Immunology Today,vol. 8, No. 9,
1987
..... .e_ Bone marrow transplantation(BMT)is undertaken in a heroic attempt to increase the cure rate in malignancyand to treat deadly metabolic disorders. After ablative therapy, patients are transfusedwith their own or with HI.A matchedor haploidenticalbone marrow. The inoculum may be "purged" to remove. residual malignant cells or mature T cells which causegraft versushost disease. Meetings such as the recent First International Workshop on Bone Marrow Purging* are important as a forum for a review of current developments, advice about the selection of techniques- many of them exquisitelyingenious and effident- and for a discussionof the exact role of bone marrowpurging in the complex process of patient management.
s a.d
Bonemarrowpurging bm 6¢ je
omas, and that of a double marker spheres of 4.5 iLm diameter (J. Ugelassay (anti-CDlO antibodies to de- stad, Trondheim) and immunotoxins. tect cALL antigen, with anti-terminal These methods (Table 2) are capable deoxynucleotidyl transferase, a nuc- of a thousand-fold elimination of lear enzyme) in acute lymphoid unwanted cells, particularly when leukaemia was demonstrated by reagents of high affinity are chosen T. Philip (Lyon) and G. Janossy (Lon- and are applied as simple cocktails. don). With these sensitive methods, Nevertheless, one or other technique optimal monoclonal antibodies (or may fail in individual cases and, as their cocktails) can be selected for Philip pointed out, the use of each patient. The investigation of alternative methods is comindividual patients with such sensi- plementary rather than competitive. tive assays is a recent development: A formal comparative study between in other studies, such as those these exciting methods is warranted Bone marrow (BM) purging is a summarized by R. Bast (Durham), as soon as those in the field are peculiar field where hard facts are the main aim was the precise stan- ready to make joint recommendahard to come by. One view is that dardization of the purging tech- tions on the basis of such pooled malignant cells may be undetectable niques themselves. For this purpose data. Paradoxically, the potentially in the marrow with conventional cell lines, rather than fresh malignant most effective agents, immunotoxmethods and although they are a cells taken from patients, can be ins, are now applied by J. Kersey small proportion of the total the used. These are model experiments (Minnesota) at a slightly suboptimal number of tumour cells inoculated which are also often referred to as concentration to avoid removing all T cells, a manoeuvre which may may be fairly large: autologous BMT 'clonogenic assays'. cause complications (see below). The without purging is thus not optimal. virtually total removal of common Another view is that the re-infusion Methods of purging There has been a tremendous ALL or B-cell lymphoma cells with of a few malignant cells may not matter because they are unlikely to development in the technology of immunotoxins causes no such conresult in overt malignancy. It can also purging (Table 2). Among the cern. Among the non-antibody medibe pointed out that the BM samples monoclonal antibodies used are taken during autologous BMT repre- those of the CD20 (B l-like) and ated techniques, a particularly imsent a mere 1-4% of the body's CD19 (B4-1ike) categories against B pressive preparative me~od with__a total BM. During BMT a considerable cells and their malignancies, those of short processing time for large scale dilution takes place. If during remis- CD19 and CDIO (cALL) against the BM manipulation, counterflow cension only a handful of leukaemic cells common form of acute lymphocytic trifugation elutriation, was discussed are left all over the body, the sm~ll leukaemia (ALL) or antibodies of by S. Noga (Baltimore). The fraction sample used for reconstitution may CD7, CD2 and CD5 against T-ALL with haemopoietic precursor cells is not contain any of those cells. Con- together with a whole cocktail of severely depleted of mature T cells sequently, autologous BM transplant antibodies made against neuroblas- (< 106 per kg reinjected T cells) and is a rational procedure, even without toma (J. Kemshead, London). The the application of this technique four most important antibody- appears to decrease graft versus host a purge. Objective measurements are re- mediated methods are complement- disease (GvHD). The immunization of quired to decide these issues, includ- mediated lysis with rabbit serum or donors one week before BM harvest ing precise and systematic