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Purpose: There is evidence that Hodgkin Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) could display some molecular and morphologic markers of cellular senescence (CS). We hypothesized that CS mechanisms may have potential prognostic relevance in cHL and investigated whether the expression of the well-established CS biomarkers p21(CIP1/WAF1) and p16(INK4a) by HRS cells might be predictive of the probability of event-free survival (EFS)., Experimental Design: The study analyzed a retrospective cohort of 147 patients and the results were validated on a cohort of 91 patients independently diagnosed and treated in a different institution. p16(INK4a) and p21(CIP1/WAF1) were categorized as dichotomous variables (< or ≥ 30% of HRS cells at diagnosis) and evaluated in univariate and multivariate analysis., Results: Both molecules were independent prognostic factors. A positive staining of one of the two molecules in more than 30% HRS cells predicted a better EFS (P < 0.01). p16(INK4a)/p21(CIP1/WAF1) together as a unique categorical variable (both <30%, either <30%, both ≥ 30%) sorted out three prognostic groups with better, intermediate, or worse outcome either overall or within I-II, bulky and advanced stages. The presence or the lack of the robust expression of p21(CIP1/WAF1) and/or p16(INK4a) defined the prognosis in our series., Conclusions: These findings point to (i) the relevance of CS-related mechanisms in cHL, and to (ii) the prognostic value of a simple, reproducible, and low-cost immunohistochemical evaluation of p16(INK4a) and p21(CIP1/WAF1) expression., (©2015 American Association for Cancer Research.)

Primary mediastinal large B-cell lymphoma is a well-defined lymphoma entity whose molecular pathogenesis is incompletely understood and also lacking well-established diagnostic markers. Recently, the presence of overlapping features between classical Hodgkin's lymphoma and primary mediastinal large B-cell lymphoma was highlighted by gene expression profiling as well as morphological studies. We investigated the expression of TP73L (commonly known as p63) isoforms in primary mediastinal large B-cell lymphoma at both protein and mRNA level, and demonstrated the exclusive presence of transactivating (TA) isoforms in all cases. We also demonstrated that TP73L is expressed in a subset of germinal center B-cells, as well as in some diffuse large B-cell lymphomas, but it is never present in classical Hodgkin lymphoma. Nodular lymphocyte predominant Hodgkin's lymphoma also showed TP73L positivity by immunohistochemistry. Isoform analysis by real-time PCR showed that TA-TP73Lalpha is the most represented in primary mediastinal large B-cell lymphoma, but TA-TP73Lgamma is the most differentially expressed in comparison to both germinal center B-cells and diffuse large B-cell lymphomas. TP73L expression proved a useful diagnostic marker of primary mediastinal large B-cell lymphoma, and gave new insights in to the molecular pathways playing a role in this lymphoma., (Modern Pathology (2005) 18, 1448-1453. doi:10.1038/modpathol.3800440; published online 13 May 2005.)

Purpose: EBV-latent membrane protein-1 (LMP-1) is often expressed in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's lymphoma (cHL), but its clinical significance is controversial. We correlated LMP-1 with presenting features, including serum interleukin 10 levels and clinical outcome., Experimental Design: Patients were eligible if they had biopsy-proven cHL, were untreated, HIV-1 negative, and had available archival tissue. LMP-1 expression was determined by immunohistochemistry., Results: We identified 577 patients with cHL with a median age of 30 years, 55% of whom were male. LMP-1 was expressed in HRS cells of 124 patients (21%) and was detected in 78 of 461 (17%) patients with nodular sclerosis compared with 44 of 112 (39%) with mixed cellularity (P < 0.001 by Fisher's exact test). Patients with tumors with LMP-1-positive HRS cells had higher serum interleukin 10 levels (P = 0.009 by Mann-Whitney test). For the 303 patients treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens, the 5-year failure-free survival (FFS) for those with LMP-1-positive tumors was 74% compared with 81% for those with LMP-1-negative tumors (P = 0.23, by log-rank test). Overall survival (OS) at 5 years for patients with LMP-1-positive tumors was 90 versus 91% for patients with LMP-1-negative tumors (P = 0.8 by log-rank test). Expression of LMP-1 was not associated with different FFS and OS in patients treated with other regimens or with radiotherapy alone., Conclusions: LMP-1 was expressed by HRS cells in 21% of cHL and correlated with mixed cellularity type and higher serum interleukin 10 levels. The presence of LMP-1 was not associated with FFS or OS in uniformly treated patients.

