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Chapter 17 Detection of small cell carcinoma in bone marrow aspirates using monoclonal antibodies and mixtures of monoclonal antibodies
Lung Cancer, 4 (1988) 76-78
76
Elsevier CHAPTER 17 DETECTION OF SMALL CELL CARCINOMA IN BONE MARROW ASPIRATES USING MONOCLONAL ANTIBODIES AND MIXTURES OF MONOCLONAL ANTIBODIES Fiona Moss 1, Lynda Bobrow2, Peter Beverley3 and Robert Souhami 4 1Department of Thoracic Medicine, Central Middlesex Hosiptal 21CRF Department of Histology, 31CRF Human Tumour Immunology Group, The Courtauld Institute, 91 Riding House Street, London, W1P 8PT. 4Department of Oncology, University College and Middlesex School of Medicine, Cower Street, London, WC1 E 6JJ. Correspondence to P C L Beverley
ABSTRACT
Four monoclonal antibodies and 4 mixtures of these were used to stain 8 control bone marrows, 8 bone marrows known to be infiltrated with SCLC and 20 cytologically negative bone marrows from patients with SCLC. A small population of haemopoietic cells stained with each of the reagents. Clumps of stained cells were seen in all cytologically positive bone marrows. Variation of antigen expression was seen but this was largely overcome by the use of mixtures of monoclonal antibodies. Clumps of stained cells were detected in 4 of 20 (20%) cytologically negative bone marrows. Monoclonal antibodies are more sensitive than routine cytological analysis at detecting SCLC infiltrating bone marrow. This method is limited both by the presence of stained single cells in the normal haemopoietic population and by variation of antigen expression by SCLC. our monoclonal antibodies and 4 mixtures of these were used to stain 8 control bone marrows, 8 bone marrows known to be infiltrated with SCLC and 20 cytologically negative bone marrows from patients with SCLC. A small population of haemopoietic cells stained with each of the reagents. Clumps of stained cells were seen in all cytologically positive bone marrows. Variation of antigen expression was seen but this was largely overcome by the use of mixtures of monoclonal antibodies. Clumps of stained cells Qwere detected in 4 of 20 (20%) cytologically negative bone marrows. Monoclonal antibodies are more sensitive than routine cytological analysis at detecting SCLC infiltrating bone marrow. This method is limited both by the presence of stained single cells in the normal haemopoietic population and by variation of antigen expression by SCLC. INTRODUCTION Small cell lung carcinoma (SCLC) frequently metastasises to bone marrow. The reported incidence of bone marrow metastasis at diagnosis ranges from 12-47 of cases 1"6. Examination of bone marrow is often carried out as part of the initial staging of disease. However this has little impact on staging as in only <2% of cases is the bone marrow the only site of extrathoracic disease 7. Furthermore there is no difference in survival between patients with and without cytological evidence of bone marrow infiltration6, 7 leading to the suggestion that bone marrow examination is unnecessary in patients with SCLC 8. Monoclonal antibodies to epithelial and neural antigens have been shown to stain SCLC in tissue section 9. If these reagents stain SCLC but not haemopoietic cells it may be possible, using immunocytochemical techniques, to detect SCLC in bone marrow at a lower density than is possible using routing cytology. This might affect disease staging and would be important for treatment
programmes
using
autologous
bone
TABLE r.~ Details of antibodies
marrow
transplantation.
Antibody
Specificity
Reference
This study compares the use of monoclonal antibodies with cytology as a method for detecting
5.2
Cytokeratins
Makin et al 11
AUA1
Epithelia
Arklie 12
SCLC in bone marrow aspirations.
