Bone marrow metastases in small cell lung cancer: detection with magnetic resonance imaging and monoclonal antibodies.

The detection of bone marrow involvement might be of prognostic value and may influence therapeutic decisions in small cell lung cancer. By unilateral bone marrow aspiration and biopsy, evidence of bone marrow metastases is seen in 15-30% of patients with this disease. Since magnetic resonance imaging of the lower body and immunostaining with monoclonal antibodies have recently been shown to be very sensitive detection methods, we investigated the value of these two techniques in detecting bone marrow involvement in 35 consecutive patients with small cell lung cancer. The results were compared to those obtained with conventional cytohistological analysis. In all cases when cytology and/or bone marrow biopsy were positive, monoclonal antibodies immunostaining and magnetic resonance imaging also detected malignant cells. Furthermore, evidence of bone marrow involvement was shown with magnetic resonance imaging and/or immunostaining in 10 of 26 cases (38%) where routine procedures were unable to detect malignant cells. In one of these 26 patients, magnetic resonance imaging and immunostaining provided the only evidence of metastatic disease. These data suggest that the rate of bone marrow metastases is underestimated by routine procedures. Further investigation is needed to determine whether or not these new non-invasive methods have prognostic value or affect therapeutic choices in small cell lung carcinoma. ImagesFigure 1Figure 2

Bone marrow (BM) involvement is common in patients with small cell lung cancer (SCLC) although it is still not clear whether it is of prognostic value in this disease. In one study , BM metastases had little prognostic value, since the difference in survival rates (8 months versus 10 for patients with no BM involvement detected) was not statistically significant. In other series, BM involvement predicted a significantly lower median duration of response and median survival time (Hirsch & Hansen, 1980). From a therapeutic point of view, BM involvement is known to increase haematological toxicity of conventional treatment (Hirsch & Hansen, 1980). Furthermore, undetectable BM tumour cells may be the cause of some of the relapses observed after intensive chemotherapy followed by autologous BM transplantation in SCLC (Spitzer et al., 1986;Humblet et al., 1987). Thus, knowledge of BM involvement at the time of diagnosis in SCLC is of great importance.
Magnetic resonance imaging (MRI) has recently been shown to be a very sensitive test for the detection of BM involvement in patients with lymphoma (Shields et al.. 1987) Correspondence: V. Trillet. Received 15 November 1988. and accepted in revised form 7 February 1989. and neuroblastoma (Couanet & Jeoffray, 1988). We investigated the use of both MRI and immunological analysis with MoAB for BM staging in patients diagnosed with SCLC, and compared the results of these two new methods with conventional cytohistological methods.

Patients and lmthods
Patients From 15 Apnrl 1987 to 15 August 1988, 35 previously untreated patients with histologically proven SCLC were referred to our institution. There were 33 men and 2 women, with a median age of 61 years. Initial staging included routine blood tests, fibreoptic bronchoscopy + biopsies, CT scan of the thorax, abdominal ultrasonography and/or CT scan, brain C( scan and radionuclide bone scan (RBS) + BM staging (see below). With all these procedures, 27 out of 35 (77%) patients were considered to have metastatic disease at the time of diagnosis.
Cvtological and histological examination of the BM In all patients, unilateral posterior iliac crest aspiration and biopsy were performed before treatment. Several smears were made from the aspirate and cytologic examination was done after Wright coloration.
The biopsies were performed according to the method described by Jamshidi & Swaim (1971) with a median length of 2.5cm and touch imprints made from each biopsy. After decalcification, pathological slices were prepared with Haematoxylin and Eosin staining before light microscopic examination.

