Genotypic analysis of pulmonary Langerhans cell histiocytosis

Genotypic Analysis of Pulmonary Langerhans Cell Histiocytosis S. DACIC, MD, PHD, C. TRUSKY, BS, A. BAKKER, BS, S. D. FINKELSTEIN, MD, AND S. A. YOUSEM, MD Reported studies show that the systemic form of Langerhans cell histiocytosis (LCH) is a clonal expansion of Langerhans cells (LC) associated with aberrant expression of several oncogenes or tumorsuppressor genes. LCH of the lung is a heterogenous group of lesions thought to be a reactive rather than neoplastic process. The histogenesis of the LCH of the lung is uncertain, and to date there are no studies investigating its underlying molecular abnormalities. We performed comparative genotypic analysis by using allelic loss (LOH) of polymorphic microsatellite markers associated with tumor suppressor genes. Fourteen cases of formalin-fixed, paraffin-embedded LCH of the lung were studied. Microdissection of a total of 26 nodules from 14 patients and paired reference lung tissue was performed under stereomicroscopic visualization. To evaluate allelic loss, we used a panel of 11 polymorphic microsatellite markers that were situated at or near tumor suppressor genes on chromosomes 1p, 1q, 3p, 5p, 9p, 17p, and 22q. The PCR products were analyzed by using

capillary electrophoresis to identify germline heterozygous alleles and LOH. Allelic loss at 1 or more tumor suppressor gene loci was identified in 19 of 24 nodules. The total fractional allelic loss (FAL) ranged from 6% (1q) to 41% (22q), with a mean of 22%. The FAL in individual cases ranged from 0 (7 nodules) to 57% (1 nodule). Fifteen discordant allelic losses at 1 to 3 chromosomal loci were identified in 8 patients with multiple synchronous nodules. Our results show that LOH of tumor suppressor genes is present in the LCH of the lung, and they indicate that the putative tumor suppressor genes situated on chromosomes 9p and 22q may play a role in the development of a subset of the LCH of the lung. HUM PATHOL 34:1345-1349. © 2003 Elsevier Inc. All rights reserved. Key words: Langerhans cell histiocytosis, lung, loss of heterozygosity. Abbreviations: LCH, Langerhans cell histiocytosis; LC, Langerhans cell; LOH, loss of heterozygosity; FAL, fractional allelic loss.

Langerhans cell histiocytosis (LCH) of the lung is an interstitial lung disease that usually affects middleaged adults, most frequently women.1 Chest radiographs in typical cases show numerous bilateral nodules, and open lung biopsy is often performed to exclude the possibility of metastatic disease. LCH of the lung usually undergoes complete spontaneous regression, and progression to advanced pulmonary fibrosis and death is uncommon.2,3 Cigarette smoking has been shown to be a strong risk factor and promoter for the development of pulmonary LCH,4,5 and the bronchiolar distribution of the pathologic lesion suggests that an inhaled antigen, such as cigarette smoke, may be involved in its pathogenesis. A therapeutic response to smoking cessation has been described in case reports,6 but no controlled studies have been reported. Pathologic findings vary with the stage of the disease. In the early stages, numerous LC accumulate adjacent to respiratory bronchioles.7 Lymphocytes, alveolar macrophages, and eosinophils are admixed. As lesions heal, cellular infiltrates are less prominent, and fibrosis results in a typical stellate scar. Emphysema and respiratory bronchiolitis are usually present in the adjacent lung parenchyma. Histologic, radiographic, and clinical criteria for diagnosis of pulmonary LCH are well established, but

its pathogenesis still remains unknown. Youkeles et al8 proposed that pulmonary LCH might result from an altered immune response to tobacco glycoprotein. Several other studies have focused on LCH as clonal disorder. Although reported studies have shown that the systemic form of LCH is a clonal expansion of LC that is associated with aberrant expression of several oncogenes or tumor-suppressor genes,9-11 it is yet unclear whether this is true of the isolated pulmonary form of LCH. A monoclonal origin has been demonstrated only in rare isolated cases of pulmonary LCH and it seems that this form of LCH is most likely a polyclonal, reactive disorder associated with cigarette smoking based on the inability to identify consistent cytogenic abnormalities, clonality studies utilizing the HUMARA assay, and the occurrence of spontaneous clinical regression.12 In this study, we assess the loss of heterozygosity (LOH) for microsatellite markers situated at or near tumor suppressor genes that are potentially involved in the development and progression of the pulmonary LCH.

