Detection of ABCC1 expression in classical Hodgkin lymphoma is associated with increased risk of treatment failure using standard chemotherapy protocols
© Greaves et al.; licensee BioMed Central Ltd. 2012
Received: 21 June 2012
Accepted: 1 August 2012
Published: 7 August 2012
The mechanisms responsible for chemoresistance in patients with refractory classical Hodgkin lymphoma (CHL) are unknown. ATP-binding cassette (ABC) transporters confer multidrug resistance in various cancers and ABCC1 overexpression has been shown to contribute to drug resistance in the CHL cell line, KMH2.
We analyzed for expression of five ABC transporters ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2 using immunohistochemistry in 103 pre-treatment tumor specimens obtained from patients with CHL. All patients received first-line standard chemotherapy with doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens. ABCC1 was expressed in Hodgkin and Reed-Sternberg (HRS) cells in 16 of 82 cases (19.5%) and ABCG2 was expressed by HRS cells in 25 of 77 cases (32.5%). All tumors were negative for ABCB1, ABCC2 and ABCC3. ABCC1 expression was associated with refractory disease (p = 0.01) and was marginally associated with poorer failure-free survival (p = 0.06). Multivariate analysis after adjusting for hemoglobin and albumin levels and age showed that patients with CHL with HRS cells positive for ABCC1 had a higher risk of not responding to treatment (HR = 2.84, 95%, CI: 1.12-7.19 p = 0.028).
Expression of ABCC1 by HRS cells in CHL patients predicts a higher risk of treatment failure and is marginally associated with poorer failure-free survival using standard frontline chemotherapy regimens.
KeywordsClassical Hodgkin lymphoma ABCC1 ATP binding cassettes Immunohistochemistry
Classical Hodgkin lymphoma (CHL) is largely a curable disease using the widely accepted current standard first-line chemotherapy regimen of doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens, with or without consolidation radiotherapy . However, approximately 20% of patients with CHL do not respond following first-line therapy, or relapse quickly, and require additional treatment with salvage chemotherapy with or without stem cell transplantation [1, 2]. A drawback to the currently used treatment modalities is their association with potentially life-threatening toxicities. In addition, patients cured of CHL have an increased lifetime relative risk of death from non CHL-related causes, presumably attributable, at least in part, to therapy . Thus, investigators continue to actively pursue novel prognostic biomarkers and therapeutic options in CHL patients with the goals of maintaining or improving survival rates as well as minimizing adverse side effects in patients with favorable prognosis . Recently, a number of biomarkers expressed by Hodgkin and Reed-Sternberg (HRS) cells as assessed in tissue samples have been proposed as being useful for predicting prognosis in CHL patients . These molecules include matrix metalloproteinase 11 (MMP11), CD20, Bcl2, MAL, HLA class II and Ki67, as well as cells within the CHL microenvironment, such as tumor-associated macrophages or subsets of tumor-infiltrating lymphocytes, including FOXP3+ regulatory T cells (Tregs) and granzyme B + T/NK cells [5–10].
The development of chemotherapy resistance by cancer cells is multifactorial . ATP binding cassette (ABC) transporters comprise a ubiquitous family of transmembrane proteins that play a physiologic role in the transport of substrates across cytoplasmic membranes. ABC transporters also play a role in multidrug resistance (MDR) in multiple tumor types by using ATP as an energy source to actively expel drug substrates from the tumor cell cytoplasm into the extracellular space . Expression of ABC transporters has been shown to correlate with response to therapy and prognosis in several hematological malignancies including acute myeloid leukemia and diffuse large B-cell lymphoma [13–15]. Although the clinical impact of ABC transporters in CHL has not been reported, several drugs used to treat CHL are known substrates of various ABC transporters [11, 16], including doxorubicin (a substrate for ABCB1, ABCC1, ABCC2, ABCC3, ABCG2), vinblastine (a substrate for ABCB1 and ABCC1) and vincristine (a substrate for ABCC1).
Steidl et al. recently showed overexpression of the ABC transporter, ABCC1 (also known as multidrug resistance protein 1 - MRP1) in the therapy-resistant CHL-derived cell line, KMH2 . They further showed that increased sensitivity of KMH2 cells to Adriamycin® toxicity by siRNA silencing of ABCC1. Prompted by this finding, we assessed for expression of five ABC transporters, ABCG2, ABCB1, ABCC1, ABCC2, and ABCC3, in untreated CHL tumor specimens. We also investigated the potential prognostic value of expression of these ABC transporters in CHL.
