Published Ahead of Print on October 27, 2014 as 10.1200/JCO.2013.50.9471 The latest version is at http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2013.50.9471
JOURNAL OF CLINICAL ONCOLOGY
Targeting Tumor-Infiltrating B Cells in Cutaneous T-Cell Lymphoma Introduction The tumor microenvironment and infiltrating immune cells are important for cancer biology and progression which has been exploited therapeutically in recent years.1-3 Whereas infiltrating T cells, macrophages, or natural-killer (NK) cells have been extensively analyzed, recent studies also suggest a role for B cells in cancer biology.4,5 Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas of which the most common subtype is mycosis fungoides (MF). At early stages (ⱕ IIA; European Organisation for Research and Treatment of Cancer [EORTC]/International Society for Cutaneous Lymphomas [ISCL]), MF usually runs an indolent course with almost normal life expectancy. However, advanced-stage MF (ⱖ IIB), the folliculotropic MF subtype (FMF) and also Se´zary syndrome (SS) are more aggressive (median survival, 13-48 months).6-8 Preclinical data suggest that chronic inflammation promotes CTCL progression with a critical role for macrophages and mast cells.9,10 Occasionally, B-cell infiltrations have been reported in CTCL11-14 and although a well-established CTCL model is lacking, B-cell deficient mice exhibited significant regressions of EL4-T-cell lymphoma grafts.15 Therefore, we analyzed diagnostic skin biopsies of 33 consecutively treated CTCL patients for infiltrating B cells (CD20/ CD79a) and reviewed respective clinical data from 1979 to 2011 (Table 1). Psoriasis and eczema tissues served as controls. Staging followed ISCL/EORTC criteria.16 Lymphoma-tissues containing ⱖ 50 B-cells/mm2 of lymphoma-infiltrate were assigned as B-cell positive. The study was approved by the institutional review board (No. 08-144).
D I A G N O S I S
I N
O N C O L O G Y
Immunohistochemistry revealed remarkable differences of CD20⫹ B-cell infiltrates between CTCL subtypes (Fig 1A) and additional flow-cytometric analysis in available frozen samples supported the B-cell nature of CD20⫹ cells (Fig 3A). Eighteen out of 33 CTCL patients (MF [n ⫽ 25], FMF [n ⫽ 5], SS [n ⫽ 3]) had significantly increased median B-cell numbers within the lymphoma infiltrate, whereas controls (psoriasis [n ⫽ 5], eczema [n ⫽ 5]) were B-cell negative (Fig 1B and Table 1). Remarkably, all FMF and SS cases showed significantly increased median B-cell numbers compared with classic MF. However, in classic MF 40% of the cases were B-cell positive. Given the fact that FMF and SS represent more aggressive subtypes, we asked whether B-cell infiltrations correspond with the clinical stage and behavior. Comparison of B-cell infiltrates in early (⬍ IIB) versus advanced (ⱖ IIB) stages revealed a significant correlation of B-cell positivity with advanced stages. This correlation not only applied to the entire CTCL cohort but also to classic MF cases solely (Fig 1C). Moreover, clinical data analyses revealed a significant correlation of B-cell infiltrates with progression-free survival (PFS). As shown in Figure 1D, patients with B-cell positive lymphoma had a significantly shortened median PFS, ie, 50 months (entire cohort) and 126 months (classic MF only). In contrast, median PFS in B-cell negative cases was not reached at the end of observation (360 months). Case Report A 77-year-old female was diagnosed in September 2009 with FMF (CD4⫹TCR␥⫹) stage IIB (pT3N0M0). The diagnosis was confirmed by a reference pathologist (W. Kempf, Zu¨rich, Switzerland). First-line therapy initiated in December 2009 consisted of systemic psoralen and ultraviolet A radiation (PUVA), interferon-alfa (IFN-␣) 3 ⫻ 3 millionIE/wk, and topic steroids. After initial remission, the lymphoma relapsed after 3 months and INF-␣ was escalated to 3 ⫻ 9
Table 1. Clinical Characteristics of Patients With CTCL and the Distribution of B-Cell Infiltrations B-Cell Infiltration Patients (no.) Characteristics
B Cell B Cell All Pos. Neg.
All patients with CTCL 33 Controls 10 Disease subtypes MF 25 FMF 5 Sézary 3
Age (years) Range (mean) P
All
B Cell Pos.
Stage (revised ISCL/EORTC classification) B Cell Neg.
⬍ II B ⬍ II B P B Cell Pos. B Cell Neg.
P
ⱖ II B ⱖ II B B Cell Pos. B Cell Neg.
P
18 0
15 10
⬍ .01
28-87 (68.2) 28-80 (63.4) 65-87 (74.0) ns 26-78 (56.9) na 26-78 (56.9)
5
15
⬍ .001
13
0
⬍ .001
10 5 3
15 0 0
37-87 (67.7) 37-80 (58.3) 65-87 (74.0) na ns ⬍ .001ⴱ 28-80 (67.4) 28-80 (67.4) 72-76 (73.7) 72-76 (73.7) na
5 na na
15 na na
⬍ .001
5 5 3
0 0 0
⬍ .001 ⬍ .001 ⬍ .001
NOTE. B-cell positivity (B cell pos.) was assigned if at least 50 B cells per mm2 lymphoma infiltrate were detectable. Statistical analysis was performed with Fishers exact test or 2 test. P values ⱕ .05 were considered statistical significant. Abbreviations: CTCL, cutaneous T-cell lymphoma; FMF, folliculotropic mycosis fungoides subtype; ISCL/EORTC, International Society for Cutaneous Lymphomas/ European Organisation for Research and Treatment of Cancer; MF, mycosis fungoides; na, not applicable; neg., negative; ns, not significant; pos, positive; Sézary, Sézary syndrome. ⴱ 2 test.