From the Department of Pathology, Division of Anatomic Pathology, University of Pittsburgh Medical Center, Presbyterian University Hospital, Pittsburgh, PA. Accepted for publication July 18, 2003. Address correspondence and reprint requests to S. Dacic, MD, PhD, Department of Pathology, University of Pittsburgh Medical Center, 200 Lothrop St. PUH A610.2, Pittsburgh, PA 15213. © 2003 Elsevier Inc. All rights reserved. 0046-8177/03/3412-0008$30.00/0 doi:10.1016/j.humpath.2003.07.14

MATERIALS AND METHODS Fourteen cases of LCH of the lung from untreated patients were obtained from the paraffin block archives of the University of Pittsburgh Medical Center and the consultation files of 1 of the authors (S.A.Y.) after obtaining internal review board consent for an anonymized study. The diagnosis in each case was confirmed by the presence of S100⫹ and CD1a⫹ LCH cells (Fig 1). No patient had evidence of extrapulmonary disease at presentation. Microdissection of a total of 26 nodules from 14 patients and paired reference lung tissue was performed as described elsewhere under stereomicroscopic visualization by using the aggregate material from two to three 4-␮m-thick unstained

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FIGURE 1. (A) Pulmonary LCH nodule composed of abundant LC with characteristic folded nucleus admixed with eosinophils, alveolar macrophages, and lymphocytes (hematoxylin and eosin, original magnification ⫻40). (B) S100 immunostaining confirms the presence of LC (immunoperoxidase, original magnification ⫻10, inset magnification ⫻40).

histologic sections.13 Microdissection was carefully performed to avoid bronchial and bronchiolar epithelium. All nodules were comprised predominantly of LCH cells, variable numbers of eosinophils, and other inflammatory cells. The cases with either early or more advanced fibrosis were not included in the study. Microdissected adjacent areas of respiratory bronchiolitis were also studied. Slides were poststained with hematoxylin and eosin to ensure accurate tissue sampling for genotypic analysis. Microdissected tissue was digested with proteinase K, and DNA was extracted as described elsewhere.14 To evaluate allelic loss, we used a panel of 9 microsatellite markers that were situated at or near tumor suppressor genes on chromosome 1q (D1S.1172), 1p (D1S.407), 5q (D5S.592, D5s.615), 9p (D9S.251), 17p (D17S.1289, D17S.974), and 22q (D22S.532, D22S.417). Fluorescent-labeled primers flanking tetranucleotide and pentanucleotide microsatellite repeat polymorphisms were used in a standard PCR, as described elsewhere.15 Amplification products were detected during capillary electrophoresis in DNA sequencer (310, Applied Biosystems, Foster City, CA). Collected data were analyzed with Genescan software. All LCH samples were run with reference lung parenchyma samples to determine microsatellite informativeness, to exclude the effect of allelic dropout, and to assess allelic imbalance in tumor samples. A case was informative for a particular allele if the maternal and paternal alleles migrated differently and was deemed noninformative if they overlapped. The allelic peak height ratios were calculated in the reference and LCH samples. The ratio between the 2 heights is defined as the allelic imbalance factor. The LCH samples were considered to have LOH if the allelic imbalance factor for the specific microsatellite marker was ⬍0.6 or ⬎1.5.16 The fractional allelic loss (FAL) in each case was defined as the number of chromosomal arms on which allelic loss was observed, divided by the number of chromosomal arms for which allelic markers were informative.17

RESULTS Of the 14 patients with LCH of the lung, 9 were women and 5 were men, with ages ranging from 41 to