To determine the clinical significance of BCL-2 expression in Hodgkin-Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD), we correlated its expression with presenting clinical and laboratory features and failure-free survival (FFS). Eligible patients were untreated and negative for HIV-1; they had biopsy-proven cHD. BCL-2 expression was determined immunohistochemically in available pretreatment tissue biopsy specimens without knowledge of clinical outcome. Tumors were considered positive if any HRS cells expressed BCL-2. We identified 707 patients with cHD, whose median age was 30 years; 54% were men. HRS cells expressed BCL-2 in 359 (65%) of 551 nodular sclerosis, 67 (47%) of 143 mixed cellularity, and all 5 lymphocyte depletion. For all patients, the 5-year FFS was 74% versus 84% for tumors with versus without BCL-2 expression (P =.0016, by log-rank test). For the 412 patients treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens, the 5-year FFS for tumors with versus without BCL-2 expression was 74% versus 88% (P =.001, by log-rank test); for the 233 patients with Ann Arbor stage I or II, FFS was 84% versus 92% (P =.04, by log-rank test); and for the 179 patients with Ann Arbor stage III or IV, FFS was 62% versus 81% (P =.006, by log-rank test). Multivariate analysis confirmed that BCL-2 expression is independently associated with inferior FFS along with age 45 or older, Ann Arbor stage IV, low serum albumin and high serum lactate dehydrogenase levels. We conclude that BCL-2 is frequently expressed by HRS cells in cHD and is associated with inferior FFS in patients treated with ABVD or equivalent regimens.

Purpose: CD20 can be expressed in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's disease (HD), but its clinical significance remains controversial. Therefore, we correlated CD20 expression with presenting features and clinical outcome of untreated patients with classical HD., Patients and Methods: Patients were eligible if they were previously untreated and human immunodeficiency virus-1 negative, had biopsy-proven classical HD, and if pretreatment paraffin-embedded tumor tissue was available. CD20 expression was determined by immunohistochemistry without knowledge of clinical outcome. A tumor was considered positive if any HRS cells expressed CD20, but other cutoffs for number of CD20-positive HRS were also investigated., Results: We identified 598 patients whose median age was 30 years and of whom 55% were male. HRS cells expressed CD20 in 132 (22%) of 598 patients with classical HD. When any percentage of CD20 expression in HRS cells was used as a cutoff, the 5-year failure-free survival (FFS) for positive versus negative tumors was 86% versus 84%, respectively, for 302 patients treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens (P =.7 by log-rank test), 74% versus 77%, respectively, for 181 patients treated with mitoxantrone, vincristine, vinblastine, and prednisone and radiotherapy (P =.7 by log-rank test), 74% versus 84%, respectively, for 54 patients treated with MOPP (P =.4 by log-rank test), and 77% versus 88% for 53 patients treated only with radiotherapy (P =.5 by log-rank test). The 5-year FFS was not statistically different when cutoffs of 5% up to 50% for CD20-positive HRS cells were used., Conclusion: CD20 is expressed by HRS cells in 22% of patients with classical HD but is not associated with different FFS after treatment with equivalent regimens.

Purpose: BAX, a proapoptotic member of the BCL-2 family of proteins, has been detected in Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin's disease (HD), but its clinical significance is unknown. Therefore, we correlated BAX expression with presenting features and clinical outcome in untreated patients with HD., Design: Patients with biopsy-proven HD were eligible if they were untreated previously and if pretreatment paraffin-embedded tumor tissue was available. BAX was detected by immunohistochemistry without knowledge of clinical features or outcome. A tumor was considered as positive if any number of HRS cells expressed BAX, but other cutoffs of BAX expression were examined for analysis of clinical outcome., Results: We identified 260 patients with HD. The median age was 31 years, and 55% were male. HRS cells expressed BAX in 181 of 195 (93%) nodular sclerosis, 47 of 48 (98%) mixed cellularity, 1 case of lymphocyte depletion, all 6 unclassified classical HD, and all 10 lymphocyte predominance tumors. Using a cutoff of 50% positive HRS cells for BAX expression, the 5-year failure-free survival (FFS) for patients with high versus low BAX expression was 83 versus 93%, respectively (P = 0.19 by Log-rank) for 116 patients treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens; it was 78 versus 79%, respectively, for 79 patients treated with mitoxantrone, vincristine, vinblastine, and prednisone and radiotherapy (P = 0.45 by Log-rank); it was 71 versus 81%, respectively, for 26 patients treated with nitrogen mustard, vincristine, prednisone, and procarbazine (P = 0.6 by Log-rank); and it was 72 versus 82% for 29 patients treated only with radiotherapy (P = 0.57 by Log-rank). The 5-year FFS was not statistically different when we used cutoffs of 20, 30, and 75% for BAX expression., Conclusion: BAX is often expressed by HRS cells in HD and does not correlate with FFS.

