relevance in MCL and suggest that targeting Akt may result in more effective therapeutic effects compared with mTOR inhibition.

Chlamydophila Psittaci Eradication With Doxycycline As First-Line Targeted Therapy for Ocular Adnexae Lymphoma: Final Results of an International Phase II Trial

PURPOSE The pathogenic association between Chlamydophila psittaci (Cp) and ocular adnexal marginal zone lymphoma (OAMZL) and the efficacy of doxycycline monotherapy have been investigated in retrospective series with variations in stage, management, and follow-up duration. To our knowledge, this is the first international phase II trial aimed at clarifying Cp prevalence and activity of first-line doxycycline in a homogeneous series of consecutive patients with newly diagnosed stage I OAMZL. PATIENTS AND METHODS Forty-seven patients were registered. Tumor tissue, conjunctival swabs, and peripheral blood from 44 patients were assessed for seven Chlamydiaceae infections by three polymerase chain reaction protocols. Thirty-four patients with measurable or parametrable disease were treated with doxycycline and assessed for chlamydial eradication and lymphoma response (primary end point). RESULTS Cp DNA was detected in biopsies of 39 patients (89%); no other Chlamydiaceae were detected. Twenty-nine patients had Cp DNA in baseline swabs and/or blood samples and were evaluable for chlamydial eradication, which was achieved in 14 patients (48%). Lymphoma regression was complete in six patients and partial in 16 (overall response rate, 65%; 95% CI, 49% to 81%); 11 had stable disease, and one had progressive disease. At a median follow-up of 37 months (range, 15 to 62 months), 20 patients remained relapse free (5-year progression-free survival [PFS] ± standard deviation, 55% ± 9%). Cp eradication was associated with improved response rate (86% v 47%; P = .02) and 5-year PFS (68% v 47%; P = .11). CONCLUSION Upfront doxycycline is a rational and active treatment for patients with stage I Cp-positive OAMZL. Lymphoma regression is consequent to Cp eradication, which can easily be monitored on conjunctival and blood samples. Prospective trials aimed at identifying more effective administration schedules for doxycycline are warranted.

Chlamydia Infection and Lymphomas: Association Beyond Ocular Adnexal Lymphomas Highlighted by Multiple Detection Methods

Purpose:Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamydia detection tools to identify Cp elementary bodies–containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. Experimental Design: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. Results: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact Cp elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyers ring. Conclusions: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated.

Simian-virus-40 footprints in human lymphoproliferative disorders of HIV- and HIV+ patients

SV40 sequences were investigated by PCR DNA amplification followed by filter hybridization in a series of human lymphoproliferative disorders obtained from human‐immunodeficiency‐virus (HIV)‐seronegative and HIV‐infected patients. Our PCR and filter‐hybridization conditions enabled us to detect SV40 sequences in the range of 10−4 to 10−2 genome equivalents per cell. In non‐Hodgkins lymphomas (NHL) from HIV− patients, SV40 footprints were found in 11 out of 79 (13.9%) samples, while in NHL from HIV+ patients SV40 DNA sequences were detected in 2/16 (12.5%). In Hodgkins disease (HD), SV40 sequences were found in 7/43 (16.3%) and 1/12 (8.3%) in HIV− and HIV+ patients respectively. A slightly higher prevalence of SV40 footprints was observed in reactive lympho‐adenopathies both in HIV− (3/9, 33.3%) and in HIV+ (6/17, 35.3%) patients. Sequence analysis of 2 NHL and 2 HD DNA samples established that the amplified PCR products belong to the SV40 sequences. SV40 prevalence and load were similar in samples from HIV‐seronegative and HIV‐infected individuals, suggesting that SV40 probably does not undergo strong reactivation phenomena in the context of HIV‐related immunosuppression. Moreover, the large T‐antigen(Tag) expression was detected by immunohistochemistry in 5/18 SV40‐DNA‐positive samples analyzed; however, few tumor cells (<1%) in 3/5 samples displayed positivity for SV40 Tag, while this viral oncoprotein was revealed in several reactive histiocytes present in all 5 SV40‐positive tissues. These results suggest that the lymphoid tissue could represent a reservoir for SV40 and may constitute the first step in understanding whether this DNA tumor polyomavirus has a role in the pathogenesis of human lymphoproliferative disorders. Int. J. Cancer 78:669–674, 1998.

Pathogenetic and histogenetic features of HIV-associated Hodgkin's disease

Compared with the cases in the general population, Hodgkins disease (HD) arising in the HIV setting shows distinctive features in terms of epidemiology, aetiopathogenesis, histopathology and clinical behaviour. Although HD does not represent an AIDS-defining condition, recent evidence consistently indicates that HIV-infected individuals have a significantly increased risk of developing HD. HIV-related HD is characterised by the preponderance of aggressive histological subtypes, advanced stage at presentation, and highly malignant clinical course. Moreover, unlike HD in the general population, the large majority of HIV-related HD cases are pathogenetically linked to Epstein-Barr virus (EBV), with rates of EBV positivity ranging from 80 to 100%. Hodgkin and Reed-Sternberg cells of these cases invariably show a strong expression of the EBV-encoded latent membrane protein-1 (LMP-1), which functions as a constitutively activated tumour necrosis factor (TNF) receptor-like molecule. Usurpation of physiologically relevant pathways by LMP-1 may lead to the simultaneous or sequential activation of signalling pathways involved in the promotion of cell activation, growth, and survival, contributing thus to most of the features of HIV-related HD.

Lymphomas occurring specifically in HIV-infected patients: from pathogenesis to pathology.

Lymphomas that develop in HIV positive patients are predominantly aggressive B-cell malignancies. The most common HIV-associated lymphomas are Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Lymphomas that occur specifically in HIV positive patients include primary effusion lymphoma (PEL) and its solid variants, plasmablastic lymphoma of the oral cavity type and lymphoma associated with Kaposi sarcoma herpesvirus (KSHV)-related multicentric Castleman disease. These lymphomas, together with BL and immunoblastic lymphoma subtypes with plasmacytoid differentiation, carry Epstein-Barr virus (EBV) infection and display a phenotype related to plasma cells. Globally, EBV is identified in the neoplastic cells of approximately 40% of HIV-associated lymphomas, but the detection of EBV varies considerably with the site of presentation and the histological subtype. EBV infection occurs in 80-100% of primary central nervous system lymphomas and PELs, 80% of DLBCLs with immunoblastic-plasmacytoid features, and 30-50% of BL-plasmacytoid. KSHV is specifically associated with PEL, which usually occurs in a setting of profound immunosuppression. Current knowledge about HIV-associated lymphomas can be summarized as follows: (1) lymphomas specifically occurring in patients with HIV infection are closely linked to other viral diseases; (2) most of these lymphomas exhibit plasmablastic differentiation.