HMFG2
Epithelia
Taylor+ Papadimitriou 13
Neuroectodermal tissue
Allan et a114
METHODS UJ13A 1) Patients Three groups of patients were tested. The first group (normal controls) was 8 patients not known to have any carcinoma (4 normal volunteers and 4 patients with lymphoma currently in remission). The second group (positive controls) was 8 patients with small cell carcinoma whose marrows were known to be infiltrated with metastatic disease. Two specimens, one taken before treatment and one after one course of chemotherapy, were available from one patient (SCBM1). The third group was 20 patients with SCLC whose bone marrows were found to be free from tumour infiltration on cytological analysis.
TABLE IB Mixtures of monoclonal antibodies Monoclonal Antibody" UJ13A °° M1
5.2 +
AUA2 +
HMFG2 +
+
+
+
+
+
M2
÷
+
M3
÷
+
M4
+
+ +
• made using equal volumes of constituent monoclonal antibodies "" used at dilution of 2p.g/ml
2) Preparation of marrOw Marrow aspirated either with a Jamshedi needle, as part of normal staging or during bone marrow harvest, was placed in a small volume of hepadnised tissue culture medium. Erythrocytes were sedimented using gravity sedimentation. Equal volumes of minimal essential medium, hydroxymethyl starch (Plasmasteril) and marrow aspirate were allowed to stand at room temperature or 10 minutes. The supernatant was collected and washed three times in minimal essential medium and then centrifuged at 3,000 r.p.m. Smears were made from the pellet, dried at room temperature for 18 hours, and either stained immediately or wrapped in silver foil and stored at -70oc.
3) Immunocy'tochemical method An immunoalkaline phosphatase technique, which included a monoclonal antibody to alkaline phosphatase (APAAP), was used 10. Rabbit anti mouse immunoglobulin (I/100) (Dako) with normal human serum (1/25) was the bridging antibody. The bridging antibody and APAAP complexes were each applied twice. Fast red TR (Sigma) with Levamisole (Sigma) in veronal acetate buffer (pH 9.2) was the substrate. For each marrow two controls were included. One stained with substrate alone (as control for endogenous phosphatase) and one without primary antibody.
+ used as undiluted supernatants
4) Monoclonal antibodies (Table la and Ib) Four monoclonal antibodies (5.2, AUAI, HMFG2 and UJ13A) and four mixtures of these monoclonal antibodies were used to stain each marrow. Three of the four monoclonal antibodies reccgnise epithelial antigens and one, UJ13A, a neural antigen. UJt3A was used at a dilution of 2 p.g/ml the rest as undiluted supernatants. 5) Assessment of staining The slides were read using a Leitz microscope. Each stained cell seen was described. Note was made of the intensity of staining, the size and shape of the cell and the presence of clumps of stained cells. The number of cells on each slide was estimated using a gdd fixed within the microscope.
RESULTS 1) Normal controls Stained cells were seen with each of the monoclonal antibodies and mixtures in at least two of the specimens tested. No clumps of stained cells were seen. The cells which stained with HMFG2 were mostly plasma ceils and would therefore have been
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
??
distinguishable from infiltrating tumour cells. It was considered that other stained cells would have been indistinguishable from infiltrating tumour carls. The size of the populations of strongly stained cells ranged from 0.3-5/104 cells counted.
2) Positive controls (Table II) In marrows from 4 of the 8 patients, clumps of stained cells were seen in slides stained with each of the monoclonal antibodies and mixtures of monoclonal antibodies. In the rest at least one reagent failed to detect clumps of stained cells. With the antibody UJ13A clumps of stained ceils were seen in bone marrows from 5 of 8 patients; no UJ13A positive cells were seen in either specimen from patient SCBMI.
3) Patients with SCLC with cytologically non-filtrated marrow Clumps of stained cells were seen in the marrow of 4 of 20 patients (20%) whose marrow were found to be free from infiltration on cytological testing. In one, clumps of stained cells were detected with 6 of 8 reagents; in one with 4 and 2 with only one. Stained single cells, without clumps of stained cells, were seen in 9 of the remaining 14 marrows; the size of these populations ranged from 0.2-5.6/104 cells counted.