Journal of Clinical Oncology, Vol 32, 2014
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Copyright 2014 by American Society of Clinical Oncology
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Theurich et al
A
MF Low B Cells
B
MF High B Cells
FMF
SS
CD20+ Infiltrating Cells
CD79a+ Infiltrating Cells
3,000 2,000 1,000
P < .05
P < .05 P < .05
CD79a+ Cells/mm² Tumor
CD20+ Cells/mm² Tumor
P < .05
P < .05
500 400 300 200 100
3,000 2,000 1,000
P < .05
400 300 200 100 0
MF
FMF
Sezary
Control
MF
B-Cell–Positive CTCL (entire cohort)
FMF
No. of Cells/mm² Tumor
No. of Cells/mm² Tumor
Control
advanced v early stages P < .05 P < .05
3,000 2,000 1,000 600 400 200
3,000 2,000 1,000
0
600 400 200 0
CD20+ < IIB
CD20+ ≥ IIB
CD79a+ < IIB
CD79a+ ≥ IIB
CD20+ < IIB
Progression-Free Survival
CD20+ ≥ IIB
Fraction Survival
CD20+ CD79a+ CD20− CD79a−
0.6
CD79a+ ≥ IIB
(classic MF only)
1.0
0.8
CD79a+ < IIB
Progression-Free Survival
(entire CTCL cohort)
1.0
Fraction Survival
Sezary
B-Cell–Positive Classic MF Only
advanced v early stages P < .05 P < .05
D
P < .05
500
0
C
P < .05
P < .001
0.4 0.2
0.8 0.6 0.4 0.2
CD20+ CD79a+ CD20− CD79a− P = .05
0
100
200
300
0
400
Time to Progression (months)
100
200
300
400
Time to Progression (months) Fig 1.
2
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Diagnosis in Oncology
A
B 06/2011
CD20
08/2011
Local 20mg
Rituximab: 20mg Day
D
8
40mg 15
22
Bexarotene 300 mg/m2
CD79a
C
1
30mg
Prior Rituximab
After 3 Weeks
After 4 Weeks
After 12 Weeks
Rituximab i.l.
FoxP3
Glucose i.l.
Prior Rituximab
E
FoxP3
After 8 Weeks
Rituximab i.l.
Glucose i.l.
CD8
CD8 Rituximab i.l.
Glucose i.l.
103
*
103 56%
400
91% T8:T4 = 0.63
102
T8:T4 = 0.05
102
300
CD4
FoxP3+ Cells/mm2
500
200 100
101
101
100
100 35%
5%
0 Rituximab
Glucose
100
101
102
103
100
101
102
103
CD8 Fig 2.
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Theurich et al
A
Skin Tumors: Identification of the B-Cell Nature of CD20+ Infiltrating Cells 103
103 CD19+CD20+
102
102
101
CD21
CD19
1,000
14%
101
500 CD45+
100
100
0 100
101
102
100
103
101
102
103
100
102
103
IgD
CD20
B
101
Skin Tumors: After 4 Cycles of Intralesional Treatment Prior
Rituximab
103 CD19+CD20−
CD19+CD20+
6%
2
10
CD19
Glucose
103
103 CD19+CD20−
11%
2
10
CD19+CD20−
CD19+CD20+
2%
0.7%
2
10
101
101
101
100
100
100
100
101
102
103
100
101
102
103
CD19+CD20+
16%
2%
100
101
102
103
CD20
C
Eosinophils
20
R
R
15
R
R
0
20
R
600
U/I
Leukocytes (%)
R
Lactate Dehydrogenase
800
10
5
400
200
0
0 0
20
40
Time After Rituximab (days)
40
Time After Rituximab (days) Fig 3.
millionIE/wk, leading to remission again. Due to INF-␣ adverse effects, methotrexate (15 mg/wk) was initiated in July 2010 intermittently combined with prednisone, PUVA, and local irradiation (2 ⫻ 4 Gy) of tumors. In November 2010, methotrexate was substituted with systemic bexarotene (300 mg/m2/d) because of generalized progres4
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sion. After a 4-month disease stabilization, lesions reoccurred including a nuchal tumor (25 ⫻ 15 ⫻ 10 mm). Treatment escalations such as total-skin electron beam irradiation or other chemotherapy were declined by the patient at that time. Evaluating treatment alternatives and based on our data, we analyzed skin biopsies of the patient which JOURNAL OF CLINICAL ONCOLOGY
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Diagnosis in Oncology
Discussion In this report, we analyzed the impact of tumor-infiltrating B cells in CTCL and found a significant correlation with advanced stages and shortened PFS. This correlation applied not only to FMF and SS with a known aggressive behavior but also to classic MF. Moreover, local depletion of B cells with rituximab led to sustained locoregional tumor remission in a patient with progressive FMF. The role of tumor-associated B cells in the tumor microenvironment is under intensive investigation.4 Several studies in solid human tumors (eg, breast, ovarian, cervical and lung cancer) suggest an improved tumor control in the presence of B cells.4,17,18 On the other hand, the majority of murine models indicate a tumor promoting effect of B cells via different mechanisms (eg, direct growth induction by lymphotoxin-, enhanced angiogenesis, or the induction of T-cellwww.jco.org
A CD24hi38hi B Cells (%)
CD24hiCD38hi B Cells in Patients With CTCL 100 80 60 40 30
P < .05
20 10
B
pB lth y
pB ea H
Skin Tumor: After 4 Cycles of Intralesional Treatment Prior
Glucose
Rituximab
103
103
CD24hiCD38hi 102
CD24
is or ia s Ps
CT
CT
CL
CL
Sk
in
pB
0
103
CD24hiCD38hi
CD24hiCD38hi
102
102
101
101
101
0
5.3% 100
100
10
100
101
102
103
100
101
102
103
4.1% 100
101
102
103
150
Tumor-Infiltrating CD24hi38hi B Cells
100
hi
CD24 38 B Cells/ 10,000 CD45+
CD38
hi
