LETTER TO THE EDITOR

Molecular evidence of a genotypically novel large T-cell lymphoma afteranti-CD4 therapy for refractory mycosis fungoides

JOHN A. PAPALAS, SIBY SEBASTIAN, & ENDI WANG

Department of Pathology, Duke University Medical Center, Durham, NC, USA

(Received 16 October 2010; revised 13 December 2010; accepted 20 December 2010)

Introduction

Zanolimumab, a human monoclonal antibody against

CD4 isolated from human antibody transgenic mice,

has been shown to have a high response rate in patients

with refractory mycosis fungoides (MF). Morbidity

associated with this form of therapy includes mainly

eczema and opportunistic infections. While transfor-

mation has been reported in association with other

monoclonal antibody treatments for MF, an aggressive

transformation in patients being treated with zanoli-

mumab has not yet been reported. We describe the

first such case of large cell transformation in a patient

on zanolimumab, with the appearance of a distinct

clone as demonstrated by T-cell receptor gamma

(TCRG) gene rearrangement assay.

The patient was a 74-year-old white male with a

history of stage IIB MF treated 4 years earlier with total

electron beam radiation therapy. The patient had also

received Targretin (bexarotene) and Vironistat (sub-

eroylanilide hydroxamic acid), but had to discontinue

use of both due to hypertriglyceridemia and persistent

peripheral neuropathy. The patient was next treated

with topical nitrogen mustard and began oral forode-

sine (Immucillin H). All medications were again

discontinued due to disease progression and he

received palliative radiation therapy. Over the next

several months, he began to notice new palpable lesions

reappearing on the scalp and anterior tibia. Over this

time period, the patient had several skin biopsies

consistent with MF. Of note, each biopsy contained at

least a 50% CD4þ T-cell component with CD8

staining in at least 20% of the infiltrate. Using

polymerase chain reaction (PCR) based TCRG gene

rearrangement assay employing BIOMED-2 primers, a

distinct 239 bp sized product of clonally rearranged

TCRG gene was detected in each of these biopsies over

a 4-year period (Figure 1; Pre-anti-CD4 treatment).

Due to the persistent refractory disease, the patient

was started on HuMax-CD4 (zanolimumab). This was

given at 14 mg/kg per dose on a roughly weekly basis.

Two months after beginning therapy (having received

six doses in total) the medication was stopped due to

the development of new larger ulcerating skin lesions

on the head, torso, and extremities. Biopsies of these

new lesions showed a dermal based lymphoid infiltrate

with medium to large sized cells consistent with large

T-cell lymphoma (LTCL) or transformed MF. The

infiltrate was predominantly CD4þwith only rare

scattered smaller CD8þ cells. No significant staining

for CD30 or CD56 was observed. Flow cytometry was

performed on fine needle aspiration biopsy material

from adrenal nodules. Debris constituted the majority

of all analyzed events, but admixed within the area of

debris was a small population of large T-cells that

expressed CD4 and CD7, but were negative for

surface CD3 and CD8. Intriguingly, the TCRG gene

rearrangement assay revealed a novel 221 bp clonal

product that was not present prior to beginning

therapy (Figure 1; Post anti-CD4 treatment).

The patient became more short of breath, with a

chest X-ray demonstrating new heterogeneous

pulmonary infiltrates, some with nodular configura-

tion. Due to concern over the rapid progression while

on therapy, the patient underwent several cycles of

apheresis to determine whether the antibody could be

removed from the bloodstream. He was started on

high-dose steroids as well, for concern over possible

diffuse alveolar hemorrhage, but he succumbed 2 days

later. Autopsy examination demonstrated dissemi-

Correspondence: John A. Papalas, MD, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710, USA.Tel: (919)684-2454.

Fax: (919)684-1856. E-mail: Papal002@mc.duke.edu

Leukemia & Lymphoma, May 2011; 52(5): 905–907

ISSN 1042-8194 print/ISSN 1029-2403 online � 2011 Informa UK, Ltd.

DOI: 10.3109/10428194.2010.551157

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nated (cutaneous and visceral) LTCL. Multiple

cutaneous skin nodules, up to 5 cm in diameter on

the head (including mucosal surfaces), trunk, abdo-

men, and both lower extremities, were identified.

Lymphomatous infiltrate was also noted in the

pancreas, adrenal glands, lungs (interstitial and alveo-

lar), kidneys, liver, stomach, heart, and bone marrow.

TCRG gene rearrangement assay performed on

autopsy specimens of lung tissue, axillary lymph

node, and a skin nodule showed the same 221 bp

clone in all these specimens (summarized in Table 1).

Additional studies employing sequence analysis re-

vealed that the 239 and 221 bp PCR products

harbored clonally rearranged TCRG gene variable

regions Vg2 and Vg5, respectively. These results

provide molecular evidence for the utilization of

different TCRG gene variable region sequences/

regions by clonal T-cells represented by PCR products

of 239 and 221 bp using the TCRG gene rearrange-

ment assay.

Zanolimumab (Genmab) is an anti-CD4 human

monoclonal immunoglobulin G1 (IgG1) antibody

(also known as HuMax-CD4) targeting the CD4

receptor, which is highly expressed on the vast majority

Figure 1. Left: Histopathology before anti-CD4 therapy demonstrating small, atypical cells which express CD4, (239 bp sized product of

clonally rearranged TCRG gene). Right: Histopathology after anti-CD4 with large atypical cells which express CD4 (distinct 221 bp sized

product of clonally rearranged TCRG gene).

Table I. Timeline of clinicopathologic and molecular progression.

Time biopsy taken Site Diagnosis CD4/CD8/CD5/CD7* Clone size (base pairs){

Pretreatment

4 years prior Left leg MF 50%/20%/30%/40% 239

3 years prior Right arm MF 50%/50%/—/40% 239

1 year prior Scalp MF 80%/20%/—/40% 239

HuMax treatment (day 0)

53 days after Chest/thigh LTCL 95%/0%/—/70% 221

Death (day 74) Chest LTCL 95/5%/—/70% 221

*Percentage of staining within the CD3-positive T-cell population.{Clones detected by T-cell gamma chain PCR using T-cell receptor gene rearrangement (TCRG) Gene Clonality Assay (InVivoScribe

Technologies) utilizing BIOMED-2 primers.

MF, mycosis fungoides; LTCL, large T-cell lymphoma; PCR, polymerase chain reaction.

906 Letter to the Editor

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of malignant T-cell lymphoma cells [1]. Recently, the

use of zanolimumab has been shown to have a high

response rate in patients with refractory MF in clinical

phase 2 studies [2]. Morbidity associated with this

form of therapy includes mainly eczema, opportunistic

infections, and rarely muscle fiber rupture [2]. While

acute transformation has been reported in association

with monoclonal antibodies targeting other cell surface

molecules [3], large cell transformation characterized

by the development of a new malignant clone has not

been reported in patients on zanolimumab.

Transformation of cutaneous T-cell lymphoma

(CTCL) to large cell lymphoma is a well-recognized

phenomenon that occurs in a minority (approximately

10–20%) of patients with CTCL [4,5]. Lack of CD30

expression, as in the current case, by the transformed

cells may be a poor prognostic indicator [6]. Currently,

little is known about the molecular relationship

between transformed large cell MF and its precursor

clone. Some investigators have shown both the

transformed large cells and the precursor lower stage

MF cells to share the same precursor clone, based on

TCRG gene rearrangement [7]. One study that

specifically addressed clonal heterogeneity in a subset

of patients with MF with serial skin biopsies found no

statistically significant correlation between the TCRG

clonal heterogeneity and clinical progression [8].

Animal models have shown a decrease in the number

of CD4þT-lymphocytes in the skin of mice treated

with zanolimumab [9]. This reduction in CD4þT-

cells is thought to be mediated in part by killing via

antibody-dependent cellular cytotoxicity [2]. Prior to

beginning therapy, the patient’s skin biopsies consisted

of a mixture of predominantly CD4þ and to a lesser

degree CD8þT-cells. CD7 expression was not noted

to be decreased to any significant extent. Skin biopsies

taken 57 days after the start of treatment revealed a

predominantly CD4þ (95%) large T-cell lymphoma.

CD8 expression was markedly diminished. Further-

more, molecular analysis revealed a new 221 bp PCR

product with the TCRG gene rearrangement assay. We

hypothesize that the 239 bp clone was effectively

eliminated by the medication, as TCRG gene rearran-

gement assay failed to detect this clone after the patient

received treatment. Furthermore, the transformed

malignancy now comprised medium to large cells, an

immunophenotypically similar but genotypically dis-

tinct T-cell neoplasm. What is less clear is whether the

new, transformed, large T-cell lymphoma was latently

suppressed by the dominant 239 bp clone, or whether it

arose de novo as either a treatment-related process or as

an unrelated transformation that was part of the natural

history of this patient’s disease. However, a close

association of transformation with HuMax-CD4 may

be suggestive of a possible culpable factor of the

medication.

In conclusion, we describe a patient treated with

zanolimumab for refractory MF who rapidly devel-

oped diffuse large cell transformation. TCRG gene

rearrangement analysis performed both on biopsies of

tissue from the new lesions developing after treatment

and on postmortem lung, lymph node, and skin

samples all disclosed the same 221 bp sized clonal

product, which is genotypically distinct from the 239

bp clone detected prior to beginning therapy. We

favor this to represent large cell transformation in a

patient with refractory MF, 2 months after treatment

with zanolimumab. Until the relationship between

transformed MF and its precursor disease is better

characterized in terms of TCRG gene rearrangement,

cases such as this underscore the importance for close

clinical and molecular monitoring of such patients as

they undergo monoclonal antibody therapy.

Acknowledgements

We would like to thank Jeffrey E. Miller PhD, Jay

Shaw PhD. and Ms. Rebecca Ringler MS. of

Invivoscribe Technologies, Inc., San Diego, Califor-

nia for performing the sequence analysis of the

TCRG gene PCR products.

Potential conflict of interest: Disclosure forms

provided by the authors are available with the full text

of this article at www.informahealthcare.com/lal.

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cutaneous T-cell lymphoma. Blood 2007;109:4655–4662.

3. Faguer S, Launay F, Ysebaert L, et al. Acute cutaneous T-cell

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