4) Comparison of monoclonal antibodies with cytology for detecting infiltrating SCLC (Table III) Monoclonal antibodies stained clumps of cells in 12 of 28 marrows. Using cytological analysis infiltrating SCLC was detected in 8. DISCUSSION
The aim of this study was to determine if staining with monoclonal antibodies was more sensitive than cytological analysis as a method for detecting infiltrating SCLC in bone marrow. This method is limited by two factors; the staining of haemopoietic cells by monoclonal antibodies and variation of expression of antigens by tumour cells. All the reagents used stained haemopoietic ceils. Whilst some of these, such as plasma cells, were easily distinguished from infiltrating tumour cells, it is clear that a single stained cell cannot be used as evidence of infiltrating tumour. Stained clumps of cells, seen only in marrows from patients with small cell carcinoma, was used as the immunocytochemical marker of SCLC infiltration.
The variation of antigen expression was largely overcome by the use of mixtures of monoclonal antibodies. The antibody UJ13A, which stains sections of small cell carcinoma consistently, was the least reliable reagent for detecting SCLC infiltrating bone marrow and used alone would have been less sensitive than routing cytology. These results reinforce the view that if monoclonal antibodies are to be used for immunodiagnosis or immunotherapy reliance should not be placed on a single reagent.
TABLE Ill Comparison of detection of small cell carcinoma in bone marrow aspirations by monoclonal antibodies and cytology
In spite of these limitations monoclonal antibodies and mixtures of monoclonal antibodies improved the detection of SCLC in bone marrow. Overall SCLC was detected by monoclonal antibodies in 43% and by cytology in 29% of patients. The improvement using monoclonal antibodies was mainly due to detection of SCLC in the marrow of patients with limited disease.
DISEASE EXTENT
Using a single monoclonal antibody, SM1, to stain bone marrow aspirations, it has been shown that the number of patients recognised as having extensive disease may be increased from 45% to 72% 15. This monoclonal antibody appears superior to the monoclonal antibodies used here. Not only was the improvement in detection of infiltrating bone marrow greater, but 6 bone marrow aspirations from normal patients did not stain. However in addition to the differences in monoclonal antibodies there are also differences in technique. Firstly a density gradient was used to separate mononuclear cells; secondly, indirect immunofluorescence was used as the antigen detection system and, thirdly, unfixed calls were stained in suspension. Any of these factors may have increased the sensitivity of the method but only 2,000 cells were counted. The positive cells detected in normal bone marrow may be a reflection of the large number of cells stained; it is more likely that small populations of cells will be revealed if larger numbers of cells are examined.
ROUTINE MONOCLONAL CYTOLOGY ANTIBOOIES
TOTAL EX/WIINEO
LIMITED
0
3
8
EXTENSIVE
8
9
20
TOTAL
8
12
28
REFERENCES Eagan, R.T., Maurer, L.H., Forcier, R.J., Tunoh, M. (1974). Small cell carcinoma of the lung: staging, paraneoplastic syndrome, treatment and survival. Cancer 33: 527-532. Anner, RM., Drewinko, B. (1977). Frequency and significance of bone marrow involvement by metastatic solid tumour. Cancer 39: 13371344. Hirsch, F.R., Hansen, H.H., Dombernowsky, P., Hainau, B. (1977). Bone marrow examination in the staging of small cell anaplastic carcinoma of the lung with special reference to subtyping. Cancer 39: 2563-2567. Hirsch, F.R.. Hansen, H.H. (1980). Bone marrow involvement in small cell carcinoma of the lung. Cancer 46:206-211.
Lawrence, J.B., Eleff, M., Behm, F.G., Johnston, C.L. (1984). Bone marrow examination in small cell carcinoma of the lung. Cancer 53: 21882190.
