JOURNAL OF HEMATOTHERAPY 1:299-301
Mary
(1992)
Ann Liebert, Inc., Publishers
Editorial To
Purge or Not to Purge Is Not the Question
issue of purging is purging, per se, and the only questions are when, how, what, and where to purge. To propose head-to-head purging versus nonpurging studies, or to offer data from a variety of non-concurrent studies in support of either side of the purging issue, is as specious as it is premature. To appreciate fundamental differences, one must first understand the nature of the processes. And purging techniques come in a variety of processes. The proliferation of purging technology grew out of early 1960s animal studies designed to identify means of increasing the number of available allogeneic. donors. Those efforts became the anläge of our present understanding of the role of T lymphocytes in the control or prevention of graft-versus-\\osi disease (GvHD). Thirty years later, we continue to grapple with GvHD, but mainly in terms of control methods that include immunological and/or physical selection/deletion or pharmacologie suppression/dispersion. An equally important outgrowth of the GvHD studies was the recognition of a graft versus leukemia (GvL) process within, but apparently separable from, the GvHD universe. Thus, purging methodology solely for GvHD control had to be revisited in terms of both GvL and engraftment. At present, pre-BMT (purging) control of GvHD falls into three major categories: (i) pan T depletion via soybean lectin agglutination with sheep red cell rosetting, (ii) elutriation with coiinterflow centrifugation, and (iii) monoclonal antibody targeting with in situ destruction (complement or ricin) or attraction (magnetic microspheres). GvHD control is fundamental to success in the treatment of the nonmalignant hematological disorders, e.g., storage defects, immune deficiencies, hemoglobinopathies, and congenital or acquired
The
cytopenias.
Newer purging methodologies consisting of gene deletion and substitution carried out via electroporation followed by transfection may well obviate concerns of graft failure in the nonmalignant marrow disorders. This approach in somewhat modified form will also apply to certain of the marrow malignancies, with gene manipulation following upon negative selection, e.g., unwanted cell removal will precede DNA selection. A major concern relative to gene splicing is the potential for late-occurring diseases directly attributable to viral transfection; and in this regard, metabolically inert, nonmutagenic material, such as methotrexate, appears to be a benign vector substitute. Efforts to add to the donor pool (less than one-third of available siblings are HLA identical) by using haplo-identical or matched unrelated donors, paralleled studies designed to test the feasibility of autologous marrow—and, subsequently, peripheral blood to serve the same purpose. Similar studies were also designed for use in enhancing marrow recovery in megadose therapy-treated patients with a variety of solid tumors. The net effect was an exponential leap in purging research. Today, somewhat more than two-thirds of all marrow transplants are accompanied by some type of purging maneuver. Autologous bone marrow transplantation (ABMT), unlike allogeneic transplantation (alloBMT), very likely requires removal of every last malignant cell prior to reinfusion. In alloBMT, elimination of residual (host) tumor cells are very likely addressed by donor induced GvL. In ABMT, such is not the case, and purging, therefore, is designed to reduce the tumor burden without significant loss in the totipotent cell pool. The purging techniques, which do not lend themselves well to large burden cytoreduction, i.e., greater than a 5% tumor mass, fall into four categories: pharmacologie, biophysical, physical, and immunologie, as well
299
GROSS combinations of the same. Among the more common pharmacologie modalities, all of which produce in are the activated cyclophosphamides (mafosfamide, 4-HC, etc.), glucocorticoids, cisplatins, doxorubicin, arabinofuranosylcytosine, deoxycytidine, etopiside, vincristine, and alkyl-lysophospholipids. This negative selection approach rarely yields a greater than a 4 log depletion of the targeted cells; and specificity is somewhat less than precise. Stem cell losses, especially following the use of combination drug purging, are considerable; and time to marrow recovery is invariably prolonged (rarely less than 38-42 days) and, on very rare occasions, infinite. Dependence on granulocyte-macrophage or erythroid colony-forming units (GM-CFU, E-CFU) in culture as a predictor of response time is unreliable with pharmacologie purging. Far more certain is reliance on number of CFU-L. The best examples of the effective application of such agents are the studies carried out with mafosfamide or 4-HC, the former for AML and ALL, the latter solely for AML. The reasons for the differences in disease response between two such essentially similar agents are unclear. Although the volume of studies does not approach that of mafosfamide or 4-HC, the least toxic pharmacologie agents appear to be the alkyl-lysophospholipids, which .have enjoyed success in acute lymphocytic leukemia (ALL) purging. Radioisotopes and photolytic agents are the best examples of biophysical purging technologies. Elutriation is the single best example of a purely physical modality. Because of the technical difficulties in controlling emission, radionuclide purging continues to be a promising, albeit still laboratory-based, process. Photoradiation procedures, on the other hand, have been successfully used in clinical trials. There are two main groups of photolytic agents: merocyanine 540 (MC-540) and dihematoporphyrin ether (DHE). Light activation is a function of the particular photolytic agent, and,in turn, tumor cell cytotoxicity is dependent upon the mode of light activation. Common to both groups of agents, however, is preferential localization and subsequent photoexcitation in tumor cells following exposure to ionizing irradiation. Successful application of these techniques has been carried out in AMBT for Hodgkin's disease, non-Hodgkin's disease, acute myeloid leukemia (AML), ALL, and neuroblastoma. Further trials are clearly appropriate. Elutriation is effective predominantly in density separation of populations of like cells. In this regard its application is limited to T-cell separation as a means of GvHD control, where its efficacy has been well demonstrated in large-scale studies carried out in a few highly organized programs. The margin of success is dependent on a narrow line that addresses pan-T-cell removal for control of GvHD without untoward