Expression of the human germinal center–associated lymphoma (HGAL) protein identifies a subset of classic Hodgkin lymphoma of germinal center derivation and improved survival

Patient selection

A total of 145 informative cases of cHL, 72 from the Cleveland Clinic Foundation and 73 from the Nebraska Lymphoma Study Group, constitute this study cohort. Institutional review board (IRB) approval was obtained from all participating institutions. All patients were staged at initial diagnosis according to the modified Ann Arbor staging system. The following clinical and laboratory data from the time of diagnosis were collected: age, sex, stage, presence or absence of B symptoms or bulky disease (defined as mediastinal widening of at least one third of the thoracic diameter or a mass of at least 10 cm in maximum dimension), number of involved sites, ESR, and subtype of histology. The IPS was computed based on the following 7 designated parameters for high-risk disease: age at least 45 years, male sex, stage IV disease, white blood count (WBC) at least 15 × 10⁹/L (15 000/μL), lymphocyte count less than 0.6 × 10⁹/L (600/μL), serum albumin less than 40 g/L (4 g/dL), and hemoglobin less than 105 g/L (10.5 g/dL). Patients were categorized into a low-risk group if up to 2 adverse factors were present and a high-risk group if 3 or more adverse factors were present. In addition, patients with early stage (I and II) were stratified into favorable- and unfavorable-risk subgroups based on the number of involved regions on the same side of the diaphragm (1 to 3 or more than 3), evidence of bulky disease, and presence of B symptoms, as previously suggested.¹¹ Data on ESR at presentation were available only for a limited number of early-stage patients and thus were not used for risk stratification of patients. None of the patients had a known history of HIV infection or other forms of immunosuppression.

All patients were treated with curative intent. In general, patients with early-stage disease received radiation therapy alone or combination chemotherapy with or without radiation therapy. Patients with advanced-stage disease received combination chemotherapy with or without radiation therapy. Patients with limited-stage disease who either demonstrated resistance or relapsed following initial response to radiation therapy were retreated with combination chemotherapy. Patients who failed initial chemotherapy received salvage chemotherapy or high-dose chemotherapy with autologous stem cell transplantation. Of 120 patients treated with chemotherapy, 30% initially received ChlVPP (chlorambucil, vinblastine, procarbazine, and prednisone), 23% received ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine), 23% received MOPP (mechlorethamine, vincristine, procarbazine, and prednisone [3%] or MOPP/ABV or ABVD hybrid [20%]), and 17% received ChlVPP/ABV. Nine patients (7%) received other therapies. Follow-up information was obtained from the medical records of the Cleveland Clinic Foundation and the Nebraska Lymphoma Study Group. Availability of tissue, clinical follow-up information, and having undergone treatment with curative intent were considered inclusion criteria for this study. The studies were approved by the IRB at Cleveland Clinic and the University of Nebraska. All of the patients signed informed consent, in accordance with the Declaration of Helsinki and IRB instructions.

Tissue microarray construction

Tissue microarrays (TMAs) were constructed using a tissue arrayer (Beecher Instruments, Silver Spring, MD) by a previously described method.¹⁶ Standardized methods for tissue fixation (10% buffered formalin) and processing for paraffin-embedded sections were used at all participating centers. Tissue cores were selected for TMA by characteristic morphology based on examination of hematoxylin and eosin (H & E)–stained sections without prior knowledge of immunohistologic stains of individual cases. Two cores of 1.5 mm were taken from all paraffin blocks to maximize inclusion of Hodgkin cells on selected cores. Sections of 4 to 5 μm thickness were cut from the TMAS and placed on glass slides, which were then baked for 1 hour at 60°C before immunohistochemistry was performed.

Immunohistochemistry

Primary antibodies directed against proteins HGAL, BCL6, CD10, MUM1/IRF4, BCL2, Blimp1, LMP2A, and pSTAT6 were used in this study. The conditions used for immunohistologic staining are summarized in Table 1. Serial 4 to 5 μm–thick sections from paraffin-embedded lymphoid tissue or TMAs were deparaffinized in xylene and hydrated in a series of graded alcohols. Heat-induced antigen retrieval was carried out by microwave pretreatment in the appropriate buffer as indicated for each antibody. Detection was carried out using the DAKO Envision system (DAKO, Carpinteria, CA). Staining for all antibodies was optimized on normal paraffin-embedded tonsil sections. For the HGAL antibody, a tyramide amplification system was used (DAKO Catalyzed Signal Amplification System; DAKO). Scoring criteria as a percent of Hodgkin cells for each antibody are listed in Table 1. These cutoffs were chosen, and the scoring of the cases was performed before examining correlation with clinical end points. Cases from both institutions were scored independently by 2 observers (Y.N. and E.D.H.).