The most important aspect of this type of study, which attempts to improve tumour detection cannot be answered by the data presented here. That is whether or not the improvement in staging will improve the prediction of outcome. Routine examination of bone marrow in patients with SCLC has been shown not to be worthwhile investigation. Monoclonal antibodies clearly improve detection of SCLC infiltrating bone marrow but the prognostic value of this approach needs to be tested using the most reliable monoclonal antibodies or mixtures of monoclonal antibodies with the most reliable method of bone marrow separation and staining. The results of this study suggest that most monoclonal antibodies will stain at least a small population of haemopoietic cells and that care needs to be taken in interpreting the significance of single stained cells.
Kelly, B.M., Morris, J.F., Harwood, B.P., Bruya, T.E. (1984). Methods and prognostic valuse of bone marrow examination in small cell carcinoma of the lung. Cancer 53: 99-102. Idhe, D.C., Makuch, R.W., Carney, D.N. et al (1981). Prognostic implications of stage of disease and sites of metastases with small cell carcinoma of the lung treated with combination chemotherapy. Am. Rev. Res. Dis. 123: 500507.
TABLE II No. of strongly stained cells seen (expressed as No/104 cells counted) in bone marrow patients with cytologically proven SCLC marrow infiltration. Specimen No.
Monoclonal Antibodies 5.2
SCBMla °
132+
HMFG2 AUA1 258.3+
643.8+
Mixtures of Monoclonal Antibodies UJ31A M1 M2 M3 0
645+
402+
578+
M4 564+
SCBmlb*
1.7+
4.0+
23.2+
0
47.5+
41.7+
36+
5.5+
SCBM2
205+
352+
241 +
258+
292+
244+
184+
236+
SCMB3
1.6+
1.5+
1.1+
0
4.5+
0.6+
1.9+
0,3+
SCBM4
0
3.0+
6.5+
2.7+
13.1+
18.7+
10.4+
42+
SCBM5
93.9+
43.3+
91.7+
6.25+
101+
87.2+
104+
93+
SCBM6
65.7+
66.5+
96.5+
64.2+
85.2+"
313+
159+
273+
SCMB7
7.2+
2.2+
9.1+
1.4+
12.4+
39.6+
7.1+
5.8+
SCMB8
0
0
12+
0
3.5+
0
0
0
+ = clumps of positive cells identified = specimen from the same patient (a) before treatment (b) after I course of combination chemotherapy.
78 Campling, B., Quirt, I., Deboer, G. et al (1986). Is bone marrow examination in lung cancer really necessary? Ann. Int. Med. 105: 508-512. 9.
Moss, F.M., Bobrow, L.G., Sheppard, M.N, et al (1986). Expression of epithelial and nueral antigens in small cell and non-small cell lung carcinoma. J. Pathol. 149: 103-11.
10. Cordell, J.L., Falini, B., Erber, W.N. et al (1984). Immunoenzymatic labelling ot monoclonal antibodies using immunocomplexes of alkaline phosphatase and monoclonal anti-alkaline
phosphatase. 219-229.
J. Histochem. Cytochem. 32:
11. Makin, C.A., Bobrow, L.G., Bodmer, W.F., (1984). Monoclonal antibodies to cytokeratin for use in routine cytology, J. Clin. Path. 37: 975983. Studies of the human 12. Arklie, J. (1981). epithelial cell surface using monoclonal antibodies. D. Phil. Thesis: University ot Oxford. 13. Taylof-Papadimitriou, J., Paterson, J.A., Ark[ie, J. et al (1981). Monoclonal antibodies to
epithelium specific components of the human mitk fat globule membrane: production and reaction with cells in culture, Int. J. Cancer 28: 17 -21. 14. Allan, P.A., Garson, A., Harper, E.L st at (1983). Biological characterisation and clinical applications of a monoclonal antibody recognising an antigen restricted to neuro ectoderman tissues. J. Cancer 31 : 591-598. 15. Stahel, R.A., Mabry, M., Skadn, A.T. et al (1985). Detection of bone marrow metastasis in small cell lung cancer by monoclonal antibody. J, Clin. Oncol. 3: 455-461.
