Explanation 1. Genetic heterogeneity of circulating cells from transplanted and non-transplanted LAM patients Blood and urine samples were collected from 65 LAM patients, 44 of which have been studied previously. LAM cells are defined genetically by TSC2 LOH and phenotypically by reactivity to specific cell surface markers. CD45 and CD235a help to identify LAM cells in blood, while CD44v6 and CD9 are used with urine. The pattern of LOH of five microsatellite markers spanning the TSC2 locus was noted. Upon examination of TSC2 LOH patterns seen in circulating cells from blood from 45 LAM patients analyzed at one visit, thirty-one patients showed LOH in both blood subpopulations. Eight had a different pattern of LOH in the CD45-, CD235a- population than in the CD45-, CD235a+ population. Therefore, 25.8% of patients that showed LOH in both populations isolated from blood had evidence of circulating cells with different LOH patterns, suggesting that a single patient may have different clones of LAM cells. LOH patterns were compared in urine and blood in 45 patients seen for one visit. Ten patients (33.3% of patients that showed LOH in urine) showed differences in LOH pattern between blood subpopulations and urine. Twenty patients were monitored for several visits. These patients also showed different patterns of allelic loss in blood subpopulations or blood versus urine over time. Thus, there may be a wide range of heterogeneity in LAM cells, both phenotypically (i.e., expression of proteins on the cell surface) and genetically (i.e., patterns of TSC2 allelic loss). We also isolated circulating LAM cells from patients after bilateral lung transplantation. The patients with circulating LAM cells also showed heterogeneity in allelic patterns. These data show for the first time that LAM patients after bilateral lung transplantation have circulating LAM cells and that these cells are genetically different. While recipient LAM cells have been found in the donor lung after single lung transplantation, the origin of the LAM cell is not known. LAM cells have been postulated to arise from AMLs, the uterus, or the axial lymphatics. It is also possible that the lung was the primary tumor source and that the circulating cells detected in these bilateral transplant patients are from micrometastases that were dormant. Cell cultures from explanted lungs from three different patients were used as a source of LAM cells. We have had success isolating TSC2 LOH cells from these mixtures based on chemokine-stimulated mobility and reactivity to anti-CD44v6 antibodies and a variety of anti-cell surface protein antibodies. Cell populations of cultures B2305R and B1705R showed retention of allele one of the D16S3395 marker 90% of the time, while allele two was retained 10% of the time. The third LAM cell mixture (BBI9054R) also showed genetic heterogeneity. Thus, heterogeneous cell mixtures are heterogeneous not only because cells with wild type TSC2 are present, but also because they contain cells with different patterns of TSC2 LOH. These cells may reflect genetic instability of cell culture or they may be representative of LAM cells present in the explanted lungs. The results of our examination of circulating and cultured LAM cells and the characterization of their genetic heterogeneity differ from previous studies of solid tissues. In this study, we looked at LAM cells from blood and found that 25.8% of those with LAM cells in both populations had different LOH patterns. Allelic patterns also differed between blood and urine and in the same body fluid over time, such that 26 of 65 (40.0%) patients analyzed showed heterogeneity in allelic patterns of isolated cells. Thus, multiple clones of LAM cells may exist in different body fluids and over time. Many human cancers have great intra-tumor heterogeneity in morphology, cell surface marker expression, and metastatic potential. Cllonal heterogeneity has been shown in breast, colon, bladder, and prostate carcinomas. The genetic heterogeneity seen in circulating LAM cells, whether due to multiclonal origin or genetic instability over time, is consistent with a more recent model of LAM, where the disease is defined as a low-grade neoplasm. 2. Detection of Tuberous Sclerosis Complex 2 (TSC2) loss of heterozygosity in circulating cells of patients with lung diseases and cancer Eighteen randomly selected patients with different lung diseases were enrolled, with diagnosis based on clinical, radiographic and histopathologic criteria. The study included patients with sarcoidosis (n = 9), pulmonary langerhans cell histiocytosis (PLCH) (n = 3), benign metastasising leiomyoma (BML) (n = 1), follicular bronchiolitis (n = 1), emphysema (n = 1), interstitial lung disease (n = 1), cystic lung disease (n = 1) and MounierKuhn syndrome (n = 1). Cells from blood and urine were sorted on the basis of cell surface markers (CD235a, CD45, CD9 and CD44v6) that have been shown previously to identify LAM cells in body fluids and cultured lung cells from LAM patients. Based on surface markers, cells were isolated from blood, and urine. Five microsatellite repeats spanning the TSC2 locus on chromosome 16 were tested (D16S521, D16S3024, D16S3395, Kg8 and D16S291. Here, we show that TSC2 LOH was detected in 2 of these 18 patients. One case of sarcoidosis (CN-02) did not show TSC2 LOH in either subpopulation from blood, but did show TSC2 LOH in the CD9-CD44v6- cell population from urine at markers D16S3395 and D16S521. This cell population is different from the CD9+CD44v6+ cell population from urine that contains LAM cells. TSC2 LOH was also detected in PLCH at marker Kg8, but only in the unsorted blood cells, and not in the two subpopulations in which LAM cells are typically found. In fact, cells presenting with TSC2 LOH in these different diseases or conditions appear to express different surface markers from LAM cells. Chromosomal alterations have been reported in sarcoidosis, PLCH, BML, asthma and COPD. We do not know if the circulating cells isolated from blood that exhibit TSC2 LOH in these other diseases are relevant to the pathogenesis or progression of those diseases. However, it would be interesting to determine the activation state of the mTOR pathway in these cells. While TSC2 LOH may not distinguish lung diseases or be diagnostic for LAM in general, the presence of TSC2 LOH in cells isolated with specific surface markers appears to be characteristic of patients with LAM. The TSC2 gene is mutated or associated with LOH in cancers in different organs and with different frequencies. Some of the cancer studies tested the same microsatellite markers used to identify circulating LAM cells. Among cancers, TSC LOH is frequently observed in lung carcinoma and LAM. TSC1 or TSC2 LOH was seen respectively in 139 or 92 cases in a total of 476 lung cancer cases; 38 cases had both TSC1 and TSC2 LOH. Interestingly, LAM and lung adenocarcinoma are more frequently found in women. Since the lung is exposed to environmental factors (e.g., pollution, air-borne oxidants), these data suggest that lung cells could be a source of metastatic cells with TSC2 gene mutations in cancers and LAM. Because LOH is an important event in the 'two-hit' hypothesis of cancer development and a frequent event in tumorigenesis, we hypothesize that TSC2 LOH could be a common event in different cancerous processes, and specific patients may benefit from rapamycin treatment. There are over 900 clinical trials currently looking at the use of rapamycin as anti-cancer agent.
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