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Chin Med J (Taipei) 1997;59:132-5.
Section of Medical Oncology, Department of Medicine, Veterans General Hospital-Taipei; and National Yang-Ming University, Taipei, Taiwan, R.O.C.
The cytologic diagnosis of lymphoid cells in pleural effusion is not readily apparent. The atypical lymphocytes in reactive process or viral infection may mimic immature lymphoblasts in lymphoblastic lymphoma. Therefore, an objective and reliable method to identify malignant lymphoid cells is important. Here we reported the application of T cell receptor (TCR) gene analysis, together with cytomorphology and immunophenotypic studies, to establish the diagnosis of T-lymphoblastic lymphoma in a 20-year-old female who presented with a mediastinal tumor and pleural effusion. The diagnosis of malignant lymphoma was suspected by cytology and then confirmed by TCR beta chain (TCR beta) gene rearrangement. The rearrangement of TCR beta gene revealed by Southern blot technique implied a T- monoclonal lymphoid population which is most likely malignant. Immunophenotypic studies revealed expression of TdT and T cell antigens indicating a T lymphoblastic lymphoma. As shown in this case, antigen receptor gene analysis can be conducted with a much smaller number of cells to identify a malignant clone among a heterogeneous cell population.
[Chin Med J (Taipei) 1997;59:132-5.]
Keywords: antigen receptor gene, lymphoblastic lymphoma, T cell receptor gene
Received: December 14, 1996.
Accepted: October 3, 1996.
Address reprint requests to: Jin-Hwang Liu, M.D., Section of Medical Oncology, Veterans General Hospital-Taipei, No. 201, Section 2, Shih-Pai Road, Taipei, Taiwan, R.O.C.
Diagnosis of malignant lymphoma is usually made by histopathology. However, adequate tissue may not always be available when there is an inaccessible anatomic tumor site or other clinical difficulties. In such a condition, analysis of clonality is helpful to diagnosis [1,2]. Analysis of antigen receptor gene merits high sensitivity in detecting a lymphoid monoclone in a heterogeneous cell popupation. A lymphoid monoclonality implies a lymphoproliferative malignancy, just as most other malignant hematological disorders are of monoclonal origin. Unfortunately, this rule is rarely but occasionally confused by the fact that all that is monoclonal is not necessarily malignant. A list of clonal disorders, not necessarily malignant, includes large granular lymphocytosis [3], Sjogren's disease [4], Castleman's diseas [5], immunodeficiency-related lymphoproliferation [6], monoclonal gammanopathy of undetermined significance and other diseases. However, antigen reoptor gene rearrangement may still offer an alternative for diagnosis of a malignant lymphoproliferative disease when it is interpreted in conjunction with clinical, morphologic, and/or immunologic features. Herein, a case of malignant lymphoma which was manifested by a mediastinal mass and pleural effusion is presented. The diagnosis of malignant lymphoma suspected by cytology is solidified by evidence of TCR beta gene rearrangement as well as by immunophenotyping.
A 20 year-old female was admitted with the chief complaints of progressive dyspnea and puffy face for 2 months. Meanwhile, edema of the upper limbs had been noted for one week. The patient denied night sweating, body weight loss or fever.
At admission, she looked ill but was clearly conscious. Her blood pressure measured 124/65 mmHg; body temperature 37.4OC; respiratory rate, 22/ min and pulse rate, 126/min. Moderate cardio-pulmonary distress was manifested by orthopnea and tachycardia. Superior vena cava (SVC) syndrome was impressed by her puffy face, swollen upper limbs and engorged veins of the upper chest.
Chest film revealed anterior superior mediastinal masses and bilateral pleural effusions. Serum biochemistry showed elevated lactic dehydrogenase (LDH) up to 1223 IU/L. Sono-guided biopsy was attempted for a histological diagnosis, but failed because of the patient's restlessness. Furthermore, respiration distress prevented her from undergoing thoracotomy or mediastino-scopy. Nevertheless, the analysis of pleural effusion facilitated diagnosis-making. The white cell count of her pleural effusion was 3000/cumm. Morphologically, many anisocytic lymphoid cells with irregular nuclei and moderately prominent nucleoli were seen (Figure 1). Hence, a picture of malignant lymphoma was impressed. However, since some reactive conditions such as viral infection might have similar pictures and, in addition, the nucleoli of lymphocyte looked more prominent in pleural effusion especially in the cytospin-prepared smear, some diagnostic uncertainties remained to be excluded [7]. Some cells from the pleural effusion were also harvested by centrifugation and taken for analyses of antigen receptor gene and of immunophenotype as previously described [8]. Immunophenotyping for cells from the pleural effusion showed TdT+ cells were 90%; CD3+, 97%; CD7+, 96%; CD2+, 94%; CD4+, 0% and CD8+, 0%. This suggested a T-cell lymphoblastic Iymphoma instead of a reactive process. The DNAs extracted were digested with EcoRI, HindIII and BamHI restriction enzymes. Southern blot showed germ lines in the restriction-digested DNAs after hybridization to the immunoglobulin (Ig) heavy chain, and kappa and lambda light chain, probes. However, as shown in Figure 2, these EcoRI-, HindIII- and BamHI-digested DNAs had rearranged bands after hybridization to TCR beta probe. The TCR beta probe, provided by Dr.T. W. Mak (Ontario Cancer Institute, Toronto, Canada), contained the 0.8 Kb PstI DNA fragment encoding the constant and joining region of TCR beta.