All hematoxylin-and-eosin and immunohistochemistry images were analyzed using a Nikon Eclipse E1000M microscope (Nikon, Burlingame, CA) equipped with a 40×/0.75 NA objective lens. Images were photographed using a SPOT RT color camera (Diagnostic Instruments, Sterling Heights, MI) and Ektachrome 100 Plus EPP 135-136 film (Eastman Kodak, Rochester, NY). The resulting photographs were acquired using a Nikon Superscan 4000ED scanner, and digitized images were processed using Adobe Photoshop CS (Adobe Systems, San Jose, CA).

In situ hybridization for Epstein-Barr viral RNA

In situ hybridization was performed on 4 to 5 μm–thick formalin-fixed, paraffin-embedded sections using the INFORM EBER probe (Ventana, Tucson, AZ). Slides were stained on an automated immunostainer (Ventana Benchmark) using the Ventana ISH iVIEW detection kit. Specific nuclear signal was considered positive. An oligo(dT) control to verify intact RNA within the nuclei of the cells composing the tissue and a known positive control were performed.

Induction of HGAL expression by cytokine stimulation

Peripheral-blood mononuclear cells from healthy donors were isolated by Ficoll-Hypaque density centrifugation (Amersham Pharmacia Biotech, Piscataway, NJ). B cells were enriched to more than 90% by human B-cell enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada), as determined by fluorescence-activated cell sorter (FACS) analysis. Enriched B cells were plated at 5× 10⁶ cells per well in 6-well plates (Corning Glassworks, Corning, NY) in IMDM (Gibco BRL, Carlsbad, CA) supplemented with 2% fetal calf serum (FCS), 0.5% bovine serum albumin (Sigma, St Louis, MO), 50 μg/mL human transferrin (Sigma), 5 μg/mL bovine insulin (Sigma), and 15 μg/mL gentamicin (Cellgro-Mediatech, Herndon, VA) at 37°C in 5% CO₂. The cells were stimulated with IL-4, IL-10, IL-2, IL-13 (R&D Systems, Minneapolis, MN), or a combination of IL-4 and IL-13 for 6 hours. Concentrations of cytokines used for stimulation are shown in Figure 3.

Isolation of RNA, its quantification, and the reverse transcription (RT) reactions were performed as previously reported.¹⁷ HGAL mRNA expression was measured by real-time polymerase chain reaction (PCR) using the Applied Biosystems Assays-on-Demand Gene Expression Product on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), as we previously reported.¹⁷

Statistical analysis

Overall survival was defined as the time interval between the date of diagnosis to the date of death or last follow-up. Failure-free survival was defined as the time interval between the date of initial diagnosis and date of disease progression or death from any cause, whichever came first. The Fisher exact test was used to compare patient groups with respect to HGAL staining, and the generalized Wilcoxon rank sum test was used for univariate comparisons of overall and failure-free survival. The Cox proportional hazards model, stratified by center, was used to simultaneously assess the impact of multiple factors on these outcomes. Hierarchic clustering was used to assess the associations between HGAL, CD10, BCL6, BCL2, MUM1, Blimp1, LMP2A, pSTAT6, and EBV EBER expression.

Patient characteristics and HGAL staining

Overall, 145 informative cHL patients whose age ranged from 19 to 84 years (median, 32 years) were studied. All patients were treated with curative intent: 45% received combination chemotherapy and radiation therapy, 38% received only chemotherapy, while 17% received only radiation therapy. The follow-up period for the 145 patients ranged from 1 month to 17.6 years (median, 74.6 months). Forty patients (28%) failed initial treatment, and 31 (21%) died during the follow-up. HGAL protein was expressed in 109 (75%) cHL specimens, whereas 36 (25%) tumors lacked staining for HGAL. Of those with staining for HGAL, 71 (65%) showed strong staining, while 38 patients (35%) showed weak staining. HGAL staining was localized to the cytoplasm of large atypical cells corresponding to Hodgkin cells in all cases. Representative examples of strong, weak, and lack of HGAL staining are shown in Figure 1.