76 years (mean 54 years). All patients were cigarette smokers. There was no evidence of systemic or extrapulmonary manifestation of LCH. Two patients (cases 8 and 12) had a concomitant adenocarcinomas of the lung, 1 patient had a history of breast carcinoma treated with chemotherapy (case 5), and 1 patient had sarcoidosis (case 1). A total of 26 six nodules was microdissected from 14 patients. Table 1 shows the results of LOH of each chromosomal locus and FAL in individual cases. Allelic loss at 1 or more tumor suppressor gene loci was identified in 19 of 26 (73%) nodules, with FAL ranging from 14% (case 2) to 57% (case 4). Seven of 26 nodules (27%) did not show any evidence of allelic imbalance for tested chromosomal loci. Fifteen discordant allelic losses at 1 to 3 chromosomal loci were identified in 8 patients with multiple synchronous nodules (Fig 2). Microdissected areas of respiratory bronchiolitis and reference lung did not show allelic imbalance (data not shown). The noninformative rate and overall frequency of LOH of each chromosomal locus are summarized in Table 2. Noninformative rates for each chromosomal locus ranged from 0 (5q) to 35% (1p). Nine of 22 (41%) of informative nodules showed allelic imbalance of the chromosome 22q. This result indicates that this chromosomal locus contains a putative tumor suppressor gene that is potentially important in the pathogenesis of the pulmonary LCH. The second most frequently involved chromosomal locus was 9p (gene p16). Six of 17 informative nodules showed allelic imbalance at this particular locus, with total FAL of 36%. LOH of p53 gene was observed in 7 of 24 nodules (FAL, 30%). As opposed to systemic forms of LCH, the 1q locus was affected in only 1 nodule (FAL 6%). As mentioned earlier, 7 nodules did not show allelic loss at any tested chromosomal locus, suggesting a possible different pathogenesis or different promoter in these cases. The other possible explanation would be that

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TABLE 1. Loss of Heterozygosity of Each Chromosomal Locus and Fractional Allelic Loss (FAL) in Individual Cases of Langerhans Cell Histiocytosis of the Lung Case No. 1 Nodule 1q 1p 3p 5q 9p 17p 22q FAL (%)

2

3

4

5

6

7

8

9

10

11

12

13

14

NI NI NL NL NI NL NL 0

F F NL NL F NL NL 0

NL L L L NI NL NL 43

NL F NL L NL NL F 16

NL NL L NL NL NL L 16

NI F NL L F NL F 17

N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 N3 N1 N2 N3 N1 N2 N3 N4 NL NL NL L NI L L 43

NL NL NL NL NI L NL 25

NL F NL L NL NL NL 14

NL F L NL L NL F 25

NL NL F NL NI NL NI 0

L NL NL L NI NL NI 50

NL NL NL L F L L 57

NL F NL L L L NL 33

F NI NL NL NL L L 40

NL NI L NL NL L L 43

NI NL L NL NL NL L 17

NI NL NL NL NL NL NL 0

NI NL NL NL L NL L 29

F NL F F L NI NL 33

NL NL NL NL L L NL 33

NL NL F NL L NL NL 29

NL NI NI NL NL NL NL 0

NL NI NI L NL NL L 29

NL NI NI NL NL NL NL 0

NL NI NI NL NL NL NL 0

Abbreviations: N, nodule; L, loss of heterozygosity; NL, no loss of heterozygosity; NI, noninformative; F, failed; FAL, fractional allelic loss.

