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Chapter 14 Reactivity of workshop monoclonal antibodies with small cell lung cancer (SCLC) cell lines, normal bone marrow and marrow from SCLC patients
67
Lung Cancer, 4 (1988) 67-69 Elsevier CHAPTER 14 REACTIVITY OF WORKSHOP MONOCLONAL ANTIBODIES WITH SMALL CELL LUNG CANCER (SCLC) CELL LINES, NORMAL BONE MARROW AND MARROW FROM SCLC PATIENTS Frances G. Haylt, Lorraine W. Adams 1, and Robert C.F. Leonard 2 1imperial Cancer Research Fund Medical Oncology Unit and 2University Department of Clinical Oncology Western General Hospital Edinburgh, EH4 2XU 1" to whom correspondence should be addressed
ABSTRACT The reactivity of the workshop antibodies was studied against 3 SCLC cell lines, 3 normal bone marrows and 7 marrow aspirates from SCLC patients. All antibodies except 1, 2, 5, 6, 16, 21, 25, 29, 47, 48, 50, 51, 54 and negative controls stained the SCLC cell lines heterogeneously. Antibodies classified as neural showed strongest reactivity. Many antibodies were unreactive or had limited reactivity with normal bone marrow cells with the exception of 38, 49, 57 and 58 all of which cross-reacted strongly. Tumour cells were detected in SCLC infiltrated marrows b y antibodies with neural, SCLC and epithelial specificity. INTRODUCTION Examination of bone marrow aspirates and biopsies for evidence of tumour involvement is frequently performed as part of the initial staging or restaging procedure for patients with SCLC. Approximately 20% of all patients studied have marrow involvement detected by histological methods, although as many as 66% are reported to have bone marrow infiltration at necropsy. The frequency of marrow involvement as detected by rnonoclonal antibody reactivity has been reported to reach similar levels to those found at necropsy 1 and this is apparently a more sensitive detection method than routing cytology and histology.
for 5 minutes and either processed immediately or stored at -200C until required.
A PAP (Peroxidase-anti-peroxidase) staining
Normal Bone Marrow Marrow aspirates were obtained from patients being investigated for folate deficiency. Five ml of aspirated marrow were mixed with 5 ml of hepadnised PBS. Red cells were removed by Ficoll Hypaque separation followed by washing in RPMI. Cells were resuspended at 5 x 106- 106 cells/ml and slides were prepared as before. SCLC Patients' Bone Marrow Bone marrow aspirates were collected from patients with histologically proven SCLC at routine staging procedures. The patients were staged prior to treatment using standard clinical and radiological procedures as having limited disease in 3 cases and extensive disease in 4 cases. Six of the marrows were lightly infiltrated with tumour cells (<1% of total cells present) whereas the 7th was heavily infiltrated with tumour cells (>70% of total cells present). Red cells were removed by Ficoll Hypaque separation and slides were prepared as for the cell lines.
method was used in this study 3- Briefly, slides were fixed in acetone/methanol and endogenous peroxidase activity was removed by immersion in methanol/H202. Slides were washed in tris buffered saline (TBS) pH7.6, incubated with diluted normal sheep serum followed by the monoclonal antibodies at optimal dilution, washed, incubated with sheep anti mouse IgG in excess, further washing then PAP complex (SaraLab). The peroxidase was visualised using diaminobenzidene tetrahydrochloride, 0.6 mg/ml in 0.2 M Tris-HCL buffer, pH7.6 containing 0.01% hydrogen peroxide and imidazole. Slides were counterstained with haemotoxylin. In the case of the rat antibodies an indirect peroxidase conjugate was substituted for the sheep anti mouse IgG and the PAP. Marrow s a m p l e s required addition of normal human serum during staining to reduce non-specific background staining. Scoring of Staining In SCLC cell lines and normal bone marrow scoring was performed on the basis of percentage
TP.BLE I Reactivity of Workshop Antibodies with SCLC Cell Lines, Normal Bone Marrows and SCLC Turnout-Infiltrated Bone Marrows antibodv
We have previously noted marked heterogeneity of staining using a number of antibodies with SCLC tumour cens 2 and propose that use of a panel of antibodies, either singly or as a cocktail, would substantially increase the likelihood of detecting turn our cells in marrow aspirates especially where infiltration is sparse. With such applications in mind we have examined the reactivity of the workshop panel of antibodies with SCLC cell lines and with normal bone marrow to identify those which detect a high proportion of turnour cells but which do not react with normal bone marrow cells.