MoAB immunostaining
In 33 of 35 patients, 2-3 ml of aspirated marrow were mixed with heparin and mononuclear BM cells were immediately obtained by Ficoll separation for immunological analysis. Two MoABs of the immunoglobulin (Ig) G isotype (UJ 13 A kindly provided by J. Kemshead,11.14 kindly provided by J.C. Laurent, SANOFI) recognise antigens expressed by cells of neuroectodermal origin (Favrot et al., 1986;Rosier & Laurent, 1987), and CD45 of the IgM isotype (kindly provided by G. Janossy) recognises the panleucocyte antigen (Kemshead et al., 1983;Maritaz et al., 1988). Double immunofluorescence immunostaining Two samples (1.3 x 106 cells in suspension in 100 p1 phosphate buffer saline (PBS) with 0.1% NaN3) were incubated with the two MoABs (one for each sample) in combination with CD45. After 10 min at 24"C, samples were washed once in PBS and incubated with TRITC anti-mouse IgM specific and FITC anti-mouse IgG specific (Southern Biotechnology Associates) for O min at 24 C. Samples were then washed twice, maintained in PBS-glycerol and analysed in a fluorescent Zeiss microscope with a 40: 1 objective, a 490 nm excitation filter and a K 530 barrier filter. Cells are considered malignant when positive with at least one of the 2 anti-neuroectodermal antigens MoABs and negative with the CD45.
Alkaline phosphatase immunostaining BM cells in suspension at 6 x I0O ml 1 in PBS are cytocentrifuged into glass (100p1 per smear at 70g for 5min in a Shandon cytospin).
Immunochemical staining is performed using an indirect 3stage immunoenzymatic procedure with alkaline phosphatase (Dakopatts, Copenhagen, Denmark). Briefly, six air-dnred slides are fixed for 3min with acetone at 4-C, incubated for 60mmn with MoABs (3 for UJ 13A and 3 for 11.14) then for 30min with enzyme-conjugated rabbit anti-mouse Ig (Dakopatts) and for 30 min with enzyme-conjugated swine antirabbit Ig (Dakopatts). Washes are done with Tris buffer. The final step consists of a 15 min incubation with Naphtol-As-Mx phosphate. dimethylformamide, levamisole and fast red (Sigma Co., St Louis, USA). Slides are counterstained with Haematoxylin, mounted permanently with glycerin and evaluated under an optical microscope. Negative controls without MoAB and positive controls with MoABs recognising class I antigen on normal BM cells are included in each test.
The limit of detection of those two immunological methods is of one malignant cell in 105 normal mononuclear BM cells if 3 x 106 cells are analysed in double immunofluorescence and six smears in alkaline phosphatase (Maritaz et al., 1988;Combaret et al., 1989).
Magnetic resonance imaging All patients were studied with a 0.5T superconductive magnet (Magni Scan 5000 Thomson CGR) using a body coil and a large field of view (500mm). Ten millimetre thick contiguous slices of the pelvic bony structures including femoral heads, upper extremities of the femurs, pelvic bones (iliac. sacrum), and two or three lower lumbar vertebral bodies were obtained in the coronal plane in order to include from anterior to posterior all the BM of these structures.
A Tl-weighted spin echo sequence was performed using a TR of 500ms and a TE of 26ms. With this sequence, 16 contiguous slices are obtained within 10min of positioning the patient within the coil. With this sequence, BM in normal adults is seen as homogeneous areas of high signal intensity. MR images were analysed by one operator.
MR examinations were considered to be positive or negative depending on the presence or absence of focal areas of low signal intensity within an area of normal signal intensity corresponding to marrow fat, as already described in malignant lymphoma (Shields et al., 1987) and in SCLC (Trillet et al.. 1988).
Radionuclide bone scan (RBS) RBS was performed in 32 out of the 35 patients, 2 h after a bolus intravenous injection of 9 MBq per kg hydroxymethyldiphosphonate labelled with 99Tc. Investigators were asked to provide an unambiguous result (positive or negative) and were blinded to the findings of the four other methods.

