Detection of small-cell-lung-cancer cells in bone-marrow aspirates by monoclonal antibodies NCC-LU-243, NCC-LU-246 and MLuCl

Abstract

Despite their high response rate to treatment, virtually all patients with small-cell lung cancer (SCLC) eventually die. More accurate staging procedures may identify patients who might perhaps benefit from novel treatment strategies.Monoclonal antibodies (MAbs) recognize antigens expressed by tumor cells and have thie potential to detect small numbers of infiltrating tumor cells ;at metastatic sites. Many of the antibodies that react with SCLC have been grouped into clusters based on their pattern of reactivity with tumors and normal tissues (Souhami et al., 1987(Souhami et al., , 1991. Since SCLC frequently metastatises to bone marrow, these antibodies could be used to identify tumor cells in bone-marrow samples of patients treated for SCLC.We have used MAbs from 2 clusters (Souhami et al., 1991) to identify tumor cells in bone-marrow aspirates of patients with SCLC and we have compared the results with conventional histomorphology. PATIENTS AND METHODS Collection of .sumplesBone-marrow aspirates were taken from the posterior iliac crcst of 52 patients with histological or cytological diagnosis of SCLC. A total of 108 bone-marrow aspirates were taken: bilaterally in 48 patients (4 of thlese had also the marrow aspirate repeated) and unilaterally in 4; 84 samples were collected at diagnosis from 44 patients and 24 during different phases of the disease (Table I).Bone-marrow aspirates (3 to 5 ml) were collected in heparinized syringes, diluted with an equivalent volume of RPMI 1640 with 5% FCS (MA Bioproducts, Walkersville, MD), layered ovcr the same amount of Ficoll-Paque and centrifuged at 400g for 30 rnin at 19°C. The cell layer was diluted in 10 ml of RPMI 1640 with 5 % FCS and centrifuged at 350 g for 7 rnin at 4°C. The cell pellet was washed twice in 10 ml of RPMI 1640 with 5% FCS. Cytospin slides (500 rpm lor 4 min), pre-treated with poly-L-lysine, were then prepared for immunostaining using 200 11. 1 of a cell suspension containing about lo6 ml-l nucleated cells. For each antibody, 2 to 4 slides were prepared, and fixed in cold acetone for 10 min as wet preparations. After fixation, the cytospin slides were air-dried for 1 hr, then placed in 10% human serum (Sigma, St. Louis, MO) and immediately immunostained or stored at -20°C until required. As an external control, we stained cytospin preparations of N592 cells, of bone marrow of patients with non-neoplastic diseases and of peripheral-blood lymphocytes from healthy volunteers.Eighty-three bone-marrow biopsies were obtained, by means of the Jamshidi needle, from 50 of these patients. In 27 patients, the samples were collected bilaterally (in 3 they were also later obtained bilaterally during different phases of the disease) and in 23 unilaterally; 65 specimens were collected at diagnosis (Table 11) from 41 patients. Bone-marrow biopsies were fixed in 10% buffered formaldehyde solution for 12 hr, and then processed routinely for paraffin embedding. Histologic sections 3-11. thick were evaluated morphologically by hematoxylin-eosin stain, Giemsa stain, Gor...

In vitro detection of metastases of small-cell pulmonary carcinoma to the bone marrow using monoclonal antibodies

“…2). [8][9][10][11][12]. The results of staining of these 5 preparations are presented in Table 1 (group II, preparations Nos.…”

“…Use of MAb to surface anti- Fig. Indirect immunofluorescence, x 3000, gens of SPC cells disclosed metastases to the bone marrow in 20-50% of patients in whom they had not been detected by cytomorphological investigation [8,11]. Aggregates of small--ceil pulmonary carcinoma cells, stained with MAb H417.1 (preparation No.…”

Immunocytochemical assessment of bone marrow aspirates for monitoring response to chemotherapy in small-cell lung cancer patients

“…A growing body of evidence suggests that not only BM contamination by SCLC cells is far more frequent than previously shown by traditional cytohistological methods (Stahel et al, 1985;Leonard et al, 1990;Pasini et al, 1994bPasini et al, , 1995 but also that BM involvement may have clinical relevance (Leonard et al, 1990;Bucher et al, 1994;Pasini et al, 1994bPasini et al, , 1995. Immunocytochemistry is a convenient method for detecting BM micrometastases thanks to its simplicity, reliability and relative low cost (Leonard et al, 1990;Molino et al, 1991;Beiske et al, 1992;Skov et al, 1992;Pasini et al, 1994a), whereas other techniques, such as in vitro cultures of marrow cells (Pollard et al, 1981;Hunter et al, 1987;Everard et al, 1990) or magnetic resonance imaging (Trillet et al, 1989), are cumbersome, time-consuming and expensive.…”

“…NCAM belongs to a family of membrane-bound, homophilic calcium-dependent glycoproteins involved in both cell-cell and cell-substrate interactions (Keilhauer et al, 1985;Cunningham et al, 1987;Rutishauer et al, 1988;Acheson et al, 1991). They are also expressed in a wide variety of normal tissues, mainly of neural and mesenchymal lineage (Cunningham et al, 1987;Dalseg et al, 1989;Patel et al, 1991;Lanier et al, 1992), as well as in most neuroectodermal and some mesodermal tumours (Patel et al, 1989(Patel et al, , 1991Moolenaar et al, 1990;Tome et al, 1991;Kern et al, 1992;Miettinen and Cupo, 1993;Ledermann et al, 1994;Pasini et al, 1994aPasini et al, , 1995Nakamura et al, 1995). Several isoforms of the molecule have been described that derive from a single copy gene by alternative mRNA splicing (Cunningham et al, 1987).…”

Effects of Different Immunolabeling Techniques on the Detection of Small-cell Lung Cancer Cells in Bone Marrow

“…Evidence is accumulating not only that bone marrow microcontamination by SCLC cells is by far more common than assessed by traditional cytohistological methods (Berendsen et al 1988;Trillet et al 1989;Beiske et al 1992) but also that this involvement may have clinical relevance (Leonard et al 1990;Bucher et al 1994;Pasini et al 1994bPasini et al , 1995. In general, the immunocytochemical search for bone marrow micrometastases in SCLC patients has been performed on bone marrow aspirates (BMAs) using monoclonal antibodies (MAbs) directed against tumor-associated membrane antigens or cytokeratin polypeptides (Leonard et al 1990;Beiske et al 1992;Skov et al 1992;Pasini et al 1994a). Immunocytochemistry is the most simple, reliable, and inexpensive technique for detecting neoplastic microlocalizations in bone marrow (Molino et al 1991), whereas other reported methods, such as in vitro cultures of marrow cells (Pollard et al 1981;Hunter et al 1987;Everard et al 1990) or magnetic resonance imaging (Trillet et al 1989), are cumbersome, expensive, and time-consuming.…”