Immunocytochemistry
Neural
4 8 13 14 20 22 23 24 28 31 32 35 36 37 46 49
53
SCLC cell lines range(%) mean(%) 20-100 10-96 60-94 40-70 50-92 70-100 25-86 0-4 35-84 30-97 0-40 25-96 50-94 45-89 70-90 =0-50 I0-98
Normal marrow
=72 63 79 57 75 90 65 1 61 73 20 71 79 63 83 17 53
0 <1 0 0 <1 <1 0 010 <1
0
SCLC Marrows light heavy infiltrates infiltrates % + 82 + 87 + 79 + 58 + 87 + 79 + 78 0 + 51 47 + 62 68 ND ND + 61 + ND <1 + 37
MATERIALS AND METHODS SCLC Cell Lines Three established SCLC cell lines of the classic type were used; NCI HI87 was obtained from Dr. D. Carney, Dublin; COR L31 and COR L47 were obtained from Dr. P. Twentyman, Cambridge. The cells were harvested in log phase, washed in RPMI and dead cells were removed by centrifugation over Ficoll Hypaque. The cells were then resuspended at a concentration of 5 x 105 or 1 x 106 cells/ml in RPMI and 20 ul of the cell suspension was dropped into each well of 12 well, Teflon-coated Multispot slides. The slides were air dried, fixed in acetone/methanol at 4Oc
Epithelial
Lung
15
0-30
10
1-10
17 18 27
80-99 0-2 10-84
39 55 56 57 58 59
0-14 2-56 0-57 95-100 65-100 1-40
91 1 39 5
0 0 ND 0
21 22 98 88 14
>10 ND >50 >50 >10
3 9 38 40 43 44
29-45 50-95
37 78
8-41 45-65 4-45
21 58 24
9-65
19 42 45
16-50 10-72 0-20
+ ÷
N+D +
0
63 ND 3 1 1
80 ND 1
30
<1 >10 0 0 <1
+ + +
5 71 40 5 17 50
29 38 10
1-10 1-10 <1
ND ND ND
ND ND ND
+
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
68 positive cells in the total number of nucleated cells. Staining intensity was also noted. In the marrow from patients with SCLC, scoring was recorded as either positive or negative except for the heavily infiltrated marrow where percentages of positive cells were recorded. The morphology of the positive cells was recorded, i.e. tumour or non-turnout if recognisable. The tumour cell population in positive samples was variable, often only 1-2 tumour cells were present per well according to the level of infiltration of each individual marrow. RESULTS SCLC Cell Lines Antibodies 1,2, 5, 6, 16, 21,25, 29, 47, 48, 50, 51, 54 and the hidden negative controls 11,30 and 34 did not stain any of the SCLC cell lines. All the other antibodies stained the small cell turnout cells to a variable extent (Table I). When the reactive antibodies were separated into groups according to their reported specificity, it was noted
that neural-associated antibodies generally had high levels of reactivity with SCLC turnout cell lines (Fig la) with the exception of No. 24 (0-4% of cells positive). SCLC-associated antibodies (Fig la) showed a rather different pattern with throe antibodies 17, 57 and 58 having a very high level of activity with SCLC tumour cell lines whereas 15, 18, 27, 39, 55, 56 and 59 stained more heterogeneously. All of the epithelialassociated antibodies reacted with the SCLC cell lines although very heterogeneously as did the antibodies directed against lung determinants (Fig lb). The remaining antibodies 7, 10, 12, 26, 33 and 41, directed against miscellaneous determinants, had little activity with SCLC cell lines. Normal Bone Marrow Only antibodies which reacted strongly with SCLC cell lines were tested against normal bone marrow cells. A few were also excluded since supplies were exhausted (35,36,27). Several
la
SCLC
Neural o
100-
o *
o
• t t
t t t
t t o t
I
I "° t
SO-
t /r /z
o
o
I N S17
4 2 2 5 3 II 3 1 ~ N 1 3 M 4 6 3 7 2 3 M 1 4 0 3 ~
I
I ST :D' 58 ~ g 9 1 5 3 0 1 8
Ib 100" * Epithelial
Lung
Mlsc
Q o
SO"
I
