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Consultations in Molecular Diagnostics |
-Chain Gene Polymerase Chain Reaction to Diagnose Central Nervous System Involvement by Cutaneous T Cell Lymphoma
From the Departments of Neurology,
*
Dermatology,
and Pathology and Laboratory Medicine,
Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania
Abstract
The authors describe a patient who was suspected of having
cutaneous T cell lymphoma involvement of the brain despite repeatedly
negative cerebrospinal fluid (CSF) cytology, inconclusive flow
cytometry, and no discrete lesion for brain biopsy. The
diagnosis was made by polymerase chain reaction (PCR) analysis that
showed a monoclonal T cell receptor 
-chain gene rearrangement in the
CSF, identically sized to that present in a skin biopsy
specimen. Thus, PCR could be used early and routinely to
diagnose central nervous system spread of T cell lymphomas,
because of its potentially superior sensitivity and specificity to CSF
cytology.
Cutaneous T cell lymphoma (CTCL) is a low-grade, non-Hodgkin’s lymphoma characterized by a malignant clone of helper T lymphocytes that homes to the skin to produce pruritic and scaly patches and plaques, tumors, and erythroderma. In advanced stages, malignant cells with hyperconvoluted nuclei (ie, Sézary cells) circulate in the peripheral blood, invade lymph nodes, and, more infrequently, spread to visceral organs such as the lungs, bone, gastrointestinal tract, and central nervous system (CNS). Autopsies have shown CNS spread to the meninges and almost any part of the brain parenchyma.1 However, symptomatic CNS involvement is uncommon, as demonstrated by a review that found only 40 total reported cases and 28 reported cases in the English literature.2 The most frequent neurological symptoms include mental status changes, motor and gait disturbances, and cranial nerve deficits.2 Symptoms arise as early as 4 months and as late as 30 years from the time of initial diagnosis, with mean time to onset described as 3.8 years to 7.5 years.2, 3 Pre-mortem diagnosis is usually made by cerebrospinal fluid (CSF) cytology, although cytology is often negative and the diagnosis is made at autopsy.2, 4 Brain biopsy is an alternative diagnostic method.3, 5
We describe a patient who developed gait abnormalities, mental status
changes, and brain magnetic resonance (MR) imaging abnormalities 6
years after the initial diagnosis of CTCL. Cytology was repeatedly
negative, flow cytometry was inconclusive, and a brain biopsy was not
performed because there was no discrete lesion for biopsy. Polymerase
chain reaction (PCR) analysis of the T cell receptor (TCR) -chain
gene of the CSF showed a monoclonal rearrangement, indicative of a
monoclonal T cell population that was identical to that evident in a
skin biopsy with known tumor involvement. CNS-active chemotherapeutic
agents were subsequently initiated.
Case Report
A 70-year-old woman developed multiple erythematous and scaly patches and plaques, which were diagnosed as CTCL by skin biopsies in 1994. The disease was controlled with a combination of psoralen and ultraviolet light A, topical corticosteroids and nitrogen mustard, and intramuscular methotrexate for six years. In April 2000, she had recrudescence of cutaneous disease with tumors of the scalp and orbit.
In August 2000, she developed neurological symptoms of disorientation, poor concentration, a slow magnetic gait, and postural instability leading to multiple falls. These neurological symptoms worsened over the ensuing months and by November 2000, she was oriented only to name, was attentive only to simple tasks, and was bed-bound. An initial brain MR imaging scan in August was unrevealing, but subsequent MR scans over the next 2 months showed increased signal intensity on T2-weighted and fluid-attenuated inversion recovery images in the periventricular white matter and right centrum semiovale, extending into the right cerebral peduncle, pons, and both basal ganglia. There was minimal enhancement outlining the basal ganglia after gadolinium administration.
Initial lumbar puncture revealed 12 white blood cells per µl that
were 63% polymorphonuclear neutrophils, 17% morphologically normal
lymphocytes, 19% monocytes, and 1% eosinophils with a protein level
of 62 mg/dl. Bacterial and fungal cultures were negative. Four
additional lumbar punctures showed a similar cellular profile (Figure 1)
. The cytopreparations were Wright (Fisher Healthcare, Houston, TX),
Diff-Quik- (EM Science, Gibbstown, NJ), and Papanicolaou-stained (EM
Science) preparations. The first was initially reviewed by a
hematopathologist (A.B.), and the latter two by a cytopathologist
(C.M.), who independently came to the same conclusion, namely the
absence of atypical cells. Following the identification of
monoclonality at the molecular genetic level, both parties subsequently
reviewed all preparations in conference and the negative cytology was
reconfirmed. CSF flow cytometry on three specimens failed to identify a
significantly expanded, immunophenotypically aberrant T cell
population. On one occasion, 8% of the cells, accounting for less than
1 cell per µl were CD2+,
CD4+, and CD7-. Serum B12
and angiotensin converting enzyme (ACE) levels were normal, and a rapid
plasma reagin (RPR) and treponema pallidum antibody (TPA) were
negative. Human immunodeficiency virus serology was unreactive. PCR
amplification assays of the CSF for varicella-zoster virus, JC virus,
and cytomegalovirus were negative.