76
Elsevier CHAPTER 17 DETECTION OF SMALL CELL CARCINOMA IN BONE MARROW ASPIRATES USING MONOCLONAL ANTIBODIES AND MIXTURES OF MONOCLONAL ANTIBODIES Fiona Moss 1, Lynda Bobrow2, Peter Beverley3 and Robert Souhami 4 1Department of Thoracic Medicine, Central Middlesex Hosiptal 21CRF Department of Histology, 31CRF Human Tumour Immunology Group, The Courtauld Institute, 91 Riding House Street, London, W1P 8PT. 4Department of Oncology, University College and Middlesex School of Medicine, Cower Street, London, WC1 E 6JJ. Correspondence to P C L Beverley
ABSTRACT
Four monoclonal antibodies and 4 mixtures of these were used to stain 8 control bone marrows, 8 bone marrows known to be infiltrated with SCLC and 20 cytologically negative bone marrows from patients with SCLC. A small population of haemopoietic cells stained with each of the reagents. Clumps of stained cells were seen in all cytologically positive bone marrows. Variation of antigen expression was seen but this was largely overcome by the use of mixtures of monoclonal antibodies. Clumps of stained cells were detected in 4 of 20 (20%) cytologically negative bone marrows. Monoclonal antibodies are more sensitive than routine cytological analysis at detecting SCLC infiltrating bone marrow. This method is limited both by the presence of stained single cells in the normal haemopoietic population and by variation of antigen expression by SCLC. our monoclonal antibodies and 4 mixtures of these were used to stain 8 control bone marrows, 8 bone marrows known to be infiltrated with SCLC and 20 cytologically negative bone marrows from patients with SCLC. A small population of haemopoietic cells stained with each of the reagents. Clumps of stained cells were seen in all cytologically positive bone marrows. Variation of antigen expression was seen but this was largely overcome by the use of mixtures of monoclonal antibodies. Clumps of stained cells Qwere detected in 4 of 20 (20%) cytologically negative bone marrows. Monoclonal antibodies are more sensitive than routine cytological analysis at detecting SCLC infiltrating bone marrow. This method is limited both by the presence of stained single cells in the normal haemopoietic population and by variation of antigen expression by SCLC. INTRODUCTION Small cell lung carcinoma (SCLC) frequently metastasises to bone marrow. The reported incidence of bone marrow metastasis at diagnosis ranges from 12-47 of cases 1"6. Examination of bone marrow is often carried out as part of the initial staging of disease. However this has little impact on staging as in only <2% of cases is the bone marrow the only site of extrathoracic disease 7. Furthermore there is no difference in survival between patients with and without cytological evidence of bone marrow infiltration6, 7 leading to the suggestion that bone marrow examination is unnecessary in patients with SCLC 8. Monoclonal antibodies to epithelial and neural antigens have been shown to stain SCLC in tissue section 9. If these reagents stain SCLC but not haemopoietic cells it may be possible, using immunocytochemical techniques, to detect SCLC in bone marrow at a lower density than is possible using routing cytology. This might affect disease staging and would be important for treatment
programmes
using
autologous
bone
TABLE r.~ Details of antibodies
marrow
transplantation.
Antibody
Specificity
Reference
This study compares the use of monoclonal antibodies with cytology as a method for detecting
5.2
Cytokeratins
Makin et al 11
AUA1
Epithelia
Arklie 12
SCLC in bone marrow aspirations.
HMFG2
Epithelia
Taylor+ Papadimitriou 13
Neuroectodermal tissue
Allan et a114
METHODS UJ13A 1) Patients Three groups of patients were tested. The first group (normal controls) was 8 patients not known to have any carcinoma (4 normal volunteers and 4 patients with lymphoma currently in remission). The second group (positive controls) was 8 patients with small cell carcinoma whose marrows were known to be infiltrated with metastatic disease. Two specimens, one taken before treatment and one after one course of chemotherapy, were available from one patient (SCBM1). The third group was 20 patients with SCLC whose bone marrows were found to be free from tumour infiltration on cytological analysis.