Thus in cells from the pleural effusion, a T cell monoclone existed. In conjunction with the invasive behavior of this tumor, the diagnosis of T cell lymphoma was then evident by TCR beta gene rearrangement as well as by immunophenotyping. The possibility of malignant lymphoma of subtypes other than lymphoblastic lymphoma was also be excluded by the expression of TdT antigen. This patient then undertook lymphoblastic lymphoma-directed chemotherapy [9] and achieved complete response in two months.
Mediastinal tumor is the most frequent cause leading to a SVC syndrome which is a medical emergency [10]. Definite diagnosis of a mediastinal tumor still depends on histopathology. Tissue characterization with some non-invasive techniques, e.g. computerized axial tomography and magnetic resonance image study, is usually not sufficient for classifying malignant tumors, and frequently not even for distinguishing malignant from benign tumors. Procedures aiding histopathological diagnosis in new cases of SVC syndrome with mediastinal tumor include sonoguided tumor biopsy, mediastinoscopy and mediastinotomy [11]. However, with these procedures, some cases still remain undiagnosed [11]. Under such circumstances, cytology provides good assistance to diagnosis-making.
But in the absence of tissue texture, the distinction of lymphoma cells from reactive lymphoid cells, especially in body fluids, may not be clear [7]. In that case, immunochemistry may help make diagnosis but also may fail, especially in a heterogeneous cell population. Molecular genetic study which merits high sensitivity in picking out a specific clone from a heterogenous population offers an alternative to provide evidence of malignancy. Among the molecular studies, clone analysis is one of the most powerful approaches, especially for hematological malignancies [1,2]. For this, analysis of antigen receptor genes, Ig and TCR genes, has been employed widely to exhibit the clonality of lymphoid populations. Analysis of Ig or TCR genes by Southern blot technique allows detection of clonal populations comprising as little as 0.15% of the total cells; therefore, can provide support for the diagnosis of lymphoid neoplasm, when histologic and immunologic techniques are inconclusive [1].
A general rule is that malignant Iymphoid tumors exhibit clonal antigen receptor gene rearrangement, whereas benign reactive lymphoid hyperplasias do not. However, this rule is not absolute, although nearly all monoclonal lymphoid disorders which do not behave in a malignant fashion have a predisposition to malignant progression [12]. Therefore, the presence of an invasive clinical behavior or malignant immunophenotypic features would link more convincingly the monoclonality of lymphoproliferative disease to a malignancy.
The immunophenotypic criteria for the diagnosis of non-Hodgkin's lymphoma has been documented by many investigators [12-14]. The criteria for T-cell malignancy include evidences of monoclonality and abnormal antigen expression, such as loss of pan-T antigens and loss or coexpression of T-subset antigens. It has been reported that about 80% of T- lineage neoplasms presented these immunophenotypic abnormalities and distinguished them from benign, reactive lymphoid process.
In this case, the presence of monoclonal T cell population in pleural effusion was unequivocally demonstrated by rearrangements of TCR beta gene in 3 enzyme-restricted DNAs and the overwhelming T cell population of uniform subtype markers. Monoclonality in conjunction with the invasive behavior of this tumor made the diagnosis of malignant lymphoma unquestionable. Meanwhile, the double negativity of both CD4 and CD8 markers also suggested a malignant lymphoma. Taken together, all the features of immunophenotyping and the TCR beta gene rearrangement could fit the diagnosis of lymphoblastic lymphoma.
In summary, it was demonstrated that in the presence of invasive clinical behavior antigen receptor gene rearrangement has facilitated the diagnosis of a malignant lymphoma. The analysis of antigen receptor gene has offered an effective alternative equivalent to immunochemistry in documenting features of malignant lymphoproliferative clone. Besides, it allows detection of a lymphoproliferative clone of very small amount in a heterogeneous cell population.
Copyright: 1997, Chinese Medical Association (Taipei)