Results
In eight patients, cytological examination of the BM was positive (Table I). BM biopsy was positive in only five out of these eight patients. In one case (no. 23). BM biopsy was positive with negative cytology. Thus. BM involvement was detected with cytological and,or histological examination in 9/35 (26%) cases: MRI was positive in all nine cases. BM involvement appearing as focal areas of low signal intensity within the normal signal intensity of marrow fat (Figures la  and 2); this correlated with strongly positive immunostaining in all eight cases where it was performed with the percentage of malignant cells ranging from 10-4 to 0.6.
Of the 26 patients with negative conventional cytohistology, MRI and/or MoABs detected BM metastases in 10 (38%) cases: three had both positive MRI and immunostaining, six had positive MRI only, and one had positive immunostaining alone. MRI and immunostaining were negative in 16 of the 26 patients and in one case (no. 10) where immunostaining was not done, MRI was negative.
Five (14%) patients had extensive disease but no evidence of BM involvement using conventional and specialised techniques.
MoABs UJ 13 A and 11-14 stained more than 95% SCLC tumour samples with very homogeneous staining of malignant cells. Table II shows the concordance between immunofluorescence and alkaline phosphatase immunostairing. A limitation to the use of MoABs against SCLC on BM samples is their reactivity with very few normal cells of the NK lineage. The use of double immunofluorescence analysis permits one to accurately distinguish between those few normal cells (stained with UJ 13A, 11-14 and the anti-panleucocyte MoAB) and malignant cells negative with the anti-  Stage  ED  ED  ED  ED  ED  ED  ED  ED  ED  LD  ED  ED  ED  ED  ED  ED  ED  ED  ED  ED  ED  ED  ED  ED  LD  LD  LD  LD  LD  LD  LD  ED  ED  ED  LD   Patient   2   22  26  29  3  24  15  23  25  11  31  12  17  16  35  20  32    panleucocyte MoAB. The morphological control used with the alkaline phosphatase method also provides objective criteria for malignancy.
RBS (Table I)  In one (no. 2) of the 27 patients with extensive disease, BM was the only metastatic site and BM invasion was detectable by cytohistological examination as well as by MoAB immunostaining and MRI. Nine out of the 10 patients with positive MRI and/or MoAB examination of the BM and a negative routine examination were shown to have at least one other metastatic site at diagnosis. Thus, only one patient (no. 25) was shifted from the 'limited disease' group to the 'extensive disease' group by including these two new methods in the initial staging.

Discussion
In our series, it appears that analysis of one BM aspirate was positive in three cases where biopsy was negative, whereas there was only one example of false negative cytology compared to histological examination of the BM.
These results are consistent with the conclusions of Hirsch and co-workers (Hirsch et al., 1977) on a group of 203 SCLC patients where BM aspiration was shown to be more effective than BM biopsy in detecting BM metastases in SCLC. On the contrary, Lawrence (Lawrence et al., 1984) claimed that histological examination of the BM was superior. when long enough BM specimens were obtained, that is, with the use of the Jamshidi needle instead of the shorter Radner needle used by the Danish group. But in most instances, his patients underwent a second and even a third biopsy 'through the same skin incision from a location of a few millimeters lateral to the first'. This is in accordance with previous reports on many neoplastic diseases (Brunning et al., 1975) including SCLC (Hirsch et al., 1979) which demonstrate that the number of positive BM aspirates and biopsies increases with bilateral examination of the BM. The rate of detection of BM involvement is clearly related to the total amount of BM tissue analysed and this is thought to result from the focal pattern of BM invasion by malignant cells (Lawrence et al., 1984). However, invasive procedures such as bilateral iliac crest trephines are often considered aggressive and investigators have been encouraged to develop new non-aggressive methods of detection. Immunological analysis is a highly sensitive method and allows detection of malignant cells in roughly 50% SCLC patients using MoABs directed against SCLC cells surface antigens like SMI (Stahel et al., 1985) or MOC (Postmus, personal communication). Here, the concentration of malignant cells by Ficoll separation, the analysis of a very large number of cells and the use of objective criteria of malignancy such as morphologic control (alkaline phosphatase method) and double membrane immunostaining with two different markers (double immunofluorescence method) improves the level of detection to 10-5 cells and allows the identification of malignant cells with cytological lymphoidlike features. MoAB immunostaining allows an estimate of the proportion of malignant cells. In the four cases where MoAB immunostaining was positive while routine procedures were negative, the contamination in the aspirate was low (less than or equal to 10-3). In one of our patients, MoAB immunostaining was the only positive test.