showed significant B-cell infiltrates (Fig 2A). Therefore, the patient was enrolled into an off-label treatment with local injections of the anti-CD20 antibody rituximab after written informed consent and approval of the institutional tumor board. In June 2011, the nuchal tumor was injected weekly with rituximab in increasing doses over 1 month (Fig 2B) and, as a control, the same volume of sterile glucose 5% solution was injected into another tumor on the trunk. Bexarotene was continued as an established regimen. Two days after the first injection, the patient noticed shivering without signs of infection. Furthermore, localized erythema and pain occurred at the injection site, which disappeared some days later. After three injections, clinical regression of the nuchal tumor was detectable finally leading to sustained complete remission (Fig 2C). Interestingly, computed tomography not only confirmed tumor remission, but also showed regression of locoregional lymph nodes. Histologic and flow-cytometric analyses of the rituximab-treated tumor showed B-cell depletion but also a marked depletion of CD4⫹ lymphoma cells (Figs 3B and 2E; flow cytometry). More interestingly, FoxP3⫹ regulatory T cells (Tregs) (CD4⫹CD25highCD127low, not shown) were also significantly reduced compared to the control (Fig 2D). In contrast, a significant increase (from 5% to 35%) of tumorinfiltrating CD8⫹ cytotoxic T cells was detected after rituximab treatment (Fig 2E). In August 2011, the patient consented for systemic chemotherapy with liposomal doxorubicine (20 mg/m2 d22) because of systemic progression. After initial stabilization, FMF progressed shortly after the second cycle with additional lymphadenopathy. Notably, the rituximab-treated nuchal lesion still remained in remission. After exclusion of large-cell transformation while confirming B-cell infiltration in an inguinal lymph node, we proceeded to systemic rituximab based on the previous encouraging results. Despite an initially reduced rituximab-dose (187 mg/m2), a systemic immunologic response (fever, shivering) was observed 2 days after the first infusion (October 2011). Two weeks later, the patient reported on an improvement of previously existing pruritus and cutaneous tensions. Also, a decrease of surrogate markers such as eosinophils, lactate dehydrogenase (Fig 3C) and thymidine kinase (not shown) illustrated a response. We therefore continued with the conventional rituximab dose (375 mg/ m2) for two more cycles (repeated days 18-21) resulting in further disease stabilization (cutaneous symptoms, computed tomography [not shown]). However, similar to the previous standard treatments, the response was temporary and disease progression eventually led to a fatal outcome 2 years after initial diagnosis.
50 0 Skin Tumor Prior
Skin Tumor Rituximab
Skin Tumor Glucose
Fig 4.
mediated tolerance).19-21 In our study, several CTCL specimens with notable B-cell infiltrates also showed marked infiltrates of Tregs (data not shown). This phenomenon had also been reported in a murine breast cancer model in which B cells induced an intratumoral expansion of Tregs, and the absence of B cells correlated with improved tumor control.22,23 Similarly, targeting of CD20⫹ B cells in our patient not only led to a depletion of B cells and subsequent tumor regression, but also had a profound effect on the composition of the remaining lymphocytic infiltrate. We observed a reduction in the numbers of Tregs complemented by an increase of CD8⫹ cytotoxic T cells. This suggests that depletion of B cells triggered a cascade that led to remodelling of the tumor microenvironment and reconstitution of effective immunosurveillance.24 While T-cell subpopulations with regulatory properties have been characterized fairly well, little is known about a B-cell counterpart. Recently, a CD24highCD38high transitional B-cell population has been shown to suppress T-cell responses mediated by interleukin-10.25 Interestingly, we also found markedly increased frequencies of CD24highCD38high B cells in the peripheral blood © 2014 by American Society of Clinical Oncology
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Theurich et al
and tumors from seven CTCL patients (Fig 4A). In our patient, local rituximab treatment led to a substantial depletion of this B-cell population (Fig 4B). It therefore needs to be determined whether such B-cell subpopulations are the dominant mediator of the effects of the B-cellular infiltrate. CD20 expression has been described on certain malignant cells including CTCL.26 Here, additional CD79a tissue staining and flowcytometry independently confirmed the B-cell nature of the CD20⫹ cells (Fig 3A). Rituximab is an improved drug with a low toxicity profile and its use would therefore be of particular interest in CTCL patients who are generally older with significant comorbidities. As combinational chemo-immunotherapy has proven to be effective in several hematological malignancies, the above could be combined (eg, with liposomal doxorubicine or others) to increase efficacy. Moreover, as a quite low skin penetration has been reported for standard rituximab doses,27 it needs to be determined whether dose escalation or use of secondgeneration CD20-antibodies with lower molecular size would improve results of systemic application. Taken together, we present first data suggesting that lymphomainfiltrating B cells may impact CTCL biology and that local depletion of B cells can re-establish immunologic tumor-control. Prospective studies are warranted that address if B-cell infiltrates can be used as a biomarker and whether B-cell depletion (local or systemic) can be generally applied to B-cell–rich CTCL.