engraftment failure. Current investigations also involve the use of elutriation in association with immunemediated CD8 purging to enhance GvHD control and further lessen graft failure. The earliest immunological purging modalities consisted of combination monoclonal antibodies (MoAbs) as targeting agents and either plant ricins or complement as the killing sources. As in pharmacologie and radionuclide or photolytic purging, the net effect is in situ cell lysis. More recent immunologie techniques include the use of immunorosetting and immunomagnetic technology (MoAbs with magnetic microspheres). With the latter, the "magnetically attracted" material is recoverable for analysis or subsequent utilization. Unlike any other purging procedure, immunomagnetic purging has the capability of addressing both positive and/or negative selection. With the advent and steady accumulation of effective and highly specific MoAbs, the opportunity to use them in association with either ricin, C, or microspheres is limited only by the target type, itself. Accordingly, MoAbs et al have been successfully used in purging AML (CD14,15), ALL (CD9,10,19,20), neuroblastoma, and retinoblastoma (neural-derived MoAbs), lung and breast cancer (site-specific MoAbs), and myeloma (B-linkeage MoAbs). Moreover, it is impossible to select out the single "best" immunologie procedure, for, in different hands, the same procedure may well produce different results. In general, institutions carrying out in-depth studies usually produce the most reliable data. Accordingly, those groups carrying out long term MoAb + C purging efforts in the AMLs have as high a rate of success as those institutions involved in extensive use of 4-HC for a similar cohort of disorders. The same applies to the various B-cell lineage luekemias as well as neural crest tumors and cancers. Another newer generation genetic approach is the use of amplification probes to identify a single gene rearrangement or a combination of genetic alterations, as in disordered gene sequences or whole translocations. Oligonucleotides tend not to be ingested by normal cells, and thus insertion (and acceptance) of this nonsensical DNA material into abnormal cells invariably results in selected cell death. Larger clinical trials as
situ destruction,
are
underway.
300
EDITORIAL
Strictly interpreted positive selective applies solely to the use of stem cells for marrow reinfusion. The two techniques in this regard include the use of (i) magnetic removal using CD34+ MoAbs and microspheres followed by chymopapain release of the targeted cells, and (ii) column immunoabsorption that utilizes biotinylated anti-CD34 MoAbs and passage over avidin-coated beads. As of this date, the latter procedure has been effectively applied to both ape and human trials with successful engraftment of trilineage hematopoiesis. The ability to upgrade the MoAb-microsphere approach has been hampered by the lack of effective large-scale chymopapain splitting of antibody-coated cells. In addressing the role of both ABMT and purging, two factors deserve special mention, viz., the lack of a GvHD (GvL) response to ABMT in the hematological malignancies and the inability of pharmacologie purging to remove drug-resistant cells. In the former, GvL lack had been a serious issue, which now appears to have been resolved concomitant with the use of low-dose cyclosporin A as a means of initiating both GvL and increasing the extent of malignant cell autocytotoxicity. The same concerns apply to removal of multidrug-resistant (MDR ) cells. The single, most effective purging approach, one that is not subject to the need to overcome P-glycoprotein expression, is the use of MoAbs with complement or ricin (for in situ destruction) or with microspheres (for entrapment). Mention has not made of the use of either cord or peripheral blood in the context of purging. It is nonetheless clear that patients with disease involving the marrow have disease in the circulation as well. Peripheral blood is not a substitute for marrow purging. Its application is ideally suited to situations of compromised marrow. Cord blood, on the other hand, with modestly immunoincompetent lymphocytes, may prove to be yet an most-studied
+
for control of GvHD. in all of these efforts is the need to limit studies to those institutions involved in extensive Implicit for investigation; in the final analysis, the fundamental issue is how best to successfully exploit purging in autologous marrow reinfusion and as a means of controlling the severity of GvHD. Purging cannot be looked upon as a separate issue; and until such time as the different purging procedures are sorted out and reach investigational maturity, they cannot be marketed for either general use or for head to head studies.
alternate
means
Samuel Gross, M.D. Professor and Chief Pédiatrie
Hematology I Oncology
Director, Pédiatrie Bone Marrow
301
Transplantation Program College of Medicine University of Florida
Mary
(1992)
Ann Liebert, Inc., Publishers
Editorial To
Purge or Not to Purge Is Not the Question
issue of purging is purging, per se, and the only questions are when, how, what, and where to purge. To propose head-to-head purging versus nonpurging studies, or to offer data from a variety of non-concurrent studies in support of either side of the purging issue, is as specious as it is premature. To appreciate fundamental differences, one must first understand the nature of the processes. And purging techniques come in a variety of processes. The proliferation of purging technology grew out of early 1960s animal studies designed to identify means of increasing the number of available allogeneic. donors. Those efforts became the anläge of our present understanding of the role of T lymphocytes in the control or prevention of graft-versus-\\osi disease (GvHD). Thirty years later, we continue to grapple with GvHD, but mainly in terms of control methods that include immunological and/or physical selection/deletion or pharmacologie suppression/dispersion. An equally important outgrowth of the GvHD studies was the recognition of a graft versus leukemia (GvL) process within, but apparently separable from, the GvHD universe. Thus, purging methodology solely for GvHD control had to be revisited in terms of both GvL and engraftment. At present, pre-BMT (purging) control of GvHD falls into three major categories: (i) pan T depletion via soybean lectin agglutination with sheep red cell rosetting, (ii) elutriation with coiinterflow centrifugation, and (iii) monoclonal antibody targeting with in situ destruction (complement or ricin) or attraction (magnetic microspheres). GvHD control is fundamental to success in the treