—  SPECIALTY CONFERENCE  —

Dermatopathology

Case 6 - Primary Cutaneous CD10/CD8 Positive T-cell Lymphoma with Dual B and T Cell Rearrangement and Co-expression of CD20

Cynthia M. Magro
Ohio State University
Columbus, OH


Click on each slide thumbnail image for an enlarged view
Clinical History
The patient was a 65 year old woman who first presented in 2001 with a left posterior thigh pea sized nodule which initially resembled a mosquito bite and then continued to increase in size. She was asymptomatic at this time. She had been on multiple pychotropic drugs, antihypertensives and a lipid lowering agent prior to the development of her skin lesions. She had a skin biopsy performed which was initially interpreted as representing a T cell lymphoblastic lymphoma based on TdT and CD10 expression although the tumor cells were CD34 negative. Because there was no evidence of extracutaneous disease, it was decided to treat her with radiation. Complete regression of the tumor occurred . The left thigh tumor then relapsed in September 2003. The clinical presentation was similar in that the tumor nodules were relatively small and few in number with none exceeding 1 centimeter in size. Once again staging studies failed to disclose any evidence of extracutaneous disease. She received 4 cycles of rituxmab in January 2004 based on the aberrant expression of CD20 with no response followed by radiation which lead to lesional resolution. However the lesions recurred in the same area. She was then given CHOP which was completed in August 2004 with complete resolution of her skin nodules. She subsequently developed a recurrence in November of 2004. At the time of this recurrence, repeat staging scans as well as bone marrow biopsy continue to be negative. Her most recent exam in December 2004 revealed multiple large tumor nodules affecting the back of her thigh, the popliteal fossa, and the calf with the largest tumor measuring 3 x 4 centimeters. She has recently been started on Ontak and has shown a dramatic response to therapy, with significant regression of the tumor nodules. Th biopsies that have been obtained over the course of 3 years appeared similar, showing a diffuse infiltrate that effaced the dermal architecture. The cells were large with prominent nucleoli. Phenotypic studies showed CD2,CD3,CD5, CD8 and CD10 positivity. TdT was positive only on her first biopsy. CD20 expression was seen in all biopsies and varied in intensity from very weak to moderately intense. Molecular studies showed both TCR beta and heavy chain immunoglobulin rearrangement. The biopsy following 1 week of Ontak therapy has demonstrated a striking reduction in the density of tumor cell infiltration, with dominant localization of the residual malignant infiltrate within the panniculus.


Case 6 - Figure 1 - There is a diffuse and nodular pan dermal lymphocytic infiltrate associated with effacement of the dermal architecture.
Case 6 - Figure 2 - Higher power magnification reveals that the cells have a large pleomorphic appearance. The cells are in the 20 micron size range manifesting prominent nucleoli. There was also perineural accentuation with focal endoneurial extension.



Case 6 - Figure 3 - Phenotypic studies revealed that the infiltrate was a T cell showing CD8 positivity and weak CD20a nd CD10 expression.
Case 6 - Figure 4 - A) GeneScanning image for TCR beta gene rearrangement shows a single distinct peak of 249 base pairs in size indicative of monoclonality with no evidence of a polyclonal background. B) GeneScanning image for IgH gene rearrangement shows two distinct peaks of 91 and 102 base pairs in size indicative of a dual clonal population (oligoclonal). C) Positive control.

Comment
The potential phenotypic and molecular heterogeneity encountered in cutaneous T cell lymphoma is well exemplified by this case. Its uniqueness lies in its phenotypic profile which was that of a mature post thymic CD8 T cell lymphoma manifesting CD10 positivity and weak CD 20 expression. Furthermore from a molecular perspective, the tumor showed both B and T cell clonality demonstrated via PCR.

Firstly in regards to CD10 expression, the literature precedent describing CD10 positivity in primary cutaneous T cell lymphoma is largely limited to two reports represented by one patient with Sézary syndrome and one case of peripheral CD8 T cell lymphoma secondarily involving the skin with concurrent lymph node and leukemic involvement.

