We have identified two cell surface proteins, CD44v6 and CD9, that are useful for isolation of disseminated LAM cells from BALF, urine, and chylous effusions. LAM cells from patients with S-LAM exhibit mutations and LOH most frequently in the TSC2 locus. For the majority of patients with S-LAM, LAM cells isolated from blood, urine, BALF, or chyle of the same patients show identical TSC2 LOH patterns for specific microsatellites, although in some, LAM cells from different body fluids appeared to differ in the extent of TSC2 LOH regions, consistent with genetic heterogeneity.
In general, these findings support the hypothesis that multisystem manifestations of LAM appear to result from a metastatic process (31
). Previously in five patients with S-LAM, identical TSC2
mutations or LOH were identified in pulmonary and renal LAM lesions (13
). LAM cells from the recipient were identified in a transplanted donor lung (17
). As LAM cells were also detected in blood (19
) or chylous fluids (19
), it was hypothesized that LAM cells could migrate or metastasize via blood and/or lymphatic circulations (19
). The LAM cells found in BALF could result from release from the LAM lung nodules or perhaps shedding from the lymphatic circulation within the nodules. Patients with LAM may experience chyloptysis. LAM cells were identified in chyle and BALF may contain components of chyle.
Identification of proteins on the surface of circulating or disseminated cells has been of interest in human cancers for use potentially as therapeutic targets (32
). Gene expression microarray analysis revealed that TSC2−/−
cells grown from TSC-associated skin tumors contained highly increased mRNA levels of the tetraspanin CD9 (25
). We demonstrated here using flow cytometric analysis and immunostaining that CD9 protein was abundant on TSC2−/−
skin tumor cells (). A correlation between greater CD9 content and potential for metastasis is evident in some tumor types (e.g., bone, cervix, head and neck, stomach) (35
), although CD9 is considered to suppress metastasis by decreasing cell motility (35
). Tetraspanins are present widely among mammals and play important roles in cell morphology, motility, invasion, adhesion, and signaling (39
). Tetraspanins form complexes with other tetraspanins and a variety of transmembrane proteins at tetraspanin-enriched membrane microdomains (42
). The diverse actions of CD9 are probably due to its association with other molecules in the tetraspanin-enriched membrane microdomains. In our studies, high levels of CD9 protein correlated with cells having TSC2
Our group had reported earlier an association between the presence of CD44v6 protein and TSC2
LOH in LAM cells grown from explanted lungs (21
). In the present study, LAM cells from BALF, urine, and chylous effusions reactive with anti-CD44v6 and anti-CD9 antibodies showed TSC2
LOH. Thus, disseminated LAM cells contained prometastatic molecules that could enable their mobilization and subsequent anchorage to sites of metastasis. Phenotypic changes in metastatic cells may occur as they migrate to sites of metastasis (44
). We observed that LAM cells grown from explanted lungs (as identified with the markers CD44v6/CD44), in blood (CD235a), and in BALF, urine, and chyle (CD44v6/CD9) differed in the expression of surface proteins, suggesting that LAM cells within different microenvironments have different phenotypic characteristics, which is consistent with their phenotypic heterogeneity in different tissues. In fact, human TSC2−/−
cells are known to exhibit different morphologies in different locations. In renal AMLs, TSC2−/−
cells appear as smooth muscle, fat, and vascular cells. LAM cells in the lungs may be spindle-shaped or epithelioid.
S-LAM is considered to be associated most frequently with mutations in TSC2
. In our studies, TSC2
LOH was detected in 38 of 43 (88%) patients with S-LAM, consistent with the hypothesis that more patients with S-LAM have dysfunctional TSC2
. We did not find TSC2
LOH in five informative patients with S-LAM; two patients with S-LAM were noninformative because of their homozygosity for all five tested markers (). To increase detection of TSC2
LOH, we did SNP-based LOH analysis on these patients using two SNPs within the TSC2
gene. We identified LOH at the exon 40 polymorphism in BALF cell samples from one of three patients informative for two SNPs; blood and urine cell samples from this patient were not amplified well, probably due to low DNA amounts (data not shown). To determine whether or not these seven patients with S-LAM have TSC1
abnormalities, we further assessed TSC1
LOH but did not identify any patients with TSC1
LOH. Failure to detect TSC1
LOH could result from the fact that the second hit for TSC1
may be subtle sequence changes (e.g., point mutations, small deletions), which are not detectable by LOH analysis. It is also possible that methylation may be responsible for dysregulation of the TSC2
). The absence of TSC1
mutations in LAM cells from patients with S-LAM suggests that pulmonary disease due to this mutation may be subclinical.