TABLE IB Mixtures of monoclonal antibodies Monoclonal Antibody" UJ13A °° M1
5.2 +
AUA2 +
HMFG2 +
+
+
+
+
+
M2
÷
+
M3
÷
+
M4
+
+ +
• made using equal volumes of constituent monoclonal antibodies "" used at dilution of 2p.g/ml
2) Preparation of marrOw Marrow aspirated either with a Jamshedi needle, as part of normal staging or during bone marrow harvest, was placed in a small volume of hepadnised tissue culture medium. Erythrocytes were sedimented using gravity sedimentation. Equal volumes of minimal essential medium, hydroxymethyl starch (Plasmasteril) and marrow aspirate were allowed to stand at room temperature or 10 minutes. The supernatant was collected and washed three times in minimal essential medium and then centrifuged at 3,000 r.p.m. Smears were made from the pellet, dried at room temperature for 18 hours, and either stained immediately or wrapped in silver foil and stored at -70oc.
3) Immunocy'tochemical method An immunoalkaline phosphatase technique, which included a monoclonal antibody to alkaline phosphatase (APAAP), was used 10. Rabbit anti mouse immunoglobulin (I/100) (Dako) with normal human serum (1/25) was the bridging antibody. The bridging antibody and APAAP complexes were each applied twice. Fast red TR (Sigma) with Levamisole (Sigma) in veronal acetate buffer (pH 9.2) was the substrate. For each marrow two controls were included. One stained with substrate alone (as control for endogenous phosphatase) and one without primary antibody.
+ used as undiluted supernatants
4) Monoclonal antibodies (Table la and Ib) Four monoclonal antibodies (5.2, AUAI, HMFG2 and UJ13A) and four mixtures of these monoclonal antibodies were used to stain each marrow. Three of the four monoclonal antibodies reccgnise epithelial antigens and one, UJ13A, a neural antigen. UJt3A was used at a dilution of 2 p.g/ml the rest as undiluted supernatants. 5) Assessment of staining The slides were read using a Leitz microscope. Each stained cell seen was described. Note was made of the intensity of staining, the size and shape of the cell and the presence of clumps of stained cells. The number of cells on each slide was estimated using a gdd fixed within the microscope.
RESULTS 1) Normal controls Stained cells were seen with each of the monoclonal antibodies and mixtures in at least two of the specimens tested. No clumps of stained cells were seen. The cells which stained with HMFG2 were mostly plasma ceils and would therefore have been
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
??
distinguishable from infiltrating tumour cells. It was considered that other stained cells would have been indistinguishable from infiltrating tumour carls. The size of the populations of strongly stained cells ranged from 0.3-5/104 cells counted.
2) Positive controls (Table II) In marrows from 4 of the 8 patients, clumps of stained cells were seen in slides stained with each of the monoclonal antibodies and mixtures of monoclonal antibodies. In the rest at least one reagent failed to detect clumps of stained cells. With the antibody UJ13A clumps of stained ceils were seen in bone marrows from 5 of 8 patients; no UJ13A positive cells were seen in either specimen from patient SCBMI.
3) Patients with SCLC with cytologically non-filtrated marrow Clumps of stained cells were seen in the marrow of 4 of 20 patients (20%) whose marrow were found to be free from infiltration on cytological testing. In one, clumps of stained cells were detected with 6 of 8 reagents; in one with 4 and 2 with only one. Stained single cells, without clumps of stained cells, were seen in 9 of the remaining 14 marrows; the size of these populations ranged from 0.2-5.6/104 cells counted.