SCLC Marrows from Patients The seven marrows were screened using only the neural, SCLC and epithelial-associated antibodies that reacted with SCLC cell lines regardless of their reactivity with normal marrow. Tumour cells were detected in SCLC marrows by all the antibodies which stained SCLC cell lines except 49, 15, 55 and 56. Numbers 36, 27 and 58 could not be assessed. In the case of the heavily The similar levels of activity of antibodies directed against both neural and epithelial determinants in addition to those raised against more specific SCLC determinants support the hypothesis that SCLC tumour cells may have an origin in pulmonary epithelial cells which follow a neuroendoorine differentiation pathway 4 as opposed to an origin from macrophages within the bone marrow5. However it is possible that the expression of neural antigens reflects the differentiation status of the cells rather than lineage s and indeed, expression of antibody number 46(123C3) which is also expressed by well differentiated ovarian tumour cells can be induced in less well differentiated ovadan tumour cell lines following sodium butyrate treatment 7. This antigen is also induced by sodium butyrate treatment of the lung adenocarcinoma cell line A549 (Langdon, unpublished observation). infiltrated marrow, the staining patterns were all within the ranges obtained with these antibodies against the SCLC cell lines (Table 1). DISCUSSION General Observations The antibodies directed against neural determinants were consistently more reactive with SCLC tumour cells than any of the other antibodies, in particular numbers 22(MOC-I, 13(SL 11-14) and 46(123C3) which consistently stained 60-100% of SCLC tumour cells. Of the SCLC-associated antibodies, 17(MOC-31), 57(SWA21) and 58(SWA22) showed high reactivity and the epithelial antibody number 9(AUA1). Use of the above antibodies as a panel or in a cocktail for the detection of SCLC tumour cells in general situations should increase the sensitivity of the technique. Antibodies with potential applications for bone marrow screening and therapy
I
/k
I O 44 40 43 3 38
antibodies cross-reacted heavily with bone marrow cells i.e. at a level of >10% of total cells positive, denoting serious cross-raactions. These were 57, 58, 49, 55, 59, 38, 41,46, 15, 19 and 42. Others reacted only at a level of 1% of marrow cells:-8, 22, 23, 31, 53, 9, 44, 45. F i n a l l y antibodies with activity against SCLC cell lines which did not cross-react with marrow components:-(4, 13, 14, 20, 24, 28, 32, 37, 17, 18, 39, 3, 40, 43) comprised 8 neural, 2 SCLC and 3 epithelial-associated antibodies (Fig 1).
42 19 45
Ill,.
26 41 33 10 7 12
Fig l a + b: Range of staining reactions (black bars) observed in 3 SCLC cell lines expressed as the percentage of total cells which stained positively Above the bars are the levels of
staining observed when those antibodies are reacted with normal bone marrow cells:-0 - no staining; "<1 of cells positive;'*1-10% of cells positive; "°>10% of cells positive.
Selection of a panel of monoclonal antibodies for use in detecting low levels of marrow infiltration by tumour requires that the antibodies chosen should have strong reactivity with a high proportion of SCLC tumour cells yet be unreactive with marrow cells. Most of the antibodies directed against SCLC determinants also cross-react significantly with normal marrow components (Fig ta) so that only 17(MOC-31) could be selected from this group. In contrast many of the neuralassociated antibodies detect large proportions of tumour cells and do not cross-react with marrow
69 so that antibodies 4(CID-5/NKHI), 8(NE25), 13(SL 1 t-14), 20(NCC-LU-246), 37(123A8), 28(MOC21) and 14(TFS-4) are suitable for inclusion in the panel. From the epithelial-associated antibodies only number 50(MOv15) is useful. In effect only nine antibodies appear appropriate for use in marrow analysis but in practice antibodies which react with less than 1% of marrow components can be included but require careful consideration of morphology of positive cells and reference to know levels of staining in marrow) so adding a further six (22, 53, 31,23, 9 and 44) to the list.