analysis with human serum, antibody- stimulates antigen-specific lymphoof residual malignant cells in the BM coated, super-paramagnetic micro- cytes and some of the activated blast and the long-term follow-up of patients. The sensitivity of the Table 1. Methodsfor detectingresidualmalignantcells methods which can be used (Table 1) was summarized by L. Nadler (Bos- Conventionalhistology/haematology 2-5%a ton) in his introduction to the confer- Microscopyor FACSanalysiswith singleantibody 0.5-2%b ence. Exactly the same sensitive Ig or T-cellreceptorgenerearrangement 1-2%c methods can be used to document Doublemarkerswith TRITC/FITClabelling 0.2-1%b the efficacy of the various purging Doublemarkerswith nuclearTdT 0.02-0.1%b procedures. The screening role of Clonogenicassaysfor selededlymphomas 0.001%d clonogenic assays in B-cell lympha Blastswith unusualmorphology(Burkitt-likeL3 lymphoblastand myeloblastswith Auer-rods) be detectedin evenlowerproportions *Heldon 13-14February1987inOrlando,USA,and b may Normal stem cells in bone marrow and their malignant variants are not discriminated organizedby S. Grossand A. Gee, Divisionof Haematology-Oncology,Departmentof Pediatrics, (Campana,D. and Janossy,G. (1986)Blood68,1264) Universityof Florida.TheproceedingsappearinBone c Lowsensitivitybut characteristicfor the malignantlymphoidclone 253 d Not all malignanciesgrow in culture(Favrot,M. and Philip,T., pets.commun.) Marrow Transplantation,May 1987. (~) 1987. ElsevierPublications,Cambridge 0167 -49191871502.00
Immunology Today, vol. 8, No. 9, 1987
ainical observations The 'cleansing' of autologous BM to remove residual malignancy, and the elimination of T lymphocytes in order to prevent GvHD are approaches to quite different clinical problems. In autologous BMT for relapsed B-cell lymphoma, Nadler presented the results obtained in collaborative studies in Boston, Heidelberg and London. A total of 80 patients with a poor prognosis, were transplanted in so-called 'responsive' relapse and purging was done with CD20 (B1) antibody and rabbit complement. Nadler favours purging because in untreated BM samples there was detectable malignant cell contamination (>5%)in 14/36 patients. The procedure had minimal toxicity and only four patients died (from drugrelated complications); following easy engraftment 66% of patients are in r3mission at one year. The relative success has prompted moves to treat similar patients earlier. * Observationspresentedby J. Chang,Paterson In acute lymphoblastic leukaemia ImlJtute. Manchesterat the BoneMarrowTram- there has been one remarkable replantationMeeting,Manchester.19February1987 port from J. Kersey (Minnesota) with
cells co-separate with the graft fraction despite overall lymphocyte depletion: thus specific immunity might still be transferred. Efficient drugs, particularly cyclophosphamide derivatives such as 4HC ancl ASTA-Z, have also been used to treat the marrow and decrease potential contamination with malignant cells: the best results, shown by A, Yaeger (Baltimore) are seen in acute myeloid leukaemia (AML), These drugs are not selective against malignancy but may work as a further 'dilution factor' eliminating large fractions of both normal stem cells and leukaemic blast cells, yet normal myelopoiesis regenerates while the leukaemia, in most patients, does not. Normal precursor cells can also be distinguished from malignant cells by culture of the bone marrow with colony-stimulating factors: blast cells in AML and ALL rapidly perish, while normal BM colonies grow*,
interesting lessons to teach. Kersey's group, which with J. Ritz (Boston) and Bast pioneered autologous BMT for ALL, uses an efficient purging technique with three antibodies, CD9, CDIO and CD24 and rabbit complement. Nevertheless, in a group of 45 patients with relapsed ALL there has been a 76% cumulative relapse rate, including early relapses, since 1982. These findings, taken together, indicate that the possible benefits of excellent purging are wiped out by insufficient conditioning. Recently, however, the intensity of conditioning regimens and their anti-leukaemic efficacy may have been increased without an accompanying increase in sideeffects. In a more recent, smaller series studied by Janossy in collaboration with colleagues in Glasgow and Uppsala purging has been done in 17 patients with poor prognosis ALL using a lytic CDIO antibody with a more than lO00-fold elimination of ALL blast cells. The follow-up is short (up to 2 years) but there has been only one early relapse and virtually no drug-related toxicity.