Design and methods
Selected demographic and histologic features of 103 CHL patients
36 years (range: 13–85)
Age ≥ 45 years
Ann Arbor Stage
ABVD + rituximab
CHL Histologic Subtype:
Antibodies used for immunohistochemistry
Antibody Common Name
Normal Tissue Control
MXR, BCRP, ABC-P
Mouse monoclonal BXP-21
Santa Cruz Biotechnology Inc. Santa Cruz, CA
Mouse monoclonal G-1
Santa Cruz Biotechnology Inc. Santa Cruz, CA
Mouse monoclonal QCRL-1
Santa Cruz Biotechnology Inc. Santa Cruz, CA
Mouse monoclonal M2 III-6
Abcam Inc. Cambridge MA
Mouse monoclonal DTX1
Abcam Inc. Cambridge MA
Fisher’s exact test was used to evaluate the association of clinical response with categorical variables. The Kaplan-Meier method and log rank test were used for survival analysis. The following variables were evaluated in univariate analysis: disease stage (IV vs. I/II/III), chemotherapy (ABVD, CVP or NOVP), radiation therapy (yes and no), bone marrow metastasis (positive and negative), serum albumin (< and > 40 g/L), WBC (< and ≥ 15,000 per mm3), hemoglobin (< or > 105 g/L), lymphocytes (< and ≥ 600 per mm3 or < and ≥ 8% of WBC), gender, International Prognostic Score (IPS) (< and ≥ 3), and age (< and ≥ 45 years). Multivariate Cox proportional hazards models including variables with p value < 0.15 in univariate analysis were fitted to evaluate the association of survival with demographic and clinical factors. Variables with p values < 0.05 were considered statistically significant. S plus software 8.04 (TIBCO software Inc., Palo Alto, CA) and SAS software (SAS Institute Inc., Cary, NC) were used for statistical analysis.
Results and discussion
Multivariate analysis to evaluate the association between FFS and ABCC1
HR (95% CI)
Positive vs. negative
2.84 (1.12, 7.19)
<4 vs. > 4
1.59 (0.70, 3.63)
> = 45 vs. <45
2.14 (1.53, 0.13)
Fisher’s exact test to evaluate the association between ABCC1 and other clinical factors
Fisher's ExactTest (2-Tail)
ABVD (R-ABVD & MOPP/ABVD)
Bone marrow disease
Stage IV disease
< 105 g/l
≥ 40 g/l
< 40 g/l
<15,000 per mm3
≥15,000 per mm3
< 600 per mm3
≥ 600 per mm3
< 45 years
≥ 45 years
Expression of ABCG2 by HRS cells was not significantly associated with OS, FFS or initial response to treatment. The lack of association of ABCG2 expression with treatment refractoriness, in contrast to ABCC1, is not fully explained, and relatively little is known about the differential substrate profiles of these two proteins. However, some authors have shown that certain drugs that are poor ABCC1 substrates, such as mitoxantrone (a type 2 topoisomerase inhibitor), are associated with overexpression of ABCG2 in vitro[11, 21], and such differences may have played a role in the discordant impact of these two proteins on therapy resistance in this patient cohort.
In summary, ABCC1 and ABCG2 are expressed by HRS cells in a subset of CHL tumors. Univariate and multivariate analyses showed that expression of ABCC1 by HRS cells is associated with an increased risk of tumor progression, treatment resistance or death in CHL patients. Our findings corroborate those published by Steidl and colleagues  in the KMH2 cell line and provide evidence that expression of ABCC1 may be useful as an indicator of poorer FFS or failure to respond to therapy in CHL patients who are treated with standard regimens. Additionally, ABCC1 may serve as a potential target for therapeutic intervention by increasing susceptibility to chemotherapy.
Classical Hodgkin lymphoma
Adriamycin, bleomycin, vinblastine, and dacarbazine
Hodgkin Reed-Sternberg cells
Matrix metalloproteinase 11
ATP binding cassette
Cyclophosphamide, vinblastine, procarbazine, and prednisone
Novantrone, vincristine, vinblastine, and prednisone
White blood count
International prognostic index
Failure free survival
This work was supported by funds from the K08 Physician-Scientist Award 1 K08 CA143151-01 (NIH) (to FV) SPORE Lymphoma grant UT M.D. Anderson Cancer Center Lymphoma SPORE 1P50CA136411-01A1 (to FV). A subset of patient samples were provided with assistance from the Biospecimens Core of the Lymphoma SPORE.
*Dr Beatriz Sanchez-Espiridion collaborated in this work as a visiting scientist supported by the Department of Pathology, M.D. Anderson España and by Centro Nacional de Investigationes Oncologicas (CNIO), Madrid (Spain).