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Figure 1

HGAL protein expression in classic Hodgkin lymphoma. The spectrum of HGAL staining in cHL cases is shown. Staining is localized to the cytoplasm of large atypical cells. Staining in more than 5% but less than 30% of Hodgkin cells was defined as weak, whereas staining in 30% or more Hodgkin cells was defined as strong.

Comparative immunohistologic analysis and EBV RNA expression

CD10 was not expressed in Hodgkin cells. Occasional cases with only cytoplasmic or Golgi staining were present; however, none of the tumors exhibited membrane reactivity for CD10. BCL6 was positive in 36 of 136 (26%) cases. MUM1/IRF4 was highly expressed in most cases (127 of 138; 92%). BCL2 was positive in 44 of 140 (31%) cases. Significant staining for Blimp1 was found in 0.02% of cases (3 of 139). In several additional cases only occasional Hodgkin cells showed staining for Blimp1, but the numbers of positive cells did not warrant a positive score as defined in Table 1 In situ hybridization for EBV using the EBER probe detected EBV-specific RNA in 28 of 140 (20%) cases. Because LMP2A has been suggested to play a role in the survival of EBV-associated lymphoma cells, we analyzed LMP2A staining and found it present in 22 of 28 (79%) EBER-positive cases but in only 2 of the remaining 140 (0.02%) EBER-negative Hodgkin lymphomas. Representative images of cHL cases stained for CD10, BCL6, BCL2, MUM1, Blimp1, and EBV EBER RNA are shown (Figure 2A)

The comparative immunohistologic and in situ hybridization data are summarized in Table 2 We performed hierarchic cluster analysis to address whether there is correlation among the expression patterns of these proteins and with EBV RNA (Figure 2B). In contrast with our previous report of coclustering of HGAL with other GC markers in GC lymphocytes and DLBCL,¹⁵ HGAL clustered here on a separate branch of the dendrogram from CD10 and BCL6 proteins, whose expression is lost in many cases of cHL. Consequently, HGAL clustered on the same branch of the dendrogram as MUM1/IRF4, which is expressed in most cHLs. Both markers used to detect EBV, EBER and LMP2A, coclustered on the dendrogram shown in Figure 2B; however, there was no relationship between the expression of HGAL and EBV in these cases.

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Figure 2

Representative immunostains and EBV expression in classic Hodgkin lymphoma. (A) Typical examples of TMA cores of cHL stained for BCL6, CD10, BCL2, MUM1/IRF4, and Blimp1 and in situ hybridization for EBV EBER RNA are shown. (B) Hierarchic cluster analysis of immunohistologic and in situ hybridization data shows that HGAL clusters with MUM1/IRF4 on 1 branch of the dendrogram (orange) but not with BCL2, CD10, and BCL6 (blue) or with the 2 EBV-specific markers, LMP2A and EBER (purple), which cocluster with each other.

Cytokine signaling and pSTAT6

The cytokine milieu in cHL tissue is important for both the proliferation of Hodgkin cells and maintenance of the characteristic inflammatory environment. Because we previously demonstrated that HGAL expression is induced by IL-4,¹³ we next evaluated whether cytokines implicated in cHL pathogenesis may induce the expression of the HGAL gene. Enriched peripheral-blood lymphocytes were stimulated with IL-4, IL-10, IL-2, IL-13, or a combination of IL-4 and IL-13 for 6 hours. Only IL-4 and IL-13 were found to induce HGAL mRNA expression (Figure 3A) A combination of IL-4 and IL-13 resulted in a slightly additive induction of HGAL mRNA expression. However, incubation with higher concentrations of IL-2 and IL-10 did not lead to induction of HGAL mRNA expression (data not shown). Because pSTAT6 is a mediator of IL-4 and IL-13 cytokine signaling and is commonly implicated in induction of their target genes, we further investigated whether pSTAT6 expression correlated with cHL cases with HGAL protein expression by immunohistologic staining. Staining for pSTAT6 was present in 119 of 142 (83%) cases and was predominantly localized to the nucleus. Reclustering of pSTAT6 staining results with the previously analyzed markers revealed that pSTAT6 clustered on the same branch of the dendrogram as HGAL and MUM1/IRF4 (Figure 3B).