Table2. Examplesof techniquesfor purging Method
Agent
Target
Investigators
Antibody-mediated techniques Complement,rabbit Complement,rabbit
CD20(+CD19) CD10(CD19)
Complement,rabbit Complement,rabbit Complement,rabbit Complement,rabbit Complement.rabbit Complement,human Immunobead Immunobead Immunobead Immunotoxin(non)
CD7,CD8 Cocktailof 8 Ab CD6(+CD8) CD2,CD5,CD7 CD2,CD3 Campath-1 CD2,CD3 CD19,CD22,CD37 Cocktailof 6 Ab CDS-ricin
B lymphoma CommonALL LommonALL T-ALL T depletion T depletion T depletion T depletion T+B depletion T depletion B lymphoma Neuroblastoma T depletion
L. Nadler,T. Philip R. Bast,G. Janossy J. Kersey G. Janossy P. Martin J. Ritz,G. Janossy E. Racadota P Herveb H. Waldmannc F. Wartdal G. Kvalheim A. Gee,J. Kemshead J. Kersey
Other methods Drugs Drugs Lectin+ E-ros Physicalsize Photosensitization Cultureof BM
4 HC Bleomycin Soyabean+E Elutriation Merocyanin540
AML,ALL AML? T depletion T depletion AML? AML
A. Yaeger A. Yaeger N. Kernan S. Noga F. Sieber J Changd
4HC+CD10e 4HC+CD7+CD5 4HC+CD20
ALL T-ALL B lymphoma
J. Kersey,L. Nadler J. Kersey T. Philip
PotenlYallyinteresting combinations Drug+Ab Drug+Ab Drug+Ab
254
a Racadot,E (1987)J C/in Onco/5, 426-435 b Harve,P eta/. (1987)Blood69, 388-393 c Waldmann,H. etal. (1984)Lancet,i,483-486 d Chang,J.; Dexter,M.; Meetingon BMT,PatersonInstitute,Manchester,February1987 e Butseepoorresultsusing4HCin ALLby A. Yaeger(Baltimore)
I m m u n o l o g y Today, voL 8, No. 9, 1987
Improvements in patient conditioning and detection of minimal disease are of prime importance in autoIogous BMT in ALL. With improved conditioning further benefits due to purging may become visible. Controlled trials comparing autologous BMT with and without additional BM purging might therefore become justified. In neuroblastoma the collaboration between A. Gee (Miami) and J. Kemshead (London) has led to the establishment of BM purging facility for the southern part of the USA. So far 50 samples have been purged with immunobeads, leaving no detectable malignant contamination, The patients re-grafted more rapidly and received fewer courses of combination chemotherapy. Again, the ease of the technology justifies an earlier use of BMT although it is too early to see whether the purging itself has been beneficial. Yaeger has introduced autologous BMT with a drug (4HC)-induced purge in 40 patients with acute myeloblastic leukaemia, second and third remissions. The survival and actuarial relapse rate (53%) compare favourably with the syngeneic BMT protocols and with the results of allogeneic transplants. The same protocol gave very poor results with adult patients with ALL. This indi. . . . . . . . . . . . . . . . . purge w,-,i o,r,ylaLing agents may have a role in AML, while purge with CD10 and CD19 antibodies is more appropriate in ALL.
T-cell depletion In 1984 the first studies in man indicated that GvHD was prevented by the specific depletion of allogeneic mature T cells from a bone marrow inoculum. In fully matched conditions (donorrecipient pairs), this observation was confirmed by several groups including French investigators such as P. Herv~ and E. Racadot and their colleagues. Extensive studies of depletion with the Campath-1 monoclonal antibody also show that GvHD is no longer a problem. Nevertheless, additional complications have arisen. P. Martin (Seattle) has demonstrated that prevention of GvHD by T-cell removal is not yet associated with survival benefit for the patients, a finding which is also borne out by the data in the Bone Marrow Transplant Registry (R. Gale, Los Angeles). The new complications are increased
~'
rejection rate of the BM due to an unleashed host versus graft reaction (HvGD) and an increased relapse rate, particularly well documented in chronic granulocytic leukaemia (CGL). Increased conditioning of patients may diminish both HvGD and leukaemic relapse but the dose range without unacceptable sideeffects is very narrow. For matched BMT the appropriate GvHD reducing T-cell dose is
1987
..... .e_ Bone marrow transplantation(BMT)is undertaken in a heroic attempt to increase the cure rate in malignancyand to treat deadly metabolic disorders. After ablative therapy, patients are transfusedwith their own or with HI.A matchedor haploidenticalbone marrow. The inoculum may be "purged" to remove. residual malignant cells or mature T cells which causegraft versushost disease. Meetings such as the recent First International Workshop on Bone Marrow Purging* are important as a forum for a review of current developments, advice about the selection of techniques- many of them exquisitelyingenious and effident- and for a discussionof the exact role of bone marrowpurging in the complex process of patient management.
