B-Cell Pulmonary Lymphoma

B-Cell Pulmonary Lymphoma* Gene Rearrangement Analysis of Bronchoalveolar Lymphocytes by Polymerase Chain Reaction Bruno Philippe, MD; Marie-He´le`ne Delfau-Larue, MD; Bernard Epardeau, MD; Brigitte Autran, MD; Jean-Pierre Clauvel, MD; Jean-Pierre Farcet, MD; and Louis-Jean Couderc, MD

Study objectives: Non-Hodgkin’s lymphomas (NHLs) are clonal proliferation of B or T lymphocytes. Assessment of clonality in lymphoid proliferations uses immunochemistry and, recently, molecular biology. The aim of our study is to assess the role of immunoglobulin gene rearrangement analysis on bronchoalveolar lymphocytes to aid in the diagnosis of B-cell pulmonary NHL. Patients and methods: The study took place in a university hospital. There were seven consecutive patients with B-cell-type pulmonary lymphoma and nine control subjects. Gene rearrangement analysis using polymerase chain reaction (PCR) technique was performed on alveolar lymphocytes recovered by BAL. Results: Analysis of the immunoglobulin heavy chain gene rearrangement showed a predominant clonal alveolar lymphocyte population in six of seven patients while all control subjects showed germline pattern. Conclusions: Gene rearrangement analysis by PCR of alveolar lymphocytes would appear to be sensitive in patients with B-cell pulmonary NHL (six of seven patients) and specific (zero of nine in the control group). This simple test should be added only in the analysis of cells recovered by BAL in patients with suspected primary and secondary B-cell pulmonary NHL. (CHEST 1999; 115:1242–1247) Key words: gene rearrangement; polymerase chain reaction; pulmonary lymphoma Abbreviations: NHL 5 non-Hodgkin’s lymphomas; PCR 5 polymerase chain reaction; TCR 5 T-cell receptor

diagnosis and classification of nonC urrent Hodgkin’s lymphoma (NHL) are based on a

concept that regards lymphoma as clonal expansions of B cells or T cells frozen at various stages of differentiation.1 Accurate morphologic assessment must remain the cornerstone in the diagnosis of lymphoproliferative disorders. In difficult cases, the ancillary diagnostic techniques now available to the pathologist to assess the clonality and lineage have

*From the Departments of Pulmonary Diseases (Drs. Philippe and Couderc) and Pathology (Dr. Epardeau), Foch Hospital, Suresnes; the Laboratory of Biological Immunology (Drs. Delfau-Larue and Farcet), Henri Mondor Hospital, Cre´teil; the Laboratory of Cellular Immunology (Dr. Autran), Pitie´-Salpe´trie`re Hospital, Paris; and the Department of Clinical Immunology and Hematology (Dr. Clauvel), Saint-Louis Hospital, Paris, France. Partial support by grants from Institut National de la Sante´ et de la Recherche Me´dicale and Mutuelle Ge´ne´rale de l’Education Nationale (grant 6522). Manuscript received July 17, 1998; revision accepted November 18, 1998. Correspondence to: Bruno Philippe, MD, Hoˆpital Foch, Service de Pneumologie, 40 rue Worth, BP 36, F-92151 Suresnes Cedex, France; e-mail: [email protected] 1242

refined diagnostic accuracy. Immunophenotyping analysis enabled identification of lymphoid B cells bearing a surface marker.2 However, the evaluation of monoclonality of B-cell infiltrates may be difficult when B cells do not express surface markers, if there is an admixture of neoplastic and nonneoplastic lymphocytes, and lastly when immunostaining is attempted on paraffin-embedded tissue.3 Recent molecular biology techniques using heavy chain immunogloblin gene rearrangement analysis have been applied to diagnose nodal B-cell NHL.4 The same techniques were recently used in a few isolated cases of pulmonary NHL.5–11 Immunoglobulin gene rearrangements have been studied by restriction-digestion of DNA followed by Southern blotting, which has a relatively low sensitivity, requires large amounts of DNA from frozen tissue or fresh cytologic samples, and is slow and labor intensive.12 More recently, polymerase chain reaction (PCR) technique for amplification of the junctional regions of rearranged genes has been used to determine the clonality of lymphoid infiltrates.13 During Clinical Investigations

the process of B-cell maturation, rearrangement of the immunoglobulin heavy chain gene involving the variable (Vh), diversity (Dh), and joining (Jh) segments occurs. Through the deletion and insertion of a varying number of N region nucleotides, tremendous diversity is generated at the site of the V(D)J junction.14 PCR utilizes consensus primers to known sequences of framework region 3 (FR3) in the Vh and Jh segments, that flank the site of V(D)J junction.15 A clonal population will have identical N regions, and after PCR amplification, will yield fragments of identical size that can be visualized as a discrete band in a standard gel with a maximum sensitivity of 1023. In contrast, a polyclonal population will yield a diffused and smeared pattern on a gel owing to the large number of fragments of varying sizes. We conducted a prospective study to assess immunoglobulin heavy chain gene rearrangement analysis in alveolar lymphocytes using PCR in the diagnosis of pulmonary B-cell lymphoma.