REFERENCES
2.
3. In conclusion, 15 antibodies from the workshop collection appear suited to applications involving bone marrow and several of these have already proved successful in detecting low levels of marrow infiltration not detected by histological methods e and include 123A8(37), UJ13A(53), AUAI(9) and HMFG2(44).
Ruff, M.R. and Pert, C.B. (1984). Small cell carcinoma of the lung: Macrophage-specific antigens suggest haemopoietic stem cell origin. Science, 225: 1034-1036.
Stahel RA, Mabry M, Skarin AT, et. al. (1985). Detection of bone marrow metastasis in small cell lung cancer by monoclonal antibody. J. Clin. Oncol., 3: 455-461. Hay, F.G. and Leonard, R.C.F. (1986). Epithelial and neural antigens in human small cell lung cancer. Proc. Br. Assoc. Cancer R., Br. J. Cancer, 54: 145-146.
Cole. S.P.C., Hirski, S., McGarry, R.C. et al (1985). Differential expression of the leu-7 antigen on human lung turnout cells. Cancer Res., 45: 4285-4290. 7.
Langdon, S.P., Hay, F.G., Hawkes, M.M, el al (1987). Selective effects of sodium butyrate and short chain fatty acids on differentiation in a human ovarian carcinoma cell line. Am. Assoc. Cancer Res., Abstract.
8.
Hay, F.G., Ford,A. and Leonard, R.C.F. (1987). increased detection of micrometastases in the bone marrow of small cell lung cancer (SCLC) patients. Proc. Br. Assoc. Cancer Res., Br. J. Cancer, (in press).
Sternberger, L A . (1979). The unlabelled antibody (PAP) method. J. Histochem. Cytochem., 27: 1657-1659. Gazdar, A.F. (1984). The biology of endocrine tumours of the lung. Becker, K.L., Gazdar, A.F. (eds). The Endocrine Lung in Health and Disease. Philadelphia, Saunders, p448-459.
Lung Cancer, 4 (1988) 67-69 Elsevier CHAPTER 14 REACTIVITY OF WORKSHOP MONOCLONAL ANTIBODIES WITH SMALL CELL LUNG CANCER (SCLC) CELL LINES, NORMAL BONE MARROW AND MARROW FROM SCLC PATIENTS Frances G. Haylt, Lorraine W. Adams 1, and Robert C.F. Leonard 2 1imperial Cancer Research Fund Medical Oncology Unit and 2University Department of Clinical Oncology Western General Hospital Edinburgh, EH4 2XU 1" to whom correspondence should be addressed
ABSTRACT The reactivity of the workshop antibodies was studied against 3 SCLC cell lines, 3 normal bone marrows and 7 marrow aspirates from SCLC patients. All antibodies except 1, 2, 5, 6, 16, 21, 25, 29, 47, 48, 50, 51, 54 and negative controls stained the SCLC cell lines heterogeneously. Antibodies classified as neural showed strongest reactivity. Many antibodies were unreactive or had limited reactivity with normal bone marrow cells with the exception of 38, 49, 57 and 58 all of which cross-reacted strongly. Tumour cells were detected in SCLC infiltrated marrows b y antibodies with neural, SCLC and epithelial specificity. INTRODUCTION Examination of bone marrow aspirates and biopsies for evidence of tumour involvement is frequently performed as part of the initial staging or restaging procedure for patients with SCLC. Approximately 20% of all patients studied have marrow involvement detected by histological methods, although as many as 66% are reported to have bone marrow infiltration at necropsy. The frequency of marrow involvement as detected by rnonoclonal antibody reactivity has been reported to reach similar levels to those found at necropsy 1 and this is apparently a more sensitive detection method than routing cytology and histology.
for 5 minutes and either processed immediately or stored at -200C until required.