s a.d
Bonemarrowpurging bm 6¢ je
omas, and that of a double marker spheres of 4.5 iLm diameter (J. Ugelassay (anti-CDlO antibodies to de- stad, Trondheim) and immunotoxins. tect cALL antigen, with anti-terminal These methods (Table 2) are capable deoxynucleotidyl transferase, a nuc- of a thousand-fold elimination of lear enzyme) in acute lymphoid unwanted cells, particularly when leukaemia was demonstrated by reagents of high affinity are chosen T. Philip (Lyon) and G. Janossy (Lon- and are applied as simple cocktails. don). With these sensitive methods, Nevertheless, one or other technique optimal monoclonal antibodies (or may fail in individual cases and, as their cocktails) can be selected for Philip pointed out, the use of each patient. The investigation of alternative methods is comindividual patients with such sensi- plementary rather than competitive. tive assays is a recent development: A formal comparative study between in other studies, such as those these exciting methods is warranted Bone marrow (BM) purging is a summarized by R. Bast (Durham), as soon as those in the field are peculiar field where hard facts are the main aim was the precise stan- ready to make joint recommendahard to come by. One view is that dardization of the purging tech- tions on the basis of such pooled malignant cells may be undetectable niques themselves. For this purpose data. Paradoxically, the potentially in the marrow with conventional cell lines, rather than fresh malignant most effective agents, immunotoxmethods and although they are a cells taken from patients, can be ins, are now applied by J. Kersey small proportion of the total the used. These are model experiments (Minnesota) at a slightly suboptimal number of tumour cells inoculated which are also often referred to as concentration to avoid removing all T cells, a manoeuvre which may may be fairly large: autologous BMT 'clonogenic assays'. cause complications (see below). The without purging is thus not optimal. virtually total removal of common Another view is that the re-infusion Methods of purging There has been a tremendous ALL or B-cell lymphoma cells with of a few malignant cells may not matter because they are unlikely to development in the technology of immunotoxins causes no such conresult in overt malignancy. It can also purging (Table 2). Among the cern. Among the non-antibody medibe pointed out that the BM samples monoclonal antibodies used are taken during autologous BMT repre- those of the CD20 (B l-like) and ated techniques, a particularly imsent a mere 1-4% of the body's CD19 (B4-1ike) categories against B pressive preparative me~od with__a total BM. During BMT a considerable cells and their malignancies, those of short processing time for large scale dilution takes place. If during remis- CD19 and CDIO (cALL) against the BM manipulation, counterflow cension only a handful of leukaemic cells common form of acute lymphocytic trifugation elutriation, was discussed are left all over the body, the sm~ll leukaemia (ALL) or antibodies of by S. Noga (Baltimore). The fraction sample used for reconstitution may CD7, CD2 and CD5 against T-ALL with haemopoietic precursor cells is not contain any of those cells. Con- together with a whole cocktail of severely depleted of mature T cells sequently, autologous BM transplant antibodies made against neuroblas- (< 106 per kg reinjected T cells) and is a rational procedure, even without toma (J. Kemshead, London). The the application of this technique four most important antibody- appears to decrease graft versus host a purge. Objective measurements are re- mediated methods are complement- disease (GvHD). The immunization of quired to decide these issues, includ- mediated lysis with rabbit serum or donors one week before BM harvest ing precise and systematic analysis with human serum, antibody- stimulates antigen-specific lymphoof residual malignant cells in the BM coated, super-paramagnetic micro- cytes and some of the activated blast and the long-term follow-up of patients. The sensitivity of the Table 1. Methodsfor detectingresidualmalignantcells methods which can be used (Table 1) was summarized by L. Nadler (Bos- Conventionalhistology/haematology 