Materials and Methods Patients Seven consecutive patients had B-cell pulmonary lymphoma diagnosed between January 1, 1993 and June 30, 1997, in the Pulmonary Disease Department of Foch Hospital in the Paris, France area. Patient selection was based on a histologic diagnosis of NHL on fixed and frozen tissue sections analysis. Each slide was analyzed independently by two pathologists. Pulmonary tissue specimens were analyzed according to the revised European-American classification of lymphoid neoplasms.16 All patients underwent a thoracic-abdominal CT scan, a bone marrow biopsy, and as clinically required, fiberoptic gastroduodenal endoscopy, and a spinal tap for cerebrospinal fluid analysis. Patients with primary pulmonary lymphoma were defined as those who had no extrathoracic localization detected by baseline investigations described above and also after a 3-month follow-up period. All other patients were considered to have secondary pulmonary lymphomatous involvement. We enrolled nine control subjects with bronchogenic carcinoma (3), Felty’s syndrome (1), idiopathic pulmonary fibrosis (4), and lung transplant (1). BAL Procedure All seven patients and the nine control subjects underwent a BAL for cytologic, phenotypic, and gene rearrangement analysis. BAL was done using a fiberoptic bronchoscope wedged into the most extensively involved pulmonary segment, as seen on chest radiograph, after topical lidocaine anesthesia. Sterile saline solution was instilled in four aliquots of 50 mL and the fluid was recovered by gentle aspiration. Differential cell counts were performed on Giemsa-stained cytocentrifuged preparations. Microbiological studies were applied to all samples and included specific stains and cultures for viruses, bacteria, mycobacteria, and fungi.

Gene Rearrangement Analysis by PCR DNA was prepared by a standard proteinase K digestion and a phenol/chloroform extraction. For PCR, two concensus oligonucleotides were used, one for FR3 region, and the other, for JH region. The primer sequences were as follows: 59ACACGGC(C/T)(G/C)TGTATTACTGT-39 termed FR3A and 59-ACCTGAGGAGACGGTGACC-39 termed JHC.15,17 The reaction mixture included 250 ng genomic DNA, 30 pmol of each primer, 1.5 mM Taq polymerase, 1 mM DNA glycosylase (Uracil; Gibco BRL; Gaithersburg, MA), 5 mL of 10 3 reaction buffer, 2 mM MgCl2, 200 mM of each dATP, dGTP, dCTP, and 400 mM dUTP in a final volume of 50 mL. Samples were transferred on the thermal cycler (model 480; Perkin Elmer Cetus; Norwalk, CT). DNA glycosylase was used to control carry over contamination as described.18 Each cycle included a denaturation step (94°C for 1 min), an annealing step (55°C for 1 min), and an elongation step (72°C for 1 min). After 40 cycles, 20% of the amplified products were run on a 10% polyacrylamide gel. The T-cell receptor (TCR) gene analysis was performed by PCR and analyzed on denaturating gradient gel electrophoresis as previously described.19 Alveolar and Blood Lymphocytes Phenotyping Analysis Alveolar lymphocytes were separated from macrophage subpopulations by adherence to plastic tissue culture flasks. Peripheral blood lymphocytes were obtained simultaneously from the BAL fluid in all 10 patients. Mononuclear cells from heparinized blood were separated by centrifugation on a Ficoll hypaque gradient. The lymphocyte subsets from blood and BAL fluid were counted by indirect immunofluorescence using the following monoclonal antibodies: anti-CD3 (IOT3) (Immunotech Marseille; Luminy, France), anti-CD8 (Leu-2a), anti-CD4 (anti-Leu3a 1 3b), anti-CD19 (Leu-11b), and anti-CD57 (Becton-Dickinson; Grenoble, France) as previously reported.20