Sebastian Theurich, Max Schlaak, Harold Steguweit, Lukas C. Heukamp, Kerstin Wennhold, Peter Kurschat, Anja Rabenhorst, Karin Hartmann, Hans Schlo¨sser, Alexander Shimabukuro-Vornhagen, Udo Holtick, and Michael Hallek University Hospital of Cologne, Cologne, Germany
Rudolf Stadler, Johannes-Wessling-Hospital, Minden, Germany
Michael von Bergwelt-Baildon University Hospital of Cologne, Cologne, Germany
ACKNOWLEDGMENT
We thank Alexandra Florin, Institute of Pathology, University Hospital of Cologne, for her excellent technical expertise and support and Horst Mu¨ller, PhD, German Hodgkin Study Group, Cologne, for his kind and helpful advice in statistical analysis. Christine Neumann, MD, supported this work with her long standing clinical experience in the CTCL field. We thank Werner Kempf, MD, Zu¨rich, Switzerland, for his careful re-evaluation of difficult or rare CTCL cases as a reference pathologist. Geothy Chakupurakal, MD, critically revised the manuscript and gave us valuable advice. M.S. is supported by the German Federal Ministry of Research and Education (BMBF grant 01KN1106). Part of this work was supported by the Deutsche Forschungsgemeinschaft (German Research Council, DFG) through SFB 832, to M.H. and M.B. (graduate school and project A16), to K.H. and A.R. (project A14) and to L.C.H. (project Z1). S.T and M.S. contributed equally to this work. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Although all authors completed the disclosure declaration, the following author(s) and/or an author’s immediate family member(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are 6
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those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Michael Hallek, Roche (C) Stock Ownership: None Honoraria: Peter Kurschat, Roche; Michael Hallek, Roche Research Funding: Michael Hallek, Roche Expert Testimony: None Patents, Royalties, and Licenses: None Other Remuneration: None REFERENCES 1. Hanahan D, Weinberg RA: Hallmarks of cancer: The next generation. Cell 144:646-674, 2011 2. Kirkwood JM, Butterfield LH, Tarhini AA, et al: Immunotherapy of cancer in 2012. CA Cancer J Clin 62:309-335, 2012 3. Zitvogel L, Kroemer G: Targeting PD-1/PD-L1 interactions for cancer immunotherapy. Oncoimmunology 1:1223-1225, 2012 4. Nelson BH. CD: 20⫹ B cells: The other tumor-infiltrating lymphocytes. J Immunol 185:4977-4982, 2010 5. Sorbye SW, Kilvaer T, Valkov A, et al: High expression of CD20⫹ lymphocytes in soft tissue sarcomas is a positive prognostic indicator. Oncoimmunology 1:75-77, 2012 6. Talpur R, Singh L, Daulat S, et al: Long-term outcomes of 1,263 patients with mycosis fungoides and Sezary syndrome from 1982 to 2009. Clin Cancer Res 18:5051-5060, 2012 7. Agar NS, Wedgeworth E, Crichton S, et al: Survival outcomes and prognostic factors in mycosis fungoides/Sezary syndrome: Validation of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal. J Clin Oncol 28:47304739, 2010 8. Benner MF, Jansen PM, Vermeer MH, et al: Prognostic factors in transformed mycosis fungoides: A retrospective analysis of 100 cases. Blood 119:1643-1649, 2012 9. Wu X, Sells RE, Hwang ST: Upregulation of inflammatory cytokines and oncogenic signal pathways preceding tumor formation in a murine model of T-cell lymphoma in skin. J Invest Dermatol 131:1727-1734, 2011 10. Rabenhorst A, Schlaak M, Heukamp LC, et al: Mast cells play a protumorigenic role in primary cutaneous lymphoma. Blood 120:2042-2054, 2012 11. van der Putte SC, Toonstra J, van Wichen DF: B cells and plasma cells in mycosis fungoides: A study including cases with B cell follicle formation or a monotypical plasma cell component. Am J Dermatopathol 11:509-516, 1989 12. Wasco MJ, Fullen D, Su L, et al: The expression of MUM1 in cutaneous T-cell lymphoproliferative disorders. Hum Pathol 39:557-563, 2008 13. Mattoch IW, Fulton R, Kim Y, et al: Cutaneous peripheral T-cell lymphoma associated with a proliferation of B cells. Am J Clin Pathol 131:810-819, 2009 14. Ferrara G, Chiarelli C, Simonetti S: B-cell lymphofollicular infiltrates in mycosis fungoides. Tumori 96:487-491, 2010 15. Shah S, Divekar AA, Hilchey SP, et al: Increased rejection of primary tumors in mice lacking B cells: Inhibition of anti-tumor CTL and TH1 cytokine responses by B cells. Int J Cancer 117:574-586, 2005 16. Olsen E, Vonderheid E, Pimpinelli N, et al: Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: A proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 110:1713-1722, 2007 17. Dieu-Nosjean M-C, Antoine M, Danel C, et al: Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures. J Clin Oncol 26:4410-4417, 2008 18. Nielsen JS, Sahota RA, Milne K, et al: CD20⫹ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8⫹ T cells promote favorable prognosis in ovarian cancer. Clin Cancer Res 18:3281-3292, 2012 19. Visser KE, Korets LV, Coussens LM: De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 7:411-423, 2005 20. Ammirante M, Luo J-L, Grivennikov S, et al: B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature 464:302-305, 2010 21. Qin Z, Richter G, Schuler T, et al: B cells inhibit induction of T celldependent tumor immunity. Nat Med 4:627-630, 1998 22. Tadmor T, Zhang Y, Cho H-M, et al: The absence of B lymphocytes reduces the number and function of T-regulatory cells and enhances the anti-tumor response in a murine tumor model. Cancer Immunol Immunother 60:609-619, 2011 JOURNAL OF CLINICAL ONCOLOGY
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23. Olkhanud PB, Damdinsuren B, Bodogai M, et al: Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4(⫹) T cells to T-regulatory cells. Cancer Res 71:3505-3515, 2011 24. Schreiber RD, Old LJ, Smyth MJ: Cancer immunoediting: Integrating immunity’s roles in cancer suppression and promotion. Science 331:1565-1570, 2011 25. Blair PA, Norena LY, Flores-Borja F, et al: CD19(⫹)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32:129-140, 2010