Based on earlier reports that AMLs and pulmonary LAM cells from the same patients with S-LAM have the same TSC2
mutations and identical TSC2
LOH patterns (13
), it was hypothesized that pulmonary LAM cells and AML cells could have a common genetic origin, and LAM cells could metastasize in vivo
. Here, we described identical LOH patterns at the chromosome 16p13.3 region in LAM cells isolated from blood, urine, BALF, or chyle from the same patient in 27 of 37 (73%) cases. In 8 of 29 (23%) patients with S-LAM and 2 of 8 (25%) patients with TSC-LAM, however, LAM cells from different body fluids appeared to differ in the extent of LOH regions based on the informative microsatellites. Our data from two patients with TSC-LAM are consistent with the report that two AMLs from the same patient with TSC with multiple AMLs showed different regions of LOH on 16p13 (46
). These findings from eight patients with S-LAM are discordant with a prior report (14
) and suggest that in some patients with S-LAM, LAM cells may show genetic heterogeneity, which (a
) could result from a different second mutation in cells containing the same first mutation, or (b
) result from the introduction of new independent genetic changes in the existing LAM cells during the metastatic process, or (c
) could represent a second novel LAM cell with two different mutations. In support of the first model, skin lesions in patients with TSC appear to arise from cells with independent second mutations in the TSC2
). The second model of chromosomal instability appears to occur frequently in cancer cells (48
). The third model is least likely. Further studies would be required to define the extent of the deletion and the identification of specific genes involved as well as the mechanism(s) of LOH (e.g., mitotic nondisjunction with reduplication of the mutant chromosome).
Among five microsatellites on chromosome 16p13.3, Kg8, closer in proximity to the TSC2 gene than the other microsatellite markers (i.e., D16S291, D16S3395, D16S3024, and D16S521), was more frequently affected in patients with LAM (). In one case, ROH was observed at the Kg8 locus, but LOH was observed in microsatellite markers D16S3395 and D16521, which span a region telomeric to the TSC2 locus (Table E1, see S-LAM1). Nearly half of patients with S-LAM showed LOH of two informative microsatellites; the patterns include LOH at Kg8 and D16S3395, two adjacent microsatellites that span the TSC2 locus, and LOH at two distant microsatellites mapped centromerically (from D16S291 through Kg8) or telomerically (from D16S3395 through D16S521) to the TSC2 locus or spanning the TSC2 locus (from Kg8 through D16S521). Our data indicate that LAM cells from these patients might have loss of a larger part and a different region of chromosome 16.
An association between the presence of AMLs and lymphatic involvement and detection of TSC2 LOH in blood and urine was also assessed, but no significant differences were found between detection of TSC2 LOH in blood cell fractions and urine and the presence of lymphangioleiomyomas, adenopathy, or lymphangioleiomyomas/adenopathy in patients with LAM with or without AMLs (Figure E5). A statistically significant association between TSC2 LOH in cells from urine and the presence of AMLs in patients with LAM () suggests that circulating LAM cells are more likely to be shed from blood into the urine in patients with LAM with AMLs than those without AMLs. Alternatively, cells from AMLs might be shed directly into urine, perhaps due to necrosis within the tumors.
Overall, we found that the presence of specific surface proteins (e.g., CD44v6, CD9, CD235a) was associated with LAM cells exhibiting TSC2 LOH in blood, BALF, urine, and chyle, which supports, in most cases, a metastatic dissemination of LAM cells via blood and/or lymphatic circulatory systems. Contrary to previous observations, however, our data suggest that LAM cells may exhibit genetic as well as phenotypic heterogeneity in some of patients with S-LAM.