Results Patients There were four women and three men ranging in age from 44 to 75 years (mean, 60 years). All patients were nonsmokers. None had a known previous immunodeficiency state. They were seronegative for HIV, hepatitis C virus, and human T-cell leukemia virus type I. Six patients had secondary pulmonary lymphoma. One patient (case 6) had primary pulmonary lymphoma (Table 1). Chest radiograph showed the following: nodules (n 5 1); alveolar opacity (n 5 3); pleural effusion (n 5 1); alveolar opacity, nodules, and mediastinal lymph nodes (n 5 1); pleural effusion, nodules, and mediastinal lymph nodes (n 5 1). The diagnosis of pulmonary B-cell NHL was histologically established in all seven patients from bronchial biopsy specimens (n 5 3), pulmonary biopsy specimens (1 open lung and 1 transbronchial biopsy) (n 5 2), mediastinal lymph node biopsy specimens (n 5 1), or pleural biopsy specimens (n 5 1). Five patients had mucosa-associated lymCHEST / 115 / 5 / MAY, 1999

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Table 1—Clinical and Radiologic Features of Patients With Pulmonary NHL* Patient/Age, yr/Sex 1/44/F 2/75/F 3/58/M 4/64/F 5/62/F 6/61/M 7/66/M

Symptoms

Radiologic Features

Sicca syndrome Dyspnea, sicca syndrome, fever Weight loss Cough, dyspnea Weight loss Sicca syndrome Dyspnea, cough

Alveolar opacities in RIL Necrotic nodules in ML and RSL Alveolar opacity in LIL Bilateral alveolar opacities and nodules; mediastinal lymph nodes Alveolar opacity in LIL and lingula Bilateral nodules and right pleural effusion; mediastinal lymph nodes Left pleural effusion

*RIL 5 right inferior lobe; LIL 5 left inferior lobe; ML 5 middle lobe; RSL 5 right superior lobe; M 5 male; F 5 female.

phoid tissue lymphoma, one had B-cell lymphoma involving the marginal node zone, and one had B-cell lymphoma involving the extramarginal node zone (Table 2). Microscopic Characteristics of Lavage Fluid The characteristics of cells recovered by BAL are summarized in Table 3. Alveolar lymphocytosis, defined as an increased absolute number of alveolar lymphocytes . 100,000/mL, was noted in patient 5. The others had an alveolar cell count within normal values. No alveolar lymphocytes exhibited cytologic abnormality. Results of microbiological studies were normal in all patients. No hemosiderin-laden alveolar macrophage was observed.

Phenotypic Lymphocyte Analysis Alveolar lymphocyte subsets were analyzed in three patients (patients 3, 4, and 5) who had a sufficient number of alveolar lymphocytes recovered by BAL to perform phenotypic analysis. An increased number of CD191 cells was noted in two patients only (.15% of alveolar lymphocytes). Absolute count of blood CD191 lymphocytes was within normal range (Table 4). In all three cases, the proportion of CD31 CD81 cells was predominant. Peripheral blood T-lymphocyte subsets analysis showed an inverted CD41CD81 ratio due to an increase in CD81 cells. Discussion

Gene Rearrangement Analysis The PCR analysis of the immunoglobulin heavy chain (IgH) gene rearrangement showed the presence of a predominant clonal alveolar lymphocyte population in six of seven patients (Fig 1). Lymphocyte gene rearrangement analysis by PCR of patient 6 disclosed a polyclonal pattern. Five patients were analyzed in TCR gene rearrangement and none showed clonal rearrangement. Among the nine control subjects, no predominant clonal rearrangement of the IgH chain and TCR genes was detected in the alveolar lymphocytes.

We report herein a gene rearrangement analysis on alveolar lymphocytes from seven patients with pulmonary B-cell NHL. We found clonal rearrangement of IgH gene in six of the seven patients using a PCR technique. Neither IgH gene nor TCR rearrangement was detected in the alveolar lymphocytes from nine control subjects. Our series differs from previously reported studies by the lymphocyte gene typing technique used. We used PCR, whereas the previous studies all used Southern blot analysis.5– 8 Only three cases of clonal alveolar B-lymphocyte population recovered by BAL have been reported

Table 2—Histologic Findings* Patient

Histology

Biopsy

Extrapulmonary Lymphomatous Involvement

1 2 3 4 5 6 7

MALT MALT MALT Marginal node zone lymphoma MALT MALT Extramarginal node zone lymphoma

Lung Bronchus Bronchus Mediastinal lymph node Lung Bronchus Pleura

Parotid Orbit Stomach, bone marrow, retroperitoneal nodes Bone marrow, retroperitoneal nodes, spleen Stomach, retroperitoneal nodes None Bone marrow