4) Comparison of monoclonal antibodies with cytology for detecting infiltrating SCLC (Table III) Monoclonal antibodies stained clumps of cells in 12 of 28 marrows. Using cytological analysis infiltrating SCLC was detected in 8. DISCUSSION
The aim of this study was to determine if staining with monoclonal antibodies was more sensitive than cytological analysis as a method for detecting infiltrating SCLC in bone marrow. This method is limited by two factors; the staining of haemopoietic cells by monoclonal antibodies and variation of expression of antigens by tumour cells. All the reagents used stained haemopoietic ceils. Whilst some of these, such as plasma cells, were easily distinguished from infiltrating tumour cells, it is clear that a single stained cell cannot be used as evidence of infiltrating tumour. Stained clumps of cells, seen only in marrows from patients with small cell carcinoma, was used as the immunocytochemical marker of SCLC infiltration.
The variation of antigen expression was largely overcome by the use of mixtures of monoclonal antibodies. The antibody UJ13A, which stains sections of small cell carcinoma consistently, was the least reliable reagent for detecting SCLC infiltrating bone marrow and used alone would have been less sensitive than routing cytology. These results reinforce the view that if monoclonal antibodies are to be used for immunodiagnosis or immunotherapy reliance should not be placed on a single reagent.
TABLE Ill Comparison of detection of small cell carcinoma in bone marrow aspirations by monoclonal antibodies and cytology
In spite of these limitations monoclonal antibodies and mixtures of monoclonal antibodies improved the detection of SCLC in bone marrow. Overall SCLC was detected by monoclonal antibodies in 43% and by cytology in 29% of patients. The improvement using monoclonal antibodies was mainly due to detection of SCLC in the marrow of patients with limited disease.
DISEASE EXTENT
Using a single monoclonal antibody, SM1, to stain bone marrow aspirations, it has been shown that the number of patients recognised as having extensive disease may be increased from 45% to 72% 15. This monoclonal antibody appears superior to the monoclonal antibodies used here. Not only was the improvement in detection of infiltrating bone marrow greater, but 6 bone marrow aspirations from normal patients did not stain. However in addition to the differences in monoclonal antibodies there are also differences in technique. Firstly a density gradient was used to separate mononuclear cells; secondly, indirect immunofluorescence was used as the antigen detection system and, thirdly, unfixed calls were stained in suspension. Any of these factors may have increased the sensitivity of the method but only 2,000 cells were counted. The positive cells detected in normal bone marrow may be a reflection of the large number of cells stained; it is more likely that small populations of cells will be revealed if larger numbers of cells are examined.
ROUTINE MONOCLONAL CYTOLOGY ANTIBOOIES
TOTAL EX/WIINEO
LIMITED
0
3
8
EXTENSIVE
8
9
20
TOTAL
8
12
28
REFERENCES Eagan, R.T., Maurer, L.H., Forcier, R.J., Tunoh, M. (1974). Small cell carcinoma of the lung: staging, paraneoplastic syndrome, treatment and survival. Cancer 33: 527-532. Anner, RM., Drewinko, B. (1977). Frequency and significance of bone marrow involvement by metastatic solid tumour. Cancer 39: 13371344. Hirsch, F.R., Hansen, H.H., Dombernowsky, P., Hainau, B. (1977). Bone marrow examination in the staging of small cell anaplastic carcinoma of the lung with special reference to subtyping. Cancer 39: 2563-2567. Hirsch, F.R.. Hansen, H.H. (1980). Bone marrow involvement in small cell carcinoma of the lung. Cancer 46:206-211.
Lawrence, J.B., Eleff, M., Behm, F.G., Johnston, C.L. (1984). Bone marrow examination in small cell carcinoma of the lung. Cancer 53: 21882190.