some chromosomal loci not analyzed in this study play a role in the subset of pulmonary LCH. DISCUSSION The etiology of pulmonary LCH remains unknown, but recent progress has been made in understanding its pathogenesis. In the present study, by using multiple microdissected specimens of pulmonary LCH

with large numbers of LC, we have shown that LOH at 22q, 9p, and 17p is more frequently present than at other tested chromosomal loci. These results suggest that a certain proportion of pulmonary LCH lesions shows genetic instability. Even though the chromosomal loci of 22q, 9p, and 17p are the most frequently affected, the overall number of individual histologic lesions that are affected is still relatively low (41% at 22q, 36% at 9p, and 30% at 17p). This finding is consistent with a previously reported clonality study in which only 30% of analyzed nodules were clonal.12 Our study also showed that synchronous nodules from 8 cases showed discordant results, in which some nodules showed allelic imbalance, whereas other morphologically similar lesions with approximately similar proportion of LC did not. This observation suggests a different initiation and progression process in synchronous lesions and argues for a reactive polyclonal LC process as the cause of pulmonary LCH. Interestingly, the most frequently affected chromosomal loci are those most frequently reported as having tobacco-related molecular abnormalities.18-23 It is wellknown that even morphologically normal respiratory epithelium of smokers has mutational changes.24-26 This indicates that exposure to still-unknown antigens in tobacco smoke promotes a whole sequence of molecular events. Because the LC are dendritic cells and therefore are potent antigen-presenting cells, we can speculate that pulmonary LCH may be a result of an uncontrolled immune response initiated by antigens TABLE 2. Noninformative Rate and Frequencies of Loss of Heterozygosity at Each Chromosomal Locus Chromosome

Noninformative Rate, % (n)

Frequency of LOH, % (n)

1q 1p 3p 5q 9p 17p 22q

22 (5/23) 35 (7/20) 17 (4/23) 0 (0/25) 26 (6/23) 4 (1/26) 8 (2/24)

6 (1/18) 8 (1/13) 21 (4/19) 36 (9/25) 36 (6/17) 28 (7/25) 41 (9/22)

FIGURE 2. Allelic imbalance analysis of 3 synchronous nodules (N1 to N3) of pulmonary LCH shows a heterogenous allelic imbalance at chromosomal arm 9p (case 6). Note that N1 and N2 retained heterozygosity of the 9p, whereas N3 shows loss (arrow).

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present in tobacco. This is supported by the finding that LC in pulmonary LCH express surface molecules that are important for the activation of T lymphocytes, which are a component in the histologic lesion of the pulmonary LCH.27 Tazi et al28 proposed that the increased secretion of GM-CSF by bronchiolar epithelium that is induced by smoking is probably responsible for the initial accumulation of large numbers of LC, with autocrine production by LC themselves contributing to the maintenance of histologic lesions. In our study, 27% of nodules did not show any evidence of allelic imbalance. This finding can be the result of different initiation and progression processes in those lesions. It would be of interest to know the precise anatomic location of those lesions, because Wistuba et al,26 in their study of smoking-related molecular changes in bronchial epithelium, suggested the presence of 2 distinct anatomic compartments in the lung that have differences in the smoking-induced genetic damage. They noted that the proximal airways showed more frequent molecular abnormalities compared with peripheral small bronchioles. Because pulmonary LCH is clearly a smoking-related lesion affecting peripheral small airways, perhaps anatomic location within the lung parenchyma has an impact on the frequency of molecular abnormalities in the associated immune cells. Although the role of smoking-induced epithelial molecular changes in the pathogenesis of pulmonary LCH seems to be very important, the fact that only a very small proportion of smokers develops this lesion indicates that other factors must be required. Murakami et al29 recently reported cytogenetic and allelotypic abnormalities in LCH of bone. It seems that some chromosomal loci, such as 9p and 22q, are commonly affected in both LCH of lung and bone. However, the Murakami et al29 study also showed frequent involvement of a region 1p, suggesting the presence of a putative tumor suppressor gene, which may play a role in the pathogenesis of LCH of bone. In our study, this chromosomal locus was infrequently affected (8%). This suggests that different molecular pathways may be involved in the progression of LCH at different anatomic locations. Therefore, it will be interesting to examine LCH lesions from different anatomic locations to determine whether similar patterns of molecular alterations are involved. It would also be of particular interest to determine the sequence of molecular alterations, depending on histologic or temporal stages of the lesion. Several investigators have proposed a genetic predisposition that may also be necessary for the development of the disease.30,31 However, Vasallo et al,2 in their study that included more than 100 adults with pulmonary LCH, did not identify a single instance of familial clustering. They proposed that pulmonary LCH in adults occurs in a sporadic fashion in majority of patients. A number of investigators have reported an association of adenocarcinoma of the lung and other solid tumors before, after, or at the same time as the diagnosis of pulmonary LCH.32,33 In our study, 2 cases were