Preliminary reports suggested a prognostic significance for serum levels of soluble CD30 (sCD30) in patients with Hodgkin's disease (HD). In this study, we investigated the prognostic impact of sCD30 concentration at diagnosis in relation to the other recognized prognostic parameters in 303 patients with HD observed in three different institutions between 1984 and 1996. sCD30 levels were correlated with stage, presence of B symptoms, and tumor burden. High sCD30 levels entailed a higher risk of poor outcome, and the event-free survival (EFS) probability at 5 years for patients with sCD30 levels >/=100 and less than 100 U/mL was 59.9% (95% confidence interval [CI], 40.6% to 65.9%) and 87.5% (95% CI, 81.5% to 91.6%), respectively (P < .001). On the basis of the results of univariate analysis of 14 pretreatment characteristics, we included five prognostic factors (high sCD30 serum level, stage III-IV, B symptoms, low hemoglobin level, and age >/=50 years) into a multivariate model. High sCD30 and advanced stage were independently associated with an unfavorable prognosis. Their combined evaluation identified patients at high risk (stages III and IV and sCD30 >/=100 U/mL: EFS, 46.9%) and low risk (stages I and II with sCD30 <100 U/mL: EFS, 88. 7%) of treatment failure (P < .001). We conclude that the combined evaluation of sCD30 serum level and stage at presentation identifies patients with HD at high risk of an unfavorable outcome.

Hodgkin's disease (HD) is characterised by a complex architectural and functional derangement of involved tissues. The interactions between neoplastic cells and the heterogeneous microenvironment lead to the expression and release of different cellular messengers [cytokines, soluble (s) forms of cytokine receptors and other membrane-associated molecules] which can be detected in the circulation and evaluated as biological markers. We and others investigated several of these molecules looking for their possible role as diagnostic or prognostic parameters in patients with HD. We update here the results of serum determination of sIL-2Ralpha, sCD8, sICAM-1, sTNFRs, and sCD30 in a large series of cases from our institution. We found that their levels are generally increased at presentation and during the active phase of the disease. They correlate with stage and clinical aggressiveness and have some prognostic implication. However, we were unable to demonstrate a prognostic usefulness for their detection, with the exception for sCD30 which was found to directly correlate with disease spread and burden at presentation and, most importantly, to have an independent prognostic significance. The prognostic significance of sCD30 might derive from a crucial involvement of this molecule in the pathophysiology of HD.

The precise cellular origin and the pathogenetic mechanism(s) leading to the neoplastic transformation of anaplastic large cell lymphoma (ALCL) and the Reed-Sternberg cell of Hodgkin's disease (HD) remains largely uncertain. Classical cytogenetic analysis has shown a unique translocation involving bands 2p23 and 5q35 bands in a variable number of ALCLs. It has been recently shown that the nucleophosmin/B23 (NPM) gene (5q35) and a novel anaplastic lymphoma kinase (ALK; 2p23) are the fused genes of t(2;5). To investigate the presence and the precise frequency of NPM-ALK gene products among ALCL and HD cases, a large and well-characterized panel of ALCL (n = 49) and HD (n = 72) cases was studied using multiple strategies including reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot analysis, and immunohistochemistry. Overall, 6 (3 T and 3 null) of 49 ALCL and 3 (2 nodular sclerosis and 1 mixed cellularity) of 72 HD showed the presence of NPM-ALK transcripts by RT-PCR. NPM-ALK gene rearrangements were detected in all RT-PCR, NPM-ALK-positive ALCL by Southern blot analysis. Furthermore, in all the available cases we were able to show the presence of ALK-related protein using a specific polyclonal antiserum recognizing the cytoplasmic domain of ALK by immunohistochemistry. Our data show that NPM-ALK gene transcripts are identified in a subpopulation of ALCL, almost exclusively in T or null cell in origin, and in rare cases of HD. These findings show that some HD may be closely related to ALCL, giving us new insights on the pathogenesis and possibly biologic evolution of HD.

After many years of frustrating discussion concerning the nature of Hodgkin's and Reed-Sternberg cells and their role in the pathogenesis of Hodgkin's disease, interesting new information has recently been gathered through the use of new technologies. In light of these studies, Hodgkin's disease emerges as a heterogeneous disorder in which some separate new entities may be elucidated, such as nodular lymphocyte predominance Hodgkin's disease and anaplastic large-cell CD30+ lymphoma. In this review, we focus on the morphologic, phenotypic, molecular, and clinical features that have served to partially clarify controversial issues.