26. Martin B, Stefanato C, Whittaker S, et al: Primary cutaneous CD20-positive T-cell lymphoma. J Cutan Pathol 38:663-669, 2011 27. Amir R: Jalilian DS, Kia L, et al: Preparation, quality control and biodistribution studies of two [111In]-rituximab immunoconjugates. Sci Pharm 76:151170, 2008
DOI: 10.1200/JCO.2013.50.9471; published online ahead of print at www.jco.org on October 27, 2014
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JOURNAL OF CLINICAL ONCOLOGY
Targeting Tumor-Infiltrating B Cells in Cutaneous T-Cell Lymphoma Introduction The tumor microenvironment and infiltrating immune cells are important for cancer biology and progression which has been exploited therapeutically in recent years.1-3 Whereas infiltrating T cells, macrophages, or natural-killer (NK) cells have been extensively analyzed, recent studies also suggest a role for B cells in cancer biology.4,5 Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas of which the most common subtype is mycosis fungoides (MF). At early stages (ⱕ IIA; European Organisation for Research and Treatment of Cancer [EORTC]/International Society for Cutaneous Lymphomas [ISCL]), MF usually runs an indolent course with almost normal life expectancy. However, advanced-stage MF (ⱖ IIB), the folliculotropic MF subtype (FMF) and also Se´zary syndrome (SS) are more aggressive (median survival, 13-48 months).6-8 Preclinical data suggest that chronic inflammation promotes CTCL progression with a critical role for macrophages and mast cells.9,10 Occasionally, B-cell infiltrations have been reported in CTCL11-14 and although a well-established CTCL model is lacking, B-cell deficient mice exhibited significant regressions of EL4-T-cell lymphoma grafts.15 Therefore, we analyzed diagnostic skin biopsies of 33 consecutively treated CTCL patients for infiltrating B cells (CD20/ CD79a) and reviewed respective clinical data from 1979 to 2011 (Table 1). Psoriasis and eczema tissues served as controls. Staging followed ISCL/EORTC criteria.16 Lymphoma-tissues containing ⱖ 50 B-cells/mm2 of lymphoma-infiltrate were assigned as B-cell positive. The study was approved by the institutional review board (No. 08-144).
D I A G N O S I S
I N
O N C O L O G Y
Immunohistochemistry revealed remarkable differences of CD20⫹ B-cell infiltrates between CTCL subtypes (Fig 1A) and additional flow-cytometric analysis in available frozen samples supported the B-cell nature of CD20⫹ cells (Fig 3A). Eighteen out of 33 CTCL patients (MF [n ⫽ 25], FMF [n ⫽ 5], SS [n ⫽ 3]) had significantly increased median B-cell numbers within the lymphoma infiltrate, whereas controls (psoriasis [n ⫽ 5], eczema [n ⫽ 5]) were B-cell negative (Fig 1B and Table 1). Remarkably, all FMF and SS cases showed significantly increased median B-cell numbers compared with classic MF. However, in classic MF 40% of the cases were B-cell positive. Given the fact that FMF and SS represent more aggressive subtypes, we asked whether B-cell infiltrations correspond with the clinical stage and behavior. Comparison of B-cell infiltrates in early (⬍ IIB) versus advanced (ⱖ IIB) stages revealed a significant correlation of B-cell positivity with advanced stages. This correlation not only applied to the entire CTCL cohort but also to classic MF cases solely (Fig 1C). Moreover, clinical data analyses revealed a significant correlation of B-cell infiltrates with progression-free survival (PFS). As shown in Figure 1D, patients with B-cell positive lymphoma had a significantly shortened median PFS, ie, 50 months (entire cohort) and 126 months (classic MF only). In contrast, median PFS in B-cell negative cases was not reached at the end of observation (360 months). Case Report A 77-year-old female was diagnosed in September 2009 with FMF (CD4⫹TCR␥⫹) stage IIB (pT3N0M0). The diagnosis was confirmed by a reference pathologist (W. Kempf, Zu¨rich, Switzerland). First-line therapy initiated in December 2009 consisted of systemic psoralen and ultraviolet A radiation (PUVA), interferon-alfa (IFN-␣) 3 ⫻ 3 millionIE/wk, and topic steroids. After initial remission, the lymphoma relapsed after 3 months and INF-␣ was escalated to 3 ⫻ 9
Table 1. Clinical Characteristics of Patients With CTCL and the Distribution of B-Cell Infiltrations B-Cell Infiltration Patients (no.) Characteristics
B Cell B Cell All Pos. Neg.
All patients with CTCL 33 Controls 10 Disease subtypes MF 25 FMF 5 Sézary 3
Age (years) Range (mean) P
All
B Cell Pos.
Stage (revised ISCL/EORTC classification) B Cell Neg.
⬍ II B ⬍ II B P B Cell Pos. B Cell Neg.
P
ⱖ II B ⱖ II B B Cell Pos. B Cell Neg.
P
18 0
15 10
⬍ .01
28-87 (68.2) 28-80 (63.4) 65-87 (74.0) ns 26-78 (56.9) na 26-78 (56.9)
5
15
⬍ .001
13
0
⬍ .001
10 5 3
15 0 0
37-87 (67.7) 37-80 (58.3) 65-87 (74.0) na ns ⬍ .001ⴱ 28-80 (67.4) 28-80 (67.4) 72-76 (73.7) 72-76 (73.7) na
5 na na
15 na na
⬍ .001
5 5 3
0 0 0
⬍ .001 ⬍ .001 ⬍ .001
NOTE. B-cell positivity (B cell pos.) was assigned if at least 50 B cells per mm2 lymphoma infiltrate were detectable. Statistical analysis was performed with Fishers exact test or 2 test. P values ⱕ .05 were considered statistical significant. Abbreviations: CTCL, cutaneous T-cell lymphoma; FMF, folliculotropic mycosis fungoides subtype; ISCL/EORTC, International Society for Cutaneous Lymphomas/ European Organisation for Research and Treatment of Cancer; MF, mycosis fungoides; na, not applicable; neg., negative; ns, not significant; pos, positive; Sézary, Sézary syndrome. ⴱ 2 test.