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-chain gene rearrangement studies to identify a clonal population of
T cells were performed on cerebrospinal fluid specimens.
Two separate skin biopsy specimens from the patient, that were
histologically and immunophenotypically diagnostic of CTCL, were used
as positive controls, and a peripheral blood sample from the patient
was used as a negative control. The negative control of the patient’s
blood sample was additionally important to eliminate the possibility of
a false positive CSF result due to contamination by circulating
Sézary cells from a bloody tap. DNA was extracted from the CSF, a
paraffin-embedded skin biopsy and peripheral blood samples by standard
techniques using a commercially-available kit, and following the
manufacturer’s instructions (Qiagen, Valencia, CA). The DNA was then
quantified spectrophotometrically. All samples were similarly handled
however, the CSF specimens (5 to 10 ml) were first centrifuged to
concentrate the cellular contents and then washed with
phosphate-buffered saline before DNA extraction. PCR amplification was
performed using two sets of fluorescently labeled primers that are
complementary to the V and J regions of the TCR gene, as previously
described.6
One set of primers used to perform PCR amplification contained a single
upstream primer, a consensus to V1–8, while the second set
contained three upstream primers, one each to V9, V10, and
V11. The PCR amplification products were resolved using an automated
high-resolution capillary electrophoresis system (ABI 310; Perkin Elmer
Biosystems, Foster City, CA) with the following expected product sizes:
V1–8 primer (200 to 250 bp; midpoint, 225 bp) and V9–11 primers
(150 to 200 bp; midpoint, 175 bp). No consistent and reproducible
monoclonal peak was discerned with the V1–8 primer in either the
CSF or in the skin. Using the V9–11 primer set however, the
analysis of both the CSF and skin biopsy specimen revealed the same two
dominant amplification products of 
175 and 194 bp (Figure 2)
. These PCR products represent a biallelic, monoclonal TCR -chain
gene rearrangement, indicative of a clonal T cell population. By
contrast, the same analysis on peripheral blood DNA displayed a
polyclonal pattern, without the aforementioned dominant amplification
products, indicating that there were no detectable circulating clonal T
cells.
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The patient was treated with high doses of intravenous steroids and methotrexate which resulted in an improvement in her mental status and a return of her ability to ambulate. The methotrexate was switched to Ara-C 4 months later, because of progressive renal failure. The patient died 1 month later (5 months from the time of initial diagnosis) at another hospital, where an autopsy was not performed, precluding the exact determination of the cause of death.
Discussion
The prognosis of CTCL involvement of the CNS is poor with a mean
survival from the time of CNS disease diagnosis of about 3.9
months.2
Earlier diagnosis and treatment can potentially
improve outcome. The TCR -chain gene rearrangement PCR assay has
already been shown to be a highly sensitive technique for detecting
monoclonal T cell populations in skin biopsies of patients with CTCL,
with the ability to detect a dominant clone constituting 0.01% to
0.1% of total specimen cells.7
The added sensitivity of
this PCR-based assay provides the potential for earlier detection of
clonal T cell populations in the CSF. A single positive PCR result, in
isolation, does not imply the presence of monoclonality, with
pseudoclonality8
(due to low numbers of lymphocytes) and
canonical rearrangements9
(of the TCR -chain gene)
being potential sources of confounding results. However, the ability of
capillary electrophoresis to identify an accurate and precisely sized
"fingerprint" of the T cell clone provided a tumor-specific marker
(identical to that obtained at a remote, involved site), which allowed
for the diagnosis of CNS involvement, in the absence of
"conventional" pathological findings. In support of this was the
patient’s initial response to appropriate systemic therapy, and
subsequent clinical course. To our knowledge, this is the first such
reported case highlighting this finding.
Footnotes
Address reprint requests to Robert A. Taylor, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104. E-mail: rat{at}mail.med.upenn.edu
Accepted for publication February 26, 2002.
References
gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis. Mol Diagn 2001, 6:169-179[Medline]
9-JP rearrangements in unstimulated human /
T lymphocytes. Eur J Immunol 1992, 22:2437-2443[Medline]
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