This case as well as other prior case reports illustrate an important point which is that CD10 expression does occur in the context of malignant cutaneous T cell infiltrates other than lymphoblastic lymphoma and or angioimmunoblastic lymphadenopathy. CD10, also referred to as neutral endopeptidase peptidase, was discoveredon the surface of acute lymphoblastic leukemia cells and was held to be a tumor-specific antigen. However it has become increasingly apparent that normal cells at certain stages of maturation or function can demonstrate CD10 expression. CD10 is expressed by immature T and B cells, centroblasts and centrocytes within germinal centers of lymphoid follicles, and benign T cells undergoing apoptosis. There is a growing body of literature which suggest that CD10 expression maybe related to apoptosis. In the seminal study by Cutrona and co-workers , cells procured continuous T-cell lines which did not normally express CD10 became CD10(+) when induced into apoptosis by human immunodeficiency virus (HIV) infection and or with exposure to CD95 monoclonal antibody, etoposide, or staurosporin. In contrast addition of inhibitors of caspases blocked apoptosis and attenuated CD10 expression. Both subsets of T cells (i.e. CD4 and CD8) exhibited this phenomenon of CD10 expression upon induction of apoptosis. A characteristic feature of Burkitt's lymphoma cells is apoptosis and indeed this tumor expresses abundant CD10. As alluded to above, the neutral endopeptidase activity results in the cleavage of proteins with inflammatoryor proinflammatory activity released by dying lymphocytes. The CD10 while not directly triggering apoptosis represents a safety device to reduce the impact of the inflammatory milieu triggered by the apoptotic environment. Secondly CD10 may degrade cytokines that would normally inhibit T-cell lymphocyte apoptosis, thereby potentiating an alternative and novel pathway which promotes cell death.

The second unique aspect of this case was the co-expression of CD20 and presence of T cell receptor and heavy chain immunoglobulin rearrangement. There are a limited number of prior citations alluding to the aberrant expression of CD79a and CD20 in peripheral T cell lymphomas with only one case in the context of cutaneous lymphoma . In one case report, the disease pursued an aggressive clinical course whereby the patient died of disease 6 weeks after presentation. Immunohistochemical studies demonstrated expression of both T- and B-cell-associated antigens, including CD3, CD8, CD43, TIA-1, CD20, and CD79a. Other markers expressed by the tumor cells included CD56 and S-100. Molecular studies by polymerase chain reaction (PCR) demonstrated clonal TCR-gamma chain gene rearrangement without evidence for a clonal rearrangement of the immunoglobulin heavy chain gene. Another case report described pancytopenia and hepatosplenomegaly in a 60 year old female on the basis of a clonal proliferation of atypical lymphocytes that were weakly positive for CD20; the cells were also of the CD8 subset. While the molecular studies showed T cell clonality a germline configuration of the immunoglobulin heavy chain gene was observed. In an interesting study by Warzynski, they reported low level antibody binding capacity for CD20 in three cases of T acute lymphoblastic leukemia and emphasized that this low level expression for CD20 was in contrast to the high level antibody binding capacity seen in B cells be it benign or malignant counterparts. A fourth study investigated CD79a and CD20 reactivity in 94 extranodal non-B-cell lymphomas. One cutaneous peripheral T cell lymphoma had a CD20 positive phenotype while 3 enteropathy associated lymphomas and one NK like T cell lymphoma showed CD79 positivity although in the absence of CD20. All five B-cell marker-positive extranodal lymphomas had a CD8 phenotype. Our patient's phenotype is consistent with these previously reported cases by virtue of its CD8 positive phenotype.

The question arises as to whether or not this expression of CD20 represents a truly aberrant phenotype or malignant transformation of a subpopulation of T cells which may normally express CD20. The CD20 molecule has always been held to be a marker of B cell differentiation. It is rich in internal serine and threonine residues that provide multiple sites for phosphorylation; CD20 is phosphorylated when B cells are activated. It has been suggested that the CD20 molecular is a unique Calcium channel and or is closely associated regulator of such a channel. CD20 expression is lost during terminal B-cell differentiation into plasma cells.

Hultin and co-workers were the first to detect using flow cytometry the presence of a population of low antibody binding capacity CD20 positive T cells in the peripheral blood. Extensive three-color immunophenotypic analyses indicated that CD20dim T cells were phenotypically heterogeneous and displayed a leukocyte differentiation profile that was slightly different than that of CD20- T cells. The CD20dim T cells were more likely than CD20- T cells to be gamma/delta T cell antigen receptor positive (14% vs. 3.4%), CD8+ (57% vs. 33%), and CD45RO+ (82% vs. 51%); fewer were CD38+ (5% vs. 24%) or CD4+ (35% vs. 61%). Qunitanilla-Martinez and co-workers examined specimens of peripheral blood lymphocytes from healthy donors for evidence of a CD20+ T-cell population by using three-color immunofluorescence analysis by flow cytometry. They observed two distinct populations of CD20+ cells: one had a bright signal and expressed other B cell markers and the other had a very dim signal whereby the cells coexpressed CD3, representing 3% of all circulating lymphocytes. Two thirds of these cells were of the CD8 subclass. In the study by Algina and co-workers, a small population of cells coexpressed CD3 and CD20 dim was identified in 94% of bone specimens, representing 1.77% of marrow mononuclear cells and 6.54% of marrow lymphoid cells. CD20dim positive cells included immature B cells and CD20-positive T cells. All of these studies clearly indicate that CD20 expression in T cell lymphomas may not represent an aberrant phenotype per se but rather may be one of malignant transformation of a small population of T cells which normally express CD20. Indeed in our case the weak cytoplasmic expression of CD20 and the fact that the cells were of the CD8 subtype were consistent with this hypothesis.