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Figure 3

Cytokine signaling and pSTAT6 in classic Hodgkin lymphoma. (A) Enriched peripheral-blood lymphocytes stimulated with 100 U/mL IL-4, 50 ng/mL IL-10, 100 U/mL IL-2, 10 ng/mL IL-13, or a combination of IL-4 and IL-13 for 6 hours are shown. Only IL-4 and IL-13 induced HGAL mRNA expression, and the combination of IL-4 and IL-13 resulted in a slightly additive induction of HGAL mRNA expression. Error bars indicate SD among 3 experiments. (B) Hierarchic cluster analysis shows that pSTAT6 expression coclusters with HGAL and MUM1/IRF4 on the same branch of the dendrogram (orange), indicating a similar pattern of staining for these 3 proteins across the 145 cHL cases.

HGAL identifies a subset of cHL with improved survival

We examined the relationships between HGAL staining and patient and disease characteristics (Table 3) HGAL staining tended to correlate with younger patient age (less than 45 years, P = .05) but not with sex, stage, IPS, presence or absence of bulky disease, or histologic subtype. Among patients with early-stage disease (I or II), there was also no association with the presence or absence of B symptoms or the number of involved sites. Correlation between clinical prognostic factors, HGAL staining, and patients' survival are summarized in Table 4 The 5-year overall survival and failure-free survival rates of our cohort of 145 patients were 83% ± 3% and 76% ± 4%, respectively. The overall survival correlated with young age (less than 45 years, P < .001), low stage (stage I and II, P = .04), and low IPS (presence of 2 or fewer adverse prognostic factors, P = .002). Failure-free survival correlated with younger age (P < .001) and low IPS (P = .003). HGAL expression by the tumor cells correlated with both overall survival (P = .01) and failure-free survival (P = .05) (Figure 4A) . The estimated 5-year survival for HGAL-positive patients was 87% (95% confidence interval, 81% to 93%) compared with 70% for HGAL-negative patients (95% confidence interval, 54% to 86%).

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Figure 4

HGAL protein expression correlates with overall survival. (A) HGAL protein expression correlates with overall survival (P = .01). (B) HGAL protein expression correlates with improved failure-free survival (P = .05).

Multivariate analyses were performed including HGAL and the clinical parameters listed in Table 4. The primary analysis included all 145 patients as a single cohort and assessed the relative prognostic value of HGAL staining, IPS, and its components in the prediction of cHL outcome. However, because non-IPS parameters are commonly used for subclassification into favorable- and unfavorable-risk groups in patients with early-stage disease (I or II), we performed separate multivariate analyses in early- and advanced-stage (III or IV) HL patients with the National Comprehensive Cancer Network guidelines and the IPS prognostic factors. All analyses were stratified by medical center to adjust for any possible inherent differences between the Cleveland Clinic Foundation and the Nebraska Lymphoma Study Group. Because age and stage are integral parts of the IPS, separate multivariate analyses based on HGAL staining, age, and stage or HGAL staining and IPS risk groups were performed. Based on the analysis of all 145 patients, age or IPS were independent predictors of both overall and failure-free survival (P < .01 in all cases). There was some indication that stage may also be an independent predictor of overall survival (P = .07). HGAL expression did not have an independent impact on overall or failure-free survival in this analysis (P > .05 in all cases). It also was not an independent predictor of either outcome when early-stage disease and advanced-stage disease patients were assessed separately, even after adjusting for factors such as the presence or absence of bulky disease, B symptoms, and the number of involved sites (early-stage disease) or age or IPS (advanced-stage disease) (P ≥ .08 in all cases). Although all patients in our study were treated with curative intent, 25 patients (17%) received radiation treatment only. We therefore repeated the primary statistical analysis excluding these 25 patients and found that HGAL expression showed a similar trend in correlation with overall survival in univariate analysis (P = .05) but was not associated with failure-free survival (P = .19); it again did not have an impact on overall or failure-free survival in multivariate analysis.