associated with adenocarcinoma of the lung, and 1 case occurred after chemotherapy for breast cancer. There was no increased frequency of LOH in pulmonary LCH associated with malignancy, as one may expect. Actually, 3 of 4 nodules in a patient with synchronous adenocarcinoma of lung did not show any allelic imbalance. Because of the small number of patients and retrospective nature of this study, a definitive conclusion about relative risk of various cancers and associated molecular abnormalities that potentially could predict such association cannot be made. Cigarette smoking and prior treatment with chemotherapeutic agents may contribute to a predisposition to the development of malignant neoplasms in patients with pulmonary LCH. In summary, despite the presence of allelic imbalance affecting several chromosomal loci with putative tumor suppressor genes, the clinical course of pulmonary LCH is benign. In this regard, it is worthwhile to mention that allelic imbalances have been identified in a number of situations in the absence of overt malignancy.34,35 Current evidence suggests that the pulmonary LCH represents a reactive polyclonal process that is induced by the antigens in cigarette smoke and is thus different from the other forms of LCH that have been shown to be the result of a monoclonal proliferation of the LC with variable clinical outcome. REFERENCES 1. Ryu JH, Colby TV, Hartman TE, et al: Smoking-related interstitial lung disease: A concise review. Eur Respir 17:122-132, 2001 2. Vassallo R, Ruy JH, Schroeder DR, et al: Clinical outcomes of pulmonary Langerhans’-cell histiocytosis in adults. N Engl J Med 346:484-490, 2002 3. Howarth DM, Gilchrist GS, Mullan BP, et al: Langerhans cell histiocytosis. Diagnosis, natural history, management, and outcome. Cancer 85:2278-2290, 1999 4. Travis WD, Borok Z, Roum JH, et al: Pulmonary Langerhans cell granulomatosis (histiocytosis X). A clinicopathologic study of 48 cases. Am J Surg Pathol 10:971-986, 1993 5. Tazi A, Soler P, Hance AJ. Adult pulmonary Langerhans’ cell histiocytosis. Thorax 55:405-416, 2000 6. Von Essen S, West W, Sitorius M, et al: Complete resolution of roentgenographic changes in a patient with pulmonary histiocytosis X. Chest 98:765-767, 1990 7. Aubry MC, Wright JL, Myers JL: The pathology of smokingrelated lung diseases. Clin Chest Med 21:11-35, 2000 8. Youkeles LH, Grizzanti JN, Liao Z, et al: Decreased tobaccoglycoprotein-induced lymphocyte proliferation in vitro in pulmonary eosinophilic granuloma. Am J Respir Crit Care Med 151:145-150, 1995 9. Yu RC, Chu C, Buluwela L, et al: Clonal proliferation of Langerhans cells in Langerhans cell histiocytosis. Lancet 343:767-768, 1994 10. Willman CL, Busque L, Griffith BB, et al: Langerhans’-cell histiocytosis (histiocytosis X)—A clonal proliferative disease. N Engl J Med 331:154-160, 1994 11. Willman CL: Detection of clonal histiocytes in Langerhans cell histiocytosis: Biology and clinical significance. Br J Cancer 70: S29-S33, 1994 12. Yousem SA, Colby TV, Chen Y-Y, et al: Pulmonary Langerhans’ cell histiocytosis. Molecular analysis of clonality. Am J Surg Pathol 25:630-636, 2001 13. Wu TT, Barnes L, Bakker A, et al: K-ras-2 and p53 genotyping of intestinal-type adenocarcinoma of the nasal cavity and paranasal sinuses. Mod Pathol 9:199-204, 1996

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