A PAP (Peroxidase-anti-peroxidase) staining
Normal Bone Marrow Marrow aspirates were obtained from patients being investigated for folate deficiency. Five ml of aspirated marrow were mixed with 5 ml of hepadnised PBS. Red cells were removed by Ficoll Hypaque separation followed by washing in RPMI. Cells were resuspended at 5 x 106- 106 cells/ml and slides were prepared as before. SCLC Patients' Bone Marrow Bone marrow aspirates were collected from patients with histologically proven SCLC at routine staging procedures. The patients were staged prior to treatment using standard clinical and radiological procedures as having limited disease in 3 cases and extensive disease in 4 cases. Six of the marrows were lightly infiltrated with tumour cells (<1% of total cells present) whereas the 7th was heavily infiltrated with tumour cells (>70% of total cells present). Red cells were removed by Ficoll Hypaque separation and slides were prepared as for the cell lines.
method was used in this study 3- Briefly, slides were fixed in acetone/methanol and endogenous peroxidase activity was removed by immersion in methanol/H202. Slides were washed in tris buffered saline (TBS) pH7.6, incubated with diluted normal sheep serum followed by the monoclonal antibodies at optimal dilution, washed, incubated with sheep anti mouse IgG in excess, further washing then PAP complex (SaraLab). The peroxidase was visualised using diaminobenzidene tetrahydrochloride, 0.6 mg/ml in 0.2 M Tris-HCL buffer, pH7.6 containing 0.01% hydrogen peroxide and imidazole. Slides were counterstained with haemotoxylin. In the case of the rat antibodies an indirect peroxidase conjugate was substituted for the sheep anti mouse IgG and the PAP. Marrow s a m p l e s required addition of normal human serum during staining to reduce non-specific background staining. Scoring of Staining In SCLC cell lines and normal bone marrow scoring was performed on the basis of percentage
TP.BLE I Reactivity of Workshop Antibodies with SCLC Cell Lines, Normal Bone Marrows and SCLC Turnout-Infiltrated Bone Marrows antibodv
We have previously noted marked heterogeneity of staining using a number of antibodies with SCLC tumour cens 2 and propose that use of a panel of antibodies, either singly or as a cocktail, would substantially increase the likelihood of detecting turn our cells in marrow aspirates especially where infiltration is sparse. With such applications in mind we have examined the reactivity of the workshop panel of antibodies with SCLC cell lines and with normal bone marrow to identify those which detect a high proportion of turnour cells but which do not react with normal bone marrow cells.
Immunocytochemistry
Neural
4 8 13 14 20 22 23 24 28 31 32 35 36 37 46 49
53
SCLC cell lines range(%) mean(%) 20-100 10-96 60-94 40-70 50-92 70-100 25-86 0-4 35-84 30-97 0-40 25-96 50-94 45-89 70-90 =0-50 I0-98
Normal marrow
=72 63 79 57 75 90 65 1 61 73 20 71 79 63 83 17 53
0 <1 0 0 <1 <1 0 0
0
SCLC Marrows light heavy infiltrates infiltrates % + 82 + 87 + 79 + 58 + 87 + 79 + 78 0 + 51 47 + 62 68 ND ND + 61 + ND <1 + 37
MATERIALS AND METHODS SCLC Cell Lines Three established SCLC cell lines of the classic type were used; NCI HI87 was obtained from Dr. D. Carney, Dublin; COR L31 and COR L47 were obtained from Dr. P. Twentyman, Cambridge. The cells were harvested in log phase, washed in RPMI and dead cells were removed by centrifugation over Ficoll Hypaque. The cells were then resuspended at a concentration of 5 x 105 or 1 x 106 cells/ml in RPMI and 20 ul of the cell suspension was dropped into each well of 12 well, Teflon-coated Multispot slides. The slides were air dried, fixed in acetone/methanol at 4Oc
Epithelial
Lung
15
0-30
10
1-10
17 18 27
80-99 0-2 10-84
39 55 56 57 58 59
0-14 2-56 0-57 95-100 65-100 1-40
91 1 39 5
0 0 ND 0
21 22 98 88 14
>10 ND >50 >50 >10
3 9 38 40 43 44
29-45 50-95
37 78
8-41 45-65 4-45
21 58 24
9-65
19 42 45
16-50 10-72 0-20
+ ÷
N+D +
0
63 ND 3 1 1
80 ND 1
30
<1 >10 0 0 <1
+ + +
5 71 40 5 17 50
29 38 10
1-10 1-10 <1
ND ND ND
ND ND ND
+
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