Journal of Clinical Oncology, Vol 32, 2014
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1
Theurich et al
A
MF Low B Cells
B
MF High B Cells
FMF
SS
CD20+ Infiltrating Cells
CD79a+ Infiltrating Cells
3,000 2,000 1,000
P < .05
P < .05 P < .05
CD79a+ Cells/mm² Tumor
CD20+ Cells/mm² Tumor
P < .05
P < .05
500 400 300 200 100
3,000 2,000 1,000
P < .05
400 300 200 100 0
MF
FMF
Sezary
Control
MF
B-Cell–Positive CTCL (entire cohort)
FMF
No. of Cells/mm² Tumor
No. of Cells/mm² Tumor
Control
advanced v early stages P < .05 P < .05
3,000 2,000 1,000 600 400 200
3,000 2,000 1,000
0
600 400 200 0
CD20+ < IIB
CD20+ ≥ IIB
CD79a+ < IIB
CD79a+ ≥ IIB
CD20+ < IIB
Progression-Free Survival
CD20+ ≥ IIB
Fraction Survival
CD20+ CD79a+ CD20− CD79a−
0.6
CD79a+ ≥ IIB
(classic MF only)
1.0
0.8
CD79a+ < IIB
Progression-Free Survival
(entire CTCL cohort)
1.0
Fraction Survival
Sezary
B-Cell–Positive Classic MF Only
advanced v early stages P < .05 P < .05
D
P < .05
500
0
C
P < .05
P < .001
0.4 0.2
0.8 0.6 0.4 0.2
CD20+ CD79a+ CD20− CD79a− P = .05
0
100
200
300
0
400
Time to Progression (months)
100
200
300
400
Time to Progression (months) Fig 1.
2
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JOURNAL OF CLINICAL ONCOLOGY
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Diagnosis in Oncology
A
B 06/2011
CD20
08/2011
Local 20mg
Rituximab: 20mg Day
D
8
40mg 15
22
Bexarotene 300 mg/m2
CD79a
C
1
30mg
Prior Rituximab
After 3 Weeks
After 4 Weeks
After 12 Weeks
Rituximab i.l.
FoxP3
Glucose i.l.
Prior Rituximab
E
FoxP3
After 8 Weeks
Rituximab i.l.
Glucose i.l.
CD8
CD8 Rituximab i.l.
Glucose i.l.
103
*
103 56%
400
91% T8:T4 = 0.63
102
T8:T4 = 0.05
102
300
CD4
FoxP3+ Cells/mm2
500
200 100
101
101
100
100 35%
5%
0 Rituximab
Glucose
100
101
102
103
100
101
102
103
CD8 Fig 2.
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Theurich et al
A
Skin Tumors: Identification of the B-Cell Nature of CD20+ Infiltrating Cells 103
103 CD19+CD20+
102
102
101
CD21
CD19
1,000
14%
101
500 CD45+
100
100
0 100
101
102
100
103
101
102
103
100
102
103
IgD
CD20
B
101
Skin Tumors: After 4 Cycles of Intralesional Treatment Prior
Rituximab
103 CD19+CD20−
CD19+CD20+
6%
2
10
CD19
Glucose
103
103 CD19+CD20−
11%
2
10
CD19+CD20−
CD19+CD20+
2%
0.7%
2
10
101
101
101
100
100
100
100
101
102
103
100
101
102
103
CD19+CD20+
16%
2%
100
101
102
103
CD20
C
Eosinophils
20
R
R
15
R
R
0
20
R
600
U/I
Leukocytes (%)
R
Lactate Dehydrogenase
800
10
5
400
200
0
0 0
20
40
Time After Rituximab (days)
40
Time After Rituximab (days) Fig 3.
millionIE/wk, leading to remission again. Due to INF-␣ adverse effects, methotrexate (15 mg/wk) was initiated in July 2010 intermittently combined with prednisone, PUVA, and local irradiation (2 ⫻ 4 Gy) of tumors. In November 2010, methotrexate was substituted with systemic bexarotene (300 mg/m2/d) because of generalized progres4
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sion. After a 4-month disease stabilization, lesions reoccurred including a nuchal tumor (25 ⫻ 15 ⫻ 10 mm). Treatment escalations such as total-skin electron beam irradiation or other chemotherapy were declined by the patient at that time. Evaluating treatment alternatives and based on our data, we analyzed skin biopsies of the patient which JOURNAL OF CLINICAL ONCOLOGY
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Diagnosis in Oncology
Discussion In this report, we analyzed the impact of tumor-infiltrating B cells in CTCL and found a significant correlation with advanced stages and shortened PFS. This correlation applied not only to FMF and SS with a known aggressive behavior but also to classic MF. Moreover, local depletion of B cells with rituximab led to sustained locoregional tumor remission in a patient with progressive FMF. The role of tumor-associated B cells in the tumor microenvironment is under intensive investigation.4 Several studies in solid human tumors (eg, breast, ovarian, cervical and lung cancer) suggest an improved tumor control in the presence of B cells.4,17,18 On the other hand, the majority of murine models indicate a tumor promoting effect of B cells via different mechanisms (eg, direct growth induction by lymphotoxin-, enhanced angiogenesis, or the induction of T-cellwww.jco.org
A CD24hi38hi B Cells (%)
CD24hiCD38hi B Cells in Patients With CTCL 100 80 60 40 30
P < .05
20 10
B
pB lth y
pB ea H
Skin Tumor: After 4 Cycles of Intralesional Treatment Prior
Glucose
Rituximab
103
103
CD24hiCD38hi 102
CD24
is or ia s Ps
CT
CT
CL
CL
Sk
in
pB
0
103
CD24hiCD38hi
CD24hiCD38hi
102
102
101
101
101
0
5.3% 100
100
10
100
101
102
103
100
101
102
103
4.1% 100
101
102
103
150
Tumor-Infiltrating CD24hi38hi B Cells
100
hi
CD24 38 B Cells/ 10,000 CD45+
CD38
hi