Of the nine patients with positive MRI and negative cytology and biopsy, three showed positive immunostaining of the BM and, in the remaining six patients. the signals obtained with MRI were very similar to those observed in cytologically positive BM patients, and similar to those described as typical features of BM involvement leukaemias, lymphomas, neuroblastomas and SCLC. Furthermore. patterns of positivity (multiple focal areas of hyposignals) tend to confirm the focal nature of BM involvement and may explain the insensitivity of methods based on the examination of a single aspiration or biopsy site, including immunological analysis. MRI has the advantage of being painless. being able to scan a wide part of the body and being particularly sensitive to focal involvement. There is a need for the confirmation of malignant BM invasion in the sites of MRJ hyposigiials, in patients with normal BM aspiration and normal biopsy in one iliac crest. RBS is a highly sensitive but very non-specific test in SCLC (Levenson et al., 1981). In our study, all RBS-positive patients also had a positive MRI, except for three cases where bone metastases were detected outside the MRI field of view. In contrast, in five patients with positive MRI. RBS was negative. MRI focal areas of low intensity are located in the fat tissue, that is, in BM itself (Porter et al.. 1986). In contrast, RBS abnormalities reflect cortical bone involvement. Therefore, no comparison can be made between these two methods in terms of sensitivity and specificity. However, the lack of bone metastases detected with RBS and CT bone scan in MRI-positive areas might reflect different patterns of BM and cortical bone involvement in SCLC. According to our results with MRI, metastatic spread in the BM could be an earlier event than cortical bone involvement with SCLC. This sequence of bone invasion is concordant with the figures shown in our study: the rate of BM metastases detected with MRI (50%) is greater than the usual rate of bone metastases (30-35% in most series) (Ihde & Hansen. 1981).
The necessity of BM sampling in SCLC has recently been questioned by some authors (Campling et al.. 1986). According to our preliminary results, routine cyto-histological examination of the BM might fail to detect BM involvement in 38% of patients with SCLC and BM could be a very frequent site of detectable malignant cells in SCLC using MRI and MoAB immunostaining. In our opinion, in a disease where the presence and number of metastatic sites is known to be an important prognostic factor, this finding strongly supports routine examination of the BM with these two complementary and non-aggressive methods. Furthermore, if future results confirm the superiority of cytological and immunological examination of BM aspiration compared to biopsy, the latter aggressive method could possibly be abandoned.
The fact that some patients might be diagnosed as having metastatic instead of limited disease could be of a prognostic importance since it could improve the selection of truly 'limited stage' patients to be treated with a 'curative intent'. However, compared to the series of Carney et al. (1988) where 45% of the patients were restaged after MRI exanination of the BM, we found only one patient with apparently 'limited disease' where MRI and/or MoAB immunostaining resulted in the re-classification in the 'extensive disease' category. This is possibly due both to different patient selection (since 23% of our patients were in the 'limited disease' group versus 60% in Carney's study) and to better routine evaluation of the BM (26% cytologically and/or histologically positive BM instead of 5%).
Another major clinical application for MRI and immunostaining could be the follow-up of BM metastases shown by MRI after chemotherapy to better evaluate the response to a therapy which is known to be a major prognostic factor in this disease. This report is part of an ongoing study undertaken by our institution in order to determine prospectively in a larger series of patients the prognostic and therapeutic implications of the use of these two complementary and non-aggressive methods of tumour staging.
We wish to thank Dr T. Philip (Centre L&on Berard, Lyon, France) for his help and advice in the initiation of this work, Drs J. Armitage (University of Omaha, Nebraska), S. Narod and G.M. Lenoir