The neoplastic nature of Hodgkin's disease (HD) is suggested by several lines of evidence, including aggressive clinical course, presence of proliferating atypical cells morphologically recognized as Hodgkin's and Reed-Sternberg cells (H-RS), aneuploidy, and, in the minority of cases, clonality. Nevertheless, the etiopathogenesis of HD still remains elusive, and probably diverse. This uncertainty is partly due to the peculiar histology of HD lesions, characterised by the paucity of the putative neoplastic cell component, i.e. H-RS cells, admixed to a variety of reactive cells which prevent an exhaustive investigation at molecular level. Nevertheless, the possible involvement of different molecules with oncogenic potential has been recently suggested on the basis of immunohistological and molecular biology studies. These include oncogenes such as bcl-2 and MDM2 and anti-oncogenes such as p53. In addition, a large amount of data has accumulated on the possible role of EBV infection in HD. The colonization of lymphoid tissues by immortalized H-RS cells can account for the derangement of cytokine networks leading to microenvironmental and systemic abnormalities. In addition, a variety of soluble receptors (sIL-2R, sCD30, sTNF-R), and adhesion molecules (sICAM-1) are abnormally produced at sites of disease involvement. Some of these molecules still retain the ability to bind their ligands and can potentially contribute to the derangement of immune mechanisms observed in HD. Many of these soluble molecules can also be found in the patient's sera providing new potential prognostic and follow-up parameters in HD patients. The comparative analysis of the same molecules within the tissue, using immunohistochemistry, and in the blood, using immunochemical assays, appears as a promising informative approach.

Previous reports have suggested soluble intercellular adhesion molecule-1 as a marker of disease activity in Hodgkin's disease. In the present study we investigated serum levels of intercellular adhesion molecule-1 at diagnosis in 104 patients with Hodgkin's disease and in 77 of these patients following the achievement of complete remission (within 12 months of diagnosis). Mean serum levels at diagnosis were significantly higher in patients than in controls (P < 0.0001) and were related to advanced stages of disease (P = < 0.0001), presence of "B" symptoms (P < 0.0001), abnormality of laboratory indexes (P < 0.0001), erythrocyte sedimentation rate values (r = 0.41, P < 0.0001) and serum levels of soluble interleukin-2 receptor alpha chain (r = 0.51, P < 0.0001). Mean values in complete remission were significantly lower than at diagnosis (P = 0.003). Lower mean values at diagnosis were detected in 30 patients with advanced disease who attained complete remission, compared with 6 patients who failed to attain complete remission with standard treatment. We conclude that in Hodgkin's disease, high serum levels of soluble intercellular adhesion molecule-1 are detectable at presentation and strictly correlate with some clinical features. Response to treatment is paralleled by reduced serum levels. Larger prospective studies are needed to evaluate the possible prognostic significance of serum levels of soluble intercellular adhesion molecule-1 at diagnosis.

The possible involvement of p53 tumor suppressor gene in the pathogenesis of Hodgkin's disease (HD) is suggested by the frequent finding of abnormal accumulation of p53 protein in the nuclei of Reed-Sternberg cells and their variants (H-RS) in a large proportion of cases. This finding, besides being consistent with the presence of p53 gene mutations, might represent a consequence of the inactivating interaction between p53 and p53-binding proteins such as the product of the MDM2 cellular oncogene. We have examined an unselected series of 77 HD cases of different histologic patterns for the expression of p53 and MDM2 proteins, using specific monoclonal antibodies and sensitive immunohistochemical techniques in single- and double-marker combination. In the large majority of cases (66/77), a variable proportion of H-RS cells expressed MDM2 that was strictly confined to the nuclei. Coexpression of both MDM2 and p53 was common in the same cells. The abnormal nuclear expression of p53 and MDM2 did not seem to correlate with the presence of Epstein-Barr virus infection, as shown by the results of LMP-1 antigen expression and EBER in situ hybridization analysis. Our data suggest that the abnormal accumulation of MDM2 and p53 proteins in HD might reflect a derangement of molecular mechanisms that could play a pathogenetic role in this disease.

