Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis

Abstract

Lymphangioleiomyomatosis (LAM) is a multisystem disorder of women, characterized by cystic degeneration of the lungs, renal angiomyolipomas (AML), and lymphatic abnormalities. LAM lesions result from the proliferation of benign-appearing, smooth muscle-like LAM cells, which are characterized by loss of heterozygosity (LOH) of one of the tuberous sclerosis complex (TSC) genes. LAM cells are believed to migrate among the involved organs. Because of the apparently metastatic behavior of LAM, we tried to isolate LAM cells from body fluids. A cell fraction separated by density gradient centrifugation from blood had TSC2 LOH in 33 of 60 (55%) LAM patients. Cells with TSC2 LOH were also found in urine from 11 of 14 (79%) patients with AML and in chylous fluid from 1 of 3 (33%) patients. Identification of LAM cells with TSC2 LOH in body fluids was not correlated with severity of lung disease or extrapulmonary involvement and was found in one patient after double lung transplantation. These studies are compatible with a multisite origin for LAM cells. They establish the existence of disseminated, potentially metastatic LAM cells through a relatively simple, noninvasive procedure that should be valuable for molecular and genetic studies of somatic mutations in LAM and perhaps other metastatic diseases.

Figures

Fig. 1.

PCR analysis of a mixture of LAM cells and blood after density-gradient fractionation. (A) Allele peaks for both L16 LAM cells and NV2 blood are detected with marker D16S663. (B) Addition of L16 LAM cells that are homozygous for marker Kg8 to blood that is heterozygous for Kg8 changes the ratio of allele peak heights from 1 to 0.5. Arrows indicate positions of alleles.

Fig. 2.

Characterization of cells in the low-density fraction from blood. Representative cells from blood (A) or grown from LAM lungs (B) that reacted with Cy3-conjugated monoclonal antibodies (red) against smooth muscle actin are shown. Nuclei were stained with DAPI (blue). (Scale bar, 20 μm.) FISH is shown in C and D. (C) A LAM cell with only one TSC2 (red) allele and two copies of TSC1 (green). (D) An interphase nucleus with a normal, disomic pattern of two TSC1 and two TSC2 signals. (E) Representative PCR analyses of chromosome 16p13.3 microsatellite markers D16S521, Kg8, and D16S291 in two LAM patients. The upper traces show the peripheral blood (N) and the lower traces the low-density fraction (L) from the same patient. Patient L245 is not informative for marker D16S521. Arrows indicate the diminished peaks.

Fig. 3.

LOH at chromosome 16p13.3 locus in low-density fractions of cells from blood, followed by cell sorting. (A) Representative FACS analysis of cells labeled with monoclonal antibodies conjugated to FITC (CD235a) or APC (CD45). CD45– cells (quadrants 1 and 2) are potentially LAM cells. (B) PCR analyses of microsatellite marker Kg8 in FACS-sorted populations from quadrants shown in A. Arrows indicate positions of missing allele. (C) Photomicrograph showing a positive immunohistochemical reaction for CD235a protein in a LAM nodule (×100).

Fig. 4.

Association between LAM tissue involvement and detection of LOH in blood or urine. (Left) Percentage detection of LOH in low-density cell fractions from bloods of LAM patients with different degrees of lung involvement by high-resolution CT scans. Involvement was rated mild (Mi, n = 12), moderate (Mo, n = 8), and severe (Se, n = 14) (5). Kidney involvement is indicated by the presence (▪) or absence (□) of renal AML and the percentage of 34 LAM patients with LOH in blood or urine. *, P = 0.016 for difference between urine samples from patients with and without AML. Lymphatic involvement evaluated by high-resolution CT scan of chest or abdomen (e.g., Fig. 5D). Masses correspond to lymphangioleiomyomas, which tend to be cystic and filled with chyle when >3 cm in diameter. Data are from patients with no involvement (None, n = 7), adenopathy (Aden, n = 19), and masses (Mass, n = 21). Of the 34 patients represented in all analyses, 65% had LOH in blood samples. (Right) Cytospin preparations of cells from urine were reacted with Cy3-conjugated monoclonal antibodies against smooth muscle actin, FITC-conjugated anti-cytokeratin, or control IgG (negative).

Fig. 5.

Detection of LOH in body fluids. (A) High-resolution CT scan of the chest with pleural effusion indicated by an arrow. (B) Fluorescent PCR analysis of chromosome 16p13.3 microsatellite marker D16S521 in cells from chyle (C), low-density fraction from blood (L), and whole blood (N). Arrows indicate positions of missing alleles. (C) High-resolution CT scan of the abdomen showing retroperitoneal lymphangioleiomyomas (arrow). (D) Hematoxylin/eosin-stained section of an abdominal tumor biopsy showing fibro-adipose tissue. A and B are from patient L38, and C and D are from patient L83.