The actual function of CD20 on T lymphocytes is not known at least in humans. However in monkeys its expression on lymph node derived T lymphocytes was increased when exposed to mitogen and interleukin 2 suggesting that its expression may be associated with a state of activation. This is, a point of interest in this case given the patient's complex drug history with various immune dysregulating agents which have been shown in vitro to have mitogenic effects on lymphocytes and or suppress T suppressor function. In this particular case while the CD20 expression initially was very weak one of the more recent recurrence showed at least focally very strong expression of CD20. It has been postulated that the expression of CD20 bright and CD20 dim populations reflect modifications of the CD20 molecule. The brightness of staining reflects the binding capacity of the CD20 antigen for the CD20 antibody whereby dim CD20 cells proliferate at a higher rate than those cells showing bright expression. Others have shown T cell lymphomas expressing both CD20 bright and dim staining cells.

With respect to the dual rearrangement, heavy chain immunoglobulin rearrangement occurs very early in B cell development while the addition of CD20 occurs later in pre B cell development and still while expressing CD10 and TdT. In T cell ontogeny, precursor T cells first undergo gamma delta chain rearrangement and then undergo beta alpha chain rearrangement, subsequently becoming either CD4 or CD8 lymphocytes. Hence the cell population comprising this lymphoma is unusual in that it has phenotypic and molecular features that one associates with both a later stage pre-B cell and a mature T cell. The molecular studies in this case suggest that the heavy chain immunoglobulin rearrangement occurred subsequent to the TCR beta rearrangement given the presence of a single population of T cells while there were two disparate populations of B cells. The phenotypic and molecular profile in our case to a certain extent recapitulates the pre-B lymphocytes of PAX 5 deficient mice which are transplanted into RAG deficient hosts. Specifically these cells can differentiate into normal CD4 or CD8 lymphocytes and express a clone specific DhJh rearrangement. No further studies have been done to date on non-neoplastic dim CD20 T cells, a possible benign counterpart of CD20+ T cell lymphomas, to ascertain if these cells do exhibit heavy chain immunoglobulin rearrangement.

The differential diagnosis in this case is clearly with T cell lymphoblastic lymphoma. Indeed there are many aspects of the phenotypic profile that would be most compatible with this diagnosis including the initial expression of TdT and CD10 and B cell lineage infidelity. However subsequent biopsies failed to show TdT positivity. The classic clinical presentation of precursor T lymphoblastic leukemia/lymphoma is one of mediastinal involvement with a high peripheral blood white count. The primary and exclusive presentation in the skin would be exceptional with the literature precedent represented by 5 prior cases, all with extracutaneous dissemination either concurrently or subsequent to the cutaneous presentation. In contrast this patient despite multiple relapses has remained asymptomatic with disease remaining localized to the skin . Due to the failure to demonstrate TdT positivity on any of her subsequent recurrences and the demonstration of strong CD8 expression in the tumor cells, the ultimate categorization of her neoplasm was as a post thymic T cell lymphoma rather than a precursor T cell lymphoblastic lymphoma.

Her most recent response to denileukin diftox is of interest, emphasizing the emerging role of biologics in the treatment of refractory cutaneous T cell lymphoma. This agent is among the recombinant toxins which have been approved for the treatment of advanced primary cutaneous lymphoma. Recombinant toxins are proteins made by genetic engineering consisting of a toxin fused to a ligand which binds selectively to a target cell. Recombinant toxins used for cancer treatment generally contain either a growth factor or a recombinant fragment of a monoclonal antibody fused to a truncated bacterial toxin, derived either from Pseudomonas exotoxin or from diphtheria toxin. Denileukin diftitox, represents a recombinant protein composed of the full-length sequence of human interleukin-2 fused to the catalytic domain of diphtheria toxin. Denileukin diftitox has potent selective killing activity against activated lymphocytes that express the medium and high affinity interleukin-2 receptor (IL-2R). Entry of a single molecule of ONTAK into the cytoplasm induces apoptosis.

In conclusion the patient presented this evening has a CD10 positive primary cutaneous T cell lymphoma possibly derived from the benign counterpart of weakly CD20 positive T cells. Because this population of cells is more frequently CD8 positive, it is not surprising that this case along with the other previously reported cases are usually of the CD8 subset. There is really very limited literature regarding this subset of peripheral blood T lymphocytes. The mechanism by which CD20 expression develops has been unclear. What is interesting in our case is the fact that the neoplastic cells do show a heavy chain immunoglobulin rearrangement in addition to a T cell receptor rearrangement. Further studies are clearly needed to better define the dim CD20 positive T cells and their malignant counterparts.

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