- Lowry L, Hoskin P, Linch D: Developments in the management of Hodgkin’s lymphoma. Lancet. 2010, 375: 786-788. 10.1016/S0140-6736(09)61878-X.View ArticlePubMedGoogle Scholar
- Jona A, Younes A: Novel treatment strategies for patients with relapsed classical Hodgkin lymphoma. Blood Rev. 2010, 24: 233-238. 10.1016/j.blre.2010.08.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Aleman BM, van den Belt-Dusebout AW, Klokman WJ, Van’t Veer MB, Bartelink H, van Leeuwen FE: Long-term cause-specific mortality of patients treated for Hodgkin’s disease. J Clin Oncol. 2003, 21: 3431-3439. 10.1200/JCO.2003.07.131.View ArticlePubMedGoogle Scholar
- Hsi ED: Biologic features of Hodgkin lymphoma and the development of biologic prognostic factors in Hodgkin lymphoma: tumor and microenvironment. Leuk Lymphom. 2008, 49: 1668-1680. 10.1080/10428190802163339.View ArticleGoogle Scholar
- Diepstra A, van Imhoff GW, Karim-Kos HE, van den Berg A, te Meerman GJ, Niens M: HLA class II expression by Hodgkin Reed-Sternberg cells is an independent prognostic factor in classical Hodgkin’s lymphoma. J Clin Onco. 2007, 25: 3101-3108. 10.1200/JCO.2006.10.0917.View ArticleGoogle Scholar
- Sup SJ, Alemany CA, Pohlman B, Elson P, Malhi S, Thakkar S: Expression of bcl-2 in classical Hodgkin’s lymphoma: an independent predictor of poor outcome. J Clin Oncol. 2005, 23: 3773-3779. 10.1200/JCO.2005.04.358.View ArticlePubMedGoogle Scholar
- Hsi ED, Sup SJ, Alemany C, Tso E, Skacel M, Elson P: MAL is expressed in a subset of Hodgkin lymphoma and identifies a population of patients with poor prognosis. Am J Clin Pathol. 2006, 125: 776-782. 10.1309/98KLHRDAM5CMDHE2.View ArticlePubMedGoogle Scholar
- Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP, Viviani S, Bonfante V, Nadali G: BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosis in patients treated with ABVD or equivalent regimens. Blood. 2002, 100: 3935-3941. 10.1182/blood.V100.12.3935.View ArticlePubMedGoogle Scholar
- Montalban C, Garcia JF, Abraira V, Gonzalez-Camacho L, Morente MM, Bello JL: Influence of biologic markers on the outcome of Hodgkin’s lymphoma: a study by the Spanish Hodgkin’s Lymphoma Study Group. J Clin Oncol. 2004, 22: 1664-1673. 10.1200/JCO.2004.06.105.View ArticlePubMedGoogle Scholar
- Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T: Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med. 2010, 362: 875-885. 10.1056/NEJMoa0905680.PubMed CentralView ArticlePubMedGoogle Scholar
- Gottesman MM, Fojo T, Bates SE: Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2002, 2: 48-58. 10.1038/nrc706.View ArticlePubMedGoogle Scholar
- Allen JD, Brinkhuis RF, van Deemter L, Wijnholds J, Schinkel AH: Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance. Cancer Res. 2000, 60: 5761-5766.PubMedGoogle Scholar
- van der Kolk DM, de Vries EG, van Putten WJ, Verdonck LF, Ossenkoppele GJ, Verhoef GE: P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia. Clin Cancer Res. 2000, 6: 3205-3214.PubMedGoogle Scholar
- Leith CP, Kopecky KJ, Chen IM, Eijdems L, Slovak ML, McConnell TS: Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood. 1999, 94: 1086-1099.PubMedGoogle Scholar
- Singh RR, Kunkalla K, Qu C, Schlette E, Neelapu SS, Samaniego F: ABCG2 is a direct transcriptional target of hedgehog signaling and involved in stroma-induced drug tolerance in diffuse large B-cell lymphoma. Oncogene. 2011, 30: 4874-4886. 10.1038/onc.2011.195.PubMed CentralView ArticlePubMedGoogle Scholar
- Deeley RG, Cole SPC: Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FEBS. 2006, 580: 1103-1111. 10.1016/j.febslet.2005.12.036.View ArticleGoogle Scholar
- Steidl C, Telenius A, Shah SP, Farinha P, Barclay L, Boyle M: Genome-wide copy number analysis of Hodgkin Reed-Sternberg cells identifies recurrent imbalances with correlations to treatment outcome. Blood. 2010, 116: 418-427. 10.1182/blood-2009-12-257345.View ArticlePubMedGoogle Scholar
- Kim JE, Singh RR, Cho-Vega JH, Drakos E, Davuluri Y, Khokhar FA: Sonic hedgehog signaling proteins and ATP-binding cassette G2 are aberrantly expressed in diffuse large B-cell lymphoma. Mod Pathol. 2009, 22: 1312-1320. 10.1038/modpathol.2009.98.View ArticlePubMedGoogle Scholar
- Sanchez-Espiridion B, Sanchez-Aguilera A, Montalban C, Martin C, Martinez R, Gonzalez-Carrero J: A TaqMan low-density array to predict outcome in advanced Hodgkin’s lymphoma using paraffin-embedded samples. Clin Cancer Res. 2009, 15: 1367-1375. 10.1158/1078-0432.CCR-08-1119.View ArticlePubMedGoogle Scholar
- Carde P, Koscielny S, Franklin J, Axdorph U, Raemaekers J, Diehl V: Early response to chemotherapy: a surrogate for final outcome of Hodgkin’s disease patients that should influence initial treatment length and intensity?. Ann Oncol. 2002, 13 (Suppl 1): 86-91. 10.1093/annonc/13.S1.86.View ArticlePubMedGoogle Scholar
- Miyake K, Mickley L, Litman T, Zhan Z, Robey R, Cristensen B: Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res. 1999, 59: 8-13.PubMedGoogle Scholar
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