2-5%a ton) in his introduction to the confer- Microscopyor FACSanalysiswith singleantibody 0.5-2%b ence. Exactly the same sensitive Ig or T-cellreceptorgenerearrangement 1-2%c methods can be used to document Doublemarkerswith TRITC/FITClabelling 0.2-1%b the efficacy of the various purging Doublemarkerswith nuclearTdT 0.02-0.1%b procedures. The screening role of Clonogenicassaysfor selededlymphomas 0.001%d clonogenic assays in B-cell lympha Blastswith unusualmorphology(Burkitt-likeL3 lymphoblastand myeloblastswith Auer-rods) be detectedin evenlowerproportions *Heldon 13-14February1987inOrlando,USA,and b may Normal stem cells in bone marrow and their malignant variants are not discriminated organizedby S. Grossand A. Gee, Divisionof Haematology-Oncology,Departmentof Pediatrics, (Campana,D. and Janossy,G. (1986)Blood68,1264) Universityof Florida.TheproceedingsappearinBone c Lowsensitivitybut characteristicfor the malignantlymphoidclone 253 d Not all malignanciesgrow in culture(Favrot,M. and Philip,T., pets.commun.) Marrow Transplantation,May 1987. (~) 1987. ElsevierPublications,Cambridge 0167 -49191871502.00
Immunology Today, vol. 8, No. 9, 1987
ainical observations The 'cleansing' of autologous BM to remove residual malignancy, and the elimination of T lymphocytes in order to prevent GvHD are approaches to quite different clinical problems. In autologous BMT for relapsed B-cell lymphoma, Nadler presented the results obtained in collaborative studies in Boston, Heidelberg and London. A total of 80 patients with a poor prognosis, were transplanted in so-called 'responsive' relapse and purging was done with CD20 (B1) antibody and rabbit complement. Nadler favours purging because in untreated BM samples there was detectable malignant cell contamination (>5%)in 14/36 patients. The procedure had minimal toxicity and only four patients died (from drugrelated complications); following easy engraftment 66% of patients are in r3mission at one year. The relative success has prompted moves to treat similar patients earlier. * Observationspresentedby J. Chang,Paterson In acute lymphoblastic leukaemia ImlJtute. Manchesterat the BoneMarrowTram- there has been one remarkable replantationMeeting,Manchester.19February1987 port from J. Kersey (Minnesota) with
cells co-separate with the graft fraction despite overall lymphocyte depletion: thus specific immunity might still be transferred. Efficient drugs, particularly cyclophosphamide derivatives such as 4HC ancl ASTA-Z, have also been used to treat the marrow and decrease potential contamination with malignant cells: the best results, shown by A, Yaeger (Baltimore) are seen in acute myeloid leukaemia (AML), These drugs are not selective against malignancy but may work as a further 'dilution factor' eliminating large fractions of both normal stem cells and leukaemic blast cells, yet normal myelopoiesis regenerates while the leukaemia, in most patients, does not. Normal precursor cells can also be distinguished from malignant cells by culture of the bone marrow with colony-stimulating factors: blast cells in AML and ALL rapidly perish, while normal BM colonies grow*,
interesting lessons to teach. Kersey's group, which with J. Ritz (Boston) and Bast pioneered autologous BMT for ALL, uses an efficient purging technique with three antibodies, CD9, CDIO and CD24 and rabbit complement. Nevertheless, in a group of 45 patients with relapsed ALL there has been a 76% cumulative relapse rate, including early relapses, since 1982. These findings, taken together, indicate that the possible benefits of excellent purging are wiped out by insufficient conditioning. Recently, however, the intensity of conditioning regimens and their anti-leukaemic efficacy may have been increased without an accompanying increase in sideeffects. In a more recent, smaller series studied by Janossy in collaboration with colleagues in Glasgow and Uppsala purging has been done in 17 patients with poor prognosis ALL using a lytic CDIO antibody with a more than lO00-fold elimination of ALL blast cells. The follow-up is short (up to 2 years) but there has been only one early relapse and virtually no drug-related toxicity.