showed significant B-cell infiltrates (Fig 2A). Therefore, the patient was enrolled into an off-label treatment with local injections of the anti-CD20 antibody rituximab after written informed consent and approval of the institutional tumor board. In June 2011, the nuchal tumor was injected weekly with rituximab in increasing doses over 1 month (Fig 2B) and, as a control, the same volume of sterile glucose 5% solution was injected into another tumor on the trunk. Bexarotene was continued as an established regimen. Two days after the first injection, the patient noticed shivering without signs of infection. Furthermore, localized erythema and pain occurred at the injection site, which disappeared some days later. After three injections, clinical regression of the nuchal tumor was detectable finally leading to sustained complete remission (Fig 2C). Interestingly, computed tomography not only confirmed tumor remission, but also showed regression of locoregional lymph nodes. Histologic and flow-cytometric analyses of the rituximab-treated tumor showed B-cell depletion but also a marked depletion of CD4⫹ lymphoma cells (Figs 3B and 2E; flow cytometry). More interestingly, FoxP3⫹ regulatory T cells (Tregs) (CD4⫹CD25highCD127low, not shown) were also significantly reduced compared to the control (Fig 2D). In contrast, a significant increase (from 5% to 35%) of tumorinfiltrating CD8⫹ cytotoxic T cells was detected after rituximab treatment (Fig 2E). In August 2011, the patient consented for systemic chemotherapy with liposomal doxorubicine (20 mg/m2 d22) because of systemic progression. After initial stabilization, FMF progressed shortly after the second cycle with additional lymphadenopathy. Notably, the rituximab-treated nuchal lesion still remained in remission. After exclusion of large-cell transformation while confirming B-cell infiltration in an inguinal lymph node, we proceeded to systemic rituximab based on the previous encouraging results. Despite an initially reduced rituximab-dose (187 mg/m2), a systemic immunologic response (fever, shivering) was observed 2 days after the first infusion (October 2011). Two weeks later, the patient reported on an improvement of previously existing pruritus and cutaneous tensions. Also, a decrease of surrogate markers such as eosinophils, lactate dehydrogenase (Fig 3C) and thymidine kinase (not shown) illustrated a response. We therefore continued with the conventional rituximab dose (375 mg/ m2) for two more cycles (repeated days 18-21) resulting in further disease stabilization (cutaneous symptoms, computed tomography [not shown]). However, similar to the previous standard treatments, the response was temporary and disease progression eventually led to a fatal outcome 2 years after initial diagnosis.
50 0 Skin Tumor Prior
Skin Tumor Rituximab
Skin Tumor Glucose
Fig 4.
mediated tolerance).19-21 In our study, several CTCL specimens with notable B-cell infiltrates also showed marked infiltrates of Tregs (data not shown). This phenomenon had also been reported in a murine breast cancer model in which B cells induced an intratumoral expansion of Tregs, and the absence of B cells correlated with improved tumor control.22,23 Similarly, targeting of CD20⫹ B cells in our patient not only led to a depletion of B cells and subsequent tumor regression, but also had a profound effect on the composition of the remaining lymphocytic infiltrate. We observed a reduction in the numbers of Tregs complemented by an increase of CD8⫹ cytotoxic T cells. This suggests that depletion of B cells triggered a cascade that led to remodelling of the tumor microenvironment and reconstitution of effective immunosurveillance.24 While T-cell subpopulations with regulatory properties have been characterized fairly well, little is known about a B-cell counterpart. Recently, a CD24highCD38high transitional B-cell population has been shown to suppress T-cell responses mediated by interleukin-10.25 Interestingly, we also found markedly increased frequencies of CD24highCD38high B cells in the peripheral blood © 2014 by American Society of Clinical Oncology
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Theurich et al
and tumors from seven CTCL patients (Fig 4A). In our patient, local rituximab treatment led to a substantial depletion of this B-cell population (Fig 4B). It therefore needs to be determined whether such B-cell subpopulations are the dominant mediator of the effects of the B-cellular infiltrate. CD20 expression has been described on certain malignant cells including CTCL.26 Here, additional CD79a tissue staining and flowcytometry independently confirmed the B-cell nature of the CD20⫹ cells (Fig 3A). Rituximab is an improved drug with a low toxicity profile and its use would therefore be of particular interest in CTCL patients who are generally older with significant comorbidities. As combinational chemo-immunotherapy has proven to be effective in several hematological malignancies, the above could be combined (eg, with liposomal doxorubicine or others) to increase efficacy. Moreover, as a quite low skin penetration has been reported for standard rituximab doses,27 it needs to be determined whether dose escalation or use of secondgeneration CD20-antibodies with lower molecular size would improve results of systemic application. Taken together, we present first data suggesting that lymphomainfiltrating B cells may impact CTCL biology and that local depletion of B cells can re-establish immunologic tumor-control. Prospective studies are warranted that address if B-cell infiltrates can be used as a biomarker and whether B-cell depletion (local or systemic) can be generally applied to B-cell–rich CTCL.