The most important aspect of this type of study, which attempts to improve tumour detection cannot be answered by the data presented here. That is whether or not the improvement in staging will improve the prediction of outcome. Routine examination of bone marrow in patients with SCLC has been shown not to be worthwhile investigation. Monoclonal antibodies clearly improve detection of SCLC infiltrating bone marrow but the prognostic value of this approach needs to be tested using the most reliable monoclonal antibodies or mixtures of monoclonal antibodies with the most reliable method of bone marrow separation and staining. The results of this study suggest that most monoclonal antibodies will stain at least a small population of haemopoietic cells and that care needs to be taken in interpreting the significance of single stained cells.
Kelly, B.M., Morris, J.F., Harwood, B.P., Bruya, T.E. (1984). Methods and prognostic valuse of bone marrow examination in small cell carcinoma of the lung. Cancer 53: 99-102. Idhe, D.C., Makuch, R.W., Carney, D.N. et al (1981). Prognostic implications of stage of disease and sites of metastases with small cell carcinoma of the lung treated with combination chemotherapy. Am. Rev. Res. Dis. 123: 500507.
TABLE II No. of strongly stained cells seen (expressed as No/104 cells counted) in bone marrow patients with cytologically proven SCLC marrow infiltration. Specimen No.
Monoclonal Antibodies 5.2
SCBMla °
132+
HMFG2 AUA1 258.3+
643.8+
Mixtures of Monoclonal Antibodies UJ31A M1 M2 M3 0
645+
402+
578+
M4 564+
SCBmlb*
1.7+
4.0+
23.2+
0
47.5+
41.7+
36+
5.5+
SCBM2
205+
352+
241 +
258+
292+
244+
184+
236+
SCMB3
1.6+
1.5+
1.1+
0
4.5+
0.6+
1.9+
0,3+
SCBM4
0
3.0+
6.5+
2.7+
13.1+
18.7+
10.4+
42+
SCBM5
93.9+
43.3+
91.7+
6.25+
101+
87.2+
104+
93+
SCBM6
65.7+
66.5+
96.5+
64.2+
85.2+"
313+
159+
273+
SCMB7
7.2+
2.2+
9.1+
1.4+
12.4+
39.6+
7.1+
5.8+
SCMB8
0
0
12+
0
3.5+
0
0
0
+ = clumps of positive cells identified = specimen from the same patient (a) before treatment (b) after I course of combination chemotherapy.
78 Campling, B., Quirt, I., Deboer, G. et al (1986). Is bone marrow examination in lung cancer really necessary? Ann. Int. Med. 105: 508-512. 9.
Moss, F.M., Bobrow, L.G., Sheppard, M.N, et al (1986). Expression of epithelial and nueral antigens in small cell and non-small cell lung carcinoma. J. Pathol. 149: 103-11.
10. Cordell, J.L., Falini, B., Erber, W.N. et al (1984). Immunoenzymatic labelling ot monoclonal antibodies using immunocomplexes of alkaline phosphatase and monoclonal anti-alkaline
phosphatase. 219-229.
J. Histochem. Cytochem. 32:
11. Makin, C.A., Bobrow, L.G., Bodmer, W.F., (1984). Monoclonal antibodies to cytokeratin for use in routine cytology, J. Clin. Path. 37: 975983. Studies of the human 12. Arklie, J. (1981). epithelial cell surface using monoclonal antibodies. D. Phil. Thesis: University ot Oxford. 13. Taylof-Papadimitriou, J., Paterson, J.A., Ark[ie, J. et al (1981). Monoclonal antibodies to
epithelium specific components of the human mitk fat globule membrane: production and reaction with cells in culture, Int. J. Cancer 28: 17 -21. 14. Allan, P.A., Garson, A., Harper, E.L st at (1983). Biological characterisation and clinical applications of a monoclonal antibody recognising an antigen restricted to neuro ectoderman tissues. J. Cancer 31 : 591-598. 15. Stahel, R.A., Mabry, M., Skadn, A.T. et al (1985). Detection of bone marrow metastasis in small cell lung cancer by monoclonal antibody. J, Clin. Oncol. 3: 455-461.