Background: Previous studies suggested a possible role for the detection of soluble interleukin-2 receptor (sIL-2R) in Hodgkin disease (HD). In this study, the authors investigated, in a large series of patients, sIL-2R serum levels in relation to disease features at presentation and prognosis. Their usefulness as markers in the management of individual cases was evaluated., Methods: The sIL-2R serum levels were measured in 195 patients at diagnosis. In 72 of these patients, sIL-2R serum levels were also monitored after diagnosis. An additional 87 cases were tested only in complete remission (CR), and 25 were tested only at relapse., Results: The sIL-2R levels at diagnosis were increased (mean +/- 1222 +/- 1012 versus 331 +/- 145 U/ml in controls, P < 0.0001) and correlated with the stage and tumor burden (Stages I and II = 1058 +/- 1007, Stages III and IV = 1502 +/- 942 U/ml, P = 0.003; Stage A = 954 +/- 705, Stage B = 1880 +/- 1238 U/ml, P < 0.0001; bulky presentation = 1958 +/- 1430, nonbulky presentation = 1043 +/- 791 U/ml, P < 0.0001). Response to treatment was associated with progressive reduction of sIL-2R levels, which were normal in virtually all cases 1 year after CR. Significantly greater levels at diagnosis were found in 11 patients who experienced a poor response or progression after treatment (P = 0.004). Overall, abnormal data in CR were found in 59 of 159 patients and 9 of them subsequently experienced a relapse., Conclusions: The sIL-2R serum levels in HD correlate with features at presentation and subsequent clinical courses. Higher levels at diagnosis entail a significantly higher risk of treatment failure.

In this paper a multimarker immunohistochemical technique is described in detail which allows the quantitative evaluation in situ of cells expressing abnormal accumulation of p53 phosphoprotein together with the CD30 antigen, a well known marker of Hodgkin's and Reed-Sternberg cells. The method is useful to define quantitatively the proportion of phenotypically abnormal cells in the complex microenvironment of Hodgkin's lesions, and to compare the phenotypical derangement of p53 gene with molecular biology data obtained on the same samples.

We investigated ICAM-1/CD54 tissue immunoreactivity and serum levels of its soluble form (sICAM-1) in patients with Hodgkin's disease (HD) at diagnosis. ICAM-1 was strongly expressed in involved tissues, and sICAM-1 serum levels were higher in HD (79 patients) than in controls (P < 0.01), and in patients with more advanced or more active disease (stages III + IV vI + II: P = 0.002; stage 'B' v 'A': P < 0.0001; 'bulky' disease v non-'bulky': P = 0.042). We suggest that tissue ICAM-1 overexpression leading to increase of circulating sICAM-1 may interfere with the lymphocyte adhesion machinery thus contributing to the well-known immune derangement of HD.

The gene encoding p53 phosphoprotein, originally believed to be an oncogene, recently has been proposed as a candidate antioncogene (tumor-suppressor gene). Abnormalities of the p53 gene expression have been demonstrated in different human malignancies including carcinomas and sarcomas, but little information concerning p53 immunoreactivity in human lymphomas is so far available. In this study immunohistochemical staining for p53-protein was performed on frozen- and paraffin-embedded samples from patients with Hodgkin's (HD) and non-Hodgkin's lymphomas (NHL). No p53 immunoreactivity could be demonstrated in any cell type in nonneoplastic lymphoid samples, including germinal center cells in reactive lymph nodes and cortical thymocytes. On the other hand, a significant proportion of p53+ neoplastic cells was observed in 23 of 31 cases of HD and 17 of 68 cases of NHL. All positive lymphoma cases were diagnosed as high-grade or CD30+ anaplastic NHL. The demonstration of abnormal expression of p53 protein in these diseases can contribute to addressing unresolved issues regarding the origin and pathogenesis of HD and CD30+ anaplastic lymphomas.

In all cases of Hodgkin's disease (HD) Reed-Sternberg (RS) cells express the CD30 antigen. It has been recently demonstrated that this molecule can be released from the cell membrane of CD30+ neoplastic cells in a soluble form (sCD30), detectable in culture supernatants and body fluids. In this paper we investigated by an immunoassay the serum levels of sCD30 in 58 patients with HD, in order to define the possible relationship of this molecule to the clinical and pathological findings. sCD30 molecule was found at detectable levels in 24 out of 50 patients (48%) with active disease, whereas it was always absent in control sera and in cases in complete remission. Among the patients with active HD, the incidence and mean values of detectable levels (+/- SEM) of sCD30 were higher in cases with progressive or relapsing disease (61.5%, mean 458 +/- 190 U/ml) as compared to those at presentation (43.2%, mean 116 +/- 33 U/ml). Among the cases at presentation, detectable levels were observed more often in patients with advanced stages (III + IV: 61%) and constitutional symptoms ('B': 61.5%) than in early stages (I + II: 26%) and without symptoms ('A': 33.3%). In addition, higher mean values were found in stages III + IV (182 +/- 60 U/ml) and in 'B' cases (208 +/- 73 U/ml) than in stages I + II (65 +/- 30 U/ml) or 'A' patients (64 +/- 26 U/ml). The above findings suggest a possible role for the sCD30 as a tumour marker in HD.