Sebastian Theurich, Max Schlaak, Harold Steguweit, Lukas C. Heukamp, Kerstin Wennhold, Peter Kurschat, Anja Rabenhorst, Karin Hartmann, Hans Schlo¨sser, Alexander Shimabukuro-Vornhagen, Udo Holtick, and Michael Hallek University Hospital of Cologne, Cologne, Germany
Rudolf Stadler, Johannes-Wessling-Hospital, Minden, Germany
Michael von Bergwelt-Baildon University Hospital of Cologne, Cologne, Germany
ACKNOWLEDGMENT
We thank Alexandra Florin, Institute of Pathology, University Hospital of Cologne, for her excellent technical expertise and support and Horst Mu¨ller, PhD, German Hodgkin Study Group, Cologne, for his kind and helpful advice in statistical analysis. Christine Neumann, MD, supported this work with her long standing clinical experience in the CTCL field. We thank Werner Kempf, MD, Zu¨rich, Switzerland, for his careful re-evaluation of difficult or rare CTCL cases as a reference pathologist. Geothy Chakupurakal, MD, critically revised the manuscript and gave us valuable advice. M.S. is supported by the German Federal Ministry of Research and Education (BMBF grant 01KN1106). Part of this work was supported by the Deutsche Forschungsgemeinschaft (German Research Council, DFG) through SFB 832, to M.H. and M.B. (graduate school and project A16), to K.H. and A.R. (project A14) and to L.C.H. (project Z1). S.T and M.S. contributed equally to this work. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Although all authors completed the disclosure declaration, the following author(s) and/or an author’s immediate family member(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are 6
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those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Michael Hallek, Roche (C) Stock Ownership: None Honoraria: Peter Kurschat, Roche; Michael Hallek, Roche Research Funding: Michael Hallek, Roche Expert Testimony: None Patents, Royalties, and Licenses: None Other Remuneration: None REFERENCES 1. Hanahan D, Weinberg RA: Hallmarks of cancer: The next generation. Cell 144:646-674, 2011 2. Kirkwood JM, Butterfield LH, Tarhini AA, et al: Immunotherapy of cancer in 2012. CA Cancer J Clin 62:309-335, 2012 3. Zitvogel L, Kroemer G: Targeting PD-1/PD-L1 interactions for cancer immunotherapy. Oncoimmunology 1:1223-1225, 2012 4. Nelson BH. CD: 20⫹ B cells: The other tumor-infiltrating lymphocytes. J Immunol 185:4977-4982, 2010 5. Sorbye SW, Kilvaer T, Valkov A, et al: High expression of CD20⫹ lymphocytes in soft tissue sarcomas is a positive prognostic indicator. Oncoimmunology 1:75-77, 2012 6. Talpur R, Singh L, Daulat S, et al: Long-term outcomes of 1,263 patients with mycosis fungoides and Sezary syndrome from 1982 to 2009. Clin Cancer Res 18:5051-5060, 2012 7. Agar NS, Wedgeworth E, Crichton S, et al: Survival outcomes and prognostic factors in mycosis fungoides/Sezary syndrome: Validation of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal. J Clin Oncol 28:47304739, 2010 8. Benner MF, Jansen PM, Vermeer MH, et al: Prognostic factors in transformed mycosis fungoides: A retrospective analysis of 100 cases. Blood 119:1643-1649, 2012 9. Wu X, Sells RE, Hwang ST: Upregulation of inflammatory cytokines and oncogenic signal pathways preceding tumor formation in a murine model of T-cell lymphoma in skin. J Invest Dermatol 131:1727-1734, 2011 10. Rabenhorst A, Schlaak M, Heukamp LC, et al: Mast cells play a protumorigenic role in primary cutaneous lymphoma. Blood 120:2042-2054, 2012 11. van der Putte SC, Toonstra J, van Wichen DF: B cells and plasma cells in mycosis fungoides: A study including cases with B cell follicle formation or a monotypical plasma cell component. Am J Dermatopathol 11:509-516, 1989 12. Wasco MJ, Fullen D, Su L, et al: The expression of MUM1 in cutaneous T-cell lymphoproliferative disorders. Hum Pathol 39:557-563, 2008 13. Mattoch IW, Fulton R, Kim Y, et al: Cutaneous peripheral T-cell lymphoma associated with a proliferation of B cells. Am J Clin Pathol 131:810-819, 2009 14. Ferrara G, Chiarelli C, Simonetti S: B-cell lymphofollicular infiltrates in mycosis fungoides. Tumori 96:487-491, 2010 15. Shah S, Divekar AA, Hilchey SP, et al: Increased rejection of primary tumors in mice lacking B cells: Inhibition of anti-tumor CTL and TH1 cytokine responses by B cells. Int J Cancer 117:574-586, 2005 16. Olsen E, Vonderheid E, Pimpinelli N, et al: Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: A proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 110:1713-1722, 2007 17. Dieu-Nosjean M-C, Antoine M, Danel C, et al: Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures. J Clin Oncol 26:4410-4417, 2008 18. Nielsen JS, Sahota RA, Milne K, et al: CD20⫹ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8⫹ T cells promote favorable prognosis in ovarian cancer. Clin Cancer Res 18:3281-3292, 2012 19. Visser KE, Korets LV, Coussens LM: De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 7:411-423, 2005 20. Ammirante M, Luo J-L, Grivennikov S, et al: B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature 464:302-305, 2010 21. Qin Z, Richter G, Schuler T, et al: B cells inhibit induction of T celldependent tumor immunity. Nat Med 4:627-630, 1998 22. Tadmor T, Zhang Y, Cho H-M, et al: The absence of B lymphocytes reduces the number and function of T-regulatory cells and enhances the anti-tumor response in a murine tumor model. Cancer Immunol Immunother 60:609-619, 2011 JOURNAL OF CLINICAL ONCOLOGY
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23. Olkhanud PB, Damdinsuren B, Bodogai M, et al: Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4(⫹) T cells to T-regulatory cells. Cancer Res 71:3505-3515, 2011 24. Schreiber RD, Old LJ, Smyth MJ: Cancer immunoediting: Integrating immunity’s roles in cancer suppression and promotion. Science 331:1565-1570, 2011 25. Blair PA, Norena LY, Flores-Borja F, et al: CD19(⫹)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32:129-140, 2010
26. Martin B, Stefanato C, Whittaker S, et al: Primary cutaneous CD20-positive T-cell lymphoma. J Cutan Pathol 38:663-669, 2011 27. Amir R: Jalilian DS, Kia L, et al: Preparation, quality control and biodistribution studies of two [111In]-rituximab immunoconjugates. Sci Pharm 76:151170, 2008
DOI: 10.1200/JCO.2013.50.9471; published online ahead of print at www.jco.org on October 27, 2014
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