Table2. Examplesof techniquesfor purging Method
Agent
Target
Investigators
Antibody-mediated techniques Complement,rabbit Complement,rabbit
CD20(+CD19) CD10(CD19)
Complement,rabbit Complement,rabbit Complement,rabbit Complement,rabbit Complement.rabbit Complement,human Immunobead Immunobead Immunobead Immunotoxin(non)
CD7,CD8 Cocktailof 8 Ab CD6(+CD8) CD2,CD5,CD7 CD2,CD3 Campath-1 CD2,CD3 CD19,CD22,CD37 Cocktailof 6 Ab CDS-ricin
B lymphoma CommonALL LommonALL T-ALL T depletion T depletion T depletion T depletion T+B depletion T depletion B lymphoma Neuroblastoma T depletion
L. Nadler,T. Philip R. Bast,G. Janossy J. Kersey G. Janossy P. Martin J. Ritz,G. Janossy E. Racadota P Herveb H. Waldmannc F. Wartdal G. Kvalheim A. Gee,J. Kemshead J. Kersey
Other methods Drugs Drugs Lectin+ E-ros Physicalsize Photosensitization Cultureof BM
4 HC Bleomycin Soyabean+E Elutriation Merocyanin540
AML,ALL AML? T depletion T depletion AML? AML
A. Yaeger A. Yaeger N. Kernan S. Noga F. Sieber J Changd
4HC+CD10e 4HC+CD7+CD5 4HC+CD20
ALL T-ALL B lymphoma
J. Kersey,L. Nadler J. Kersey T. Philip
PotenlYallyinteresting combinations Drug+Ab Drug+Ab Drug+Ab
254
a Racadot,E (1987)J C/in Onco/5, 426-435 b Harve,P eta/. (1987)Blood69, 388-393 c Waldmann,H. etal. (1984)Lancet,i,483-486 d Chang,J.; Dexter,M.; Meetingon BMT,PatersonInstitute,Manchester,February1987 e Butseepoorresultsusing4HCin ALLby A. Yaeger(Baltimore)
I m m u n o l o g y Today, voL 8, No. 9, 1987
Improvements in patient conditioning and detection of minimal disease are of prime importance in autoIogous BMT in ALL. With improved conditioning further benefits due to purging may become visible. Controlled trials comparing autologous BMT with and without additional BM purging might therefore become justified. In neuroblastoma the collaboration between A. Gee (Miami) and J. Kemshead (London) has led to the establishment of BM purging facility for the southern part of the USA. So far 50 samples have been purged with immunobeads, leaving no detectable malignant contamination, The patients re-grafted more rapidly and received fewer courses of combination chemotherapy. Again, the ease of the technology justifies an earlier use of BMT although it is too early to see whether the purging itself has been beneficial. Yaeger has introduced autologous BMT with a drug (4HC)-induced purge in 40 patients with acute myeloblastic leukaemia, second and third remissions. The survival and actuarial relapse rate (53%) compare favourably with the syngeneic BMT protocols and with the results of allogeneic transplants. The same protocol gave very poor results with adult patients with ALL. This indi. . . . . . . . . . . . . . . . . purge w,-,i o,r,ylaLing agents may have a role in AML, while purge with CD10 and CD19 antibodies is more appropriate in ALL.
T-cell depletion In 1984 the first studies in man indicated that GvHD was prevented by the specific depletion of allogeneic mature T cells from a bone marrow inoculum. In fully matched conditions (donorrecipient pairs), this observation was confirmed by several groups including French investigators such as P. Herv~ and E. Racadot and their colleagues. Extensive studies of depletion with the Campath-1 monoclonal antibody also show that GvHD is no longer a problem. Nevertheless, additional complications have arisen. P. Martin (Seattle) has demonstrated that prevention of GvHD by T-cell removal is not yet associated with survival benefit for the patients, a finding which is also borne out by the data in the Bone Marrow Transplant Registry (R. Gale, Los Angeles). The new complications are increased
~'
rejection rate of the BM due to an unleashed host versus graft reaction (HvGD) and an increased relapse rate, particularly well documented in chronic granulocytic leukaemia (CGL). Increased conditioning of patients may diminish both HvGD and leukaemic relapse but the dose range without unacceptable sideeffects is very narrow. For matched BMT the appropriate GvHD reducing T-cell dose is