68 positive cells in the total number of nucleated cells. Staining intensity was also noted. In the marrow from patients with SCLC, scoring was recorded as either positive or negative except for the heavily infiltrated marrow where percentages of positive cells were recorded. The morphology of the positive cells was recorded, i.e. tumour or non-turnout if recognisable. The tumour cell population in positive samples was variable, often only 1-2 tumour cells were present per well according to the level of infiltration of each individual marrow. RESULTS SCLC Cell Lines Antibodies 1,2, 5, 6, 16, 21,25, 29, 47, 48, 50, 51, 54 and the hidden negative controls 11,30 and 34 did not stain any of the SCLC cell lines. All the other antibodies stained the small cell turnout cells to a variable extent (Table I). When the reactive antibodies were separated into groups according to their reported specificity, it was noted
that neural-associated antibodies generally had high levels of reactivity with SCLC turnout cell lines (Fig la) with the exception of No. 24 (0-4% of cells positive). SCLC-associated antibodies (Fig la) showed a rather different pattern with throe antibodies 17, 57 and 58 having a very high level of activity with SCLC tumour cell lines whereas 15, 18, 27, 39, 55, 56 and 59 stained more heterogeneously. All of the epithelialassociated antibodies reacted with the SCLC cell lines although very heterogeneously as did the antibodies directed against lung determinants (Fig lb). The remaining antibodies 7, 10, 12, 26, 33 and 41, directed against miscellaneous determinants, had little activity with SCLC cell lines. Normal Bone Marrow Only antibodies which reacted strongly with SCLC cell lines were tested against normal bone marrow cells. A few were also excluded since supplies were exhausted (35,36,27). Several
la
SCLC
Neural o
100-
o *
o
• t t
t t t
t t o t
I
I "° t
SO-
t /r /z
o
o
I N S17
4 2 2 5 3 II 3 1 ~ N 1 3 M 4 6 3 7 2 3 M 1 4 0 3 ~
I
I ST :D' 58 ~ g 9 1 5 3 0 1 8
Ib 100" * Epithelial
Lung
Mlsc
Q o
SO"
I
SCLC Marrows from Patients The seven marrows were screened using only the neural, SCLC and epithelial-associated antibodies that reacted with SCLC cell lines regardless of their reactivity with normal marrow. Tumour cells were detected in SCLC marrows by all the antibodies which stained SCLC cell lines except 49, 15, 55 and 56. Numbers 36, 27 and 58 could not be assessed. In the case of the heavily The similar levels of activity of antibodies directed against both neural and epithelial determinants in addition to those raised against more specific SCLC determinants support the hypothesis that SCLC tumour cells may have an origin in pulmonary epithelial cells which follow a neuroendoorine differentiation pathway 4 as opposed to an origin from macrophages within the bone marrow5. However it is possible that the expression of neural antigens reflects the differentiation status of the cells rather than lineage s and indeed, expression of antibody number 46(123C3) which is also expressed by well differentiated ovarian tumour cells can be induced in less well differentiated ovadan tumour cell lines following sodium butyrate treatment 7. This antigen is also induced by sodium butyrate treatment of the lung adenocarcinoma cell line A549 (Langdon, unpublished observation). infiltrated marrow, the staining patterns were all within the ranges obtained with these antibodies against the SCLC cell lines (Table 1). DISCUSSION General Observations The antibodies directed against neural determinants were consistently more reactive with SCLC tumour cells than any of the other antibodies, in particular numbers 22(MOC-I, 13(SL 11-14) and 46(123C3) which consistently stained 60-100% of SCLC tumour cells. Of the SCLC-associated antibodies, 17(MOC-31), 57(SWA21) and 58(SWA22) showed high reactivity and the epithelial antibody number 9(AUA1). Use of the above antibodies as a panel or in a cocktail for the detection of SCLC tumour cells in general situations should increase the sensitivity of the technique. Antibodies with potential applications for bone marrow screening and therapy
I
/k
I O 44 40 43 3 38
antibodies cross-reacted heavily with bone marrow cells i.e. at a level of >10% of total cells positive, denoting serious cross-raactions. These were 57, 58, 49, 55, 59, 38, 41,46, 15, 19 and 42. Others reacted only at a level of 1% of marrow cells:-8, 22, 23, 31, 53, 9, 44, 45. F i n a l l y antibodies with activity against SCLC cell lines which did not cross-react with marrow components:-(4, 13, 14, 20, 24, 28, 32, 37, 17, 18, 39, 3, 40, 43) comprised 8 neural, 2 SCLC and 3 epithelial-associated antibodies (Fig 1).
42 19 45
Ill,.
26 41 33 10 7 12
Fig l a + b: Range of staining reactions (black bars) observed in 3 SCLC cell lines expressed as the percentage of total cells which stained positively Above the bars are the levels of
staining observed when those antibodies are reacted with normal bone marrow cells:-0 - no staining; "<1 of cells positive;'*1-10% of cells positive; "°>10% of cells positive.
Selection of a panel of monoclonal antibodies for use in detecting low levels of marrow infiltration by tumour requires that the antibodies chosen should have strong reactivity with a high proportion of SCLC tumour cells yet be unreactive with marrow cells. Most of the antibodies directed against SCLC determinants also cross-react significantly with normal marrow components (Fig ta) so that only 17(MOC-31) could be selected from this group. In contrast many of the neuralassociated antibodies detect large proportions of tumour cells and do not cross-react with marrow
69 so that antibodies 4(CID-5/NKHI), 8(NE25), 13(SL 1 t-14), 20(NCC-LU-246), 37(123A8), 28(MOC21) and 14(TFS-4) are suitable for inclusion in the panel. From the epithelial-associated antibodies only number 50(MOv15) is useful. In effect only nine antibodies appear appropriate for use in marrow analysis but in practice antibodies which react with less than 1% of marrow components can be included but require careful consideration of morphology of positive cells and reference to know levels of staining in marrow) so adding a further six (22, 53, 31,23, 9 and 44) to the list.
REFERENCES
2.
3. In conclusion, 15 antibodies from the workshop collection appear suited to applications involving bone marrow and several of these have already proved successful in detecting low levels of marrow infiltration not detected by histological methods e and include 123A8(37), UJ13A(53), AUAI(9) and HMFG2(44).
Ruff, M.R. and Pert, C.B. (1984). Small cell carcinoma of the lung: Macrophage-specific antigens suggest haemopoietic stem cell origin. Science, 225: 1034-1036.
Stahel RA, Mabry M, Skarin AT, et. al. (1985). Detection of bone marrow metastasis in small cell lung cancer by monoclonal antibody. J. Clin. Oncol., 3: 455-461. Hay, F.G. and Leonard, R.C.F. (1986). Epithelial and neural antigens in human small cell lung cancer. Proc. Br. Assoc. Cancer R., Br. J. Cancer, 54: 145-146.
Cole. S.P.C., Hirski, S., McGarry, R.C. et al (1985). Differential expression of the leu-7 antigen on human lung turnout cells. Cancer Res., 45: 4285-4290. 7.
Langdon, S.P., Hay, F.G., Hawkes, M.M, el al (1987). Selective effects of sodium butyrate and short chain fatty acids on differentiation in a human ovarian carcinoma cell line. Am. Assoc. Cancer Res., Abstract.
8.
Hay, F.G., Ford,A. and Leonard, R.C.F. (1987). increased detection of micrometastases in the bone marrow of small cell lung cancer (SCLC) patients. Proc. Br. Assoc. Cancer Res., Br. J. Cancer, (in press).
Sternberger, L A . (1979). The unlabelled antibody (PAP) method. J. Histochem. Cytochem., 27: 1657-1659. Gazdar, A.F. (1984). The biology of endocrine tumours of the lung. Becker, K.L., Gazdar, A.F. (eds). The Endocrine Lung in Health and Disease. Philadelphia, Saunders, p448-459.