*MALT 5 mucosa-associated lymphoid tissue. 1244

Clinical Investigations

Table 3—Findings From BAL in Patients With B-Cell Pulmonary Lymphoma* BAL Cells Patient

Total Cells, 3103/mL

Alveolar Macrophages, %

Alveolar Lymphocytes, %

Alveolar Neutrophils, %

IgH Gene

TCR Gene

1 2 3 4 5 6 7

220 320 612 152 436 60 196

93 64 79 81 52 69 43

3 3 16 15 40 29 7

4 32 4 4 5 2 50

C C C C C P C

P P P P ND P ND

*C 5 clonal pattern; P 5 polyclonal pattern; ND 5 not determined.

previously in patients with B-cell pulmonary NHL.5–7 These cases were as follows: one low-grade NHL with pulmonary and orbital involvement; one low-grade primary pulmonary NHL; and one highgrade malignancy NHL with pulmonary, splenic, and renal involvement. Isolated cases of predominant clonal lymphoid population detected by molecular biology in tissue specimens and the pleural space were also reported in patients with pulmonary or pleural NHL.9 –11,21–24 The PCR technique has advantages over the Southern blot technique in that it is less costly, faster, does not use radioactive materials, and requires very small amounts of DNA.25 The polyclonal pattern of alveolar lymphocytes from patient 6 may have several explanations. First, the failure to detect a clonal population by PCR IgH amplification may be due to excessive junctional deletion leading to removal of nucleotides homologous to the 39 end of the FR3A or JH amplication

primer. Second, somatic mutation may arise during B-cell maturation, thus impairing PCR primers hybridization. In addition to its high sensitivity, PCR also demonstrated good specificity. No clonal rearrangement was detected in the nine control subjects who did not have NHL. However, we must remain prudent in our interpretation because our population was small and because the lack of detection of a predominant clonal alveolar lymphocyte population does not exclude the diagnosis of pulmonary lymphoma, as was apparent in patient 6. Blood contamination of BAL fluid seems unlikely because of the following: (1) cytologic analysis of lymphocytes in peripheral blood was normal in seven of seven patients; (2) no hemosiderin-laden alveolar macrophages were observed; and (3) phenotypic comparison of peripheral blood and alveolar B lymphocytes of patient 5 showed a high CD191 cell count in the alveolar compartment

Figure 1. IgH PCR-amplified products from alveolar lymphocytes. PCR-amplified products were analyzed on 10% polyacrylamide gel stained with ethidium bromide. Control subjects 1 to 3 had nonlymphomatous alveolar cells (pulmonary fibrosis, subjects 1 and 2; lung transplant, subject 3) and showed polyclonal pattern. A similar pattern was obtained from patient 6. Alveolar lymphocytes from patients 1 to 5 and 7 showed a monoclonal predominant band. CHEST / 115 / 5 / MAY, 1999

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Table 4 —Lymphocyte Subsets From Blood and BAL* Alveolar Lymphocytes, %†

Blood Lymphocytes, %†

Patient

CD31

CD41

CD81

CD191

CD31

CD41

CD81

CD191

3 4 5

69 92 60

29 20 20

43 70 40

19 2 18

ND† 63 63

ND 25 20

ND 40 44

ND 4 3

*See Table 3 for definition of abbreviations. †Percentage of total lymphocytes.

and within normal range in peripheral blood. The phenotypic analysis of alveolar lymphocytes was not performed in four patients (patients 1, 2, 6, and 7) because the number of recovered cells was not sufficient for this technique. Interestingly, the results of alveolar lymphocyte phenotyping showed an increased T-lymphocyte count that seems paradoxic in patients with B-cell lymphoma. A T-cell alveolitis was previously noted in patients suffering with B-cell pulmonary lymphoma.7,26,27 The significance of the alveolar CD8 lymphocytosis remains unknown. A CD81 lymphocytic infiltration has been observed in affected lymph nodes of a patient with nodal NHL.28 Most T cells in BAL fluid may be reactive. In conclusion, PCR may eventually permit clinical decisions to be made in the evaluation of patients suspected of having pulmonary involvement with lymphoma based on BAL specimen analysis, but up to now, it will not replace histologic tissue review as the “gold standard.” ACKNOWLEDGMENTS: The authors thank Genevie`ve Flohic for preparation of the manuscript and Dr. Anne Mary Delay for revising the English.

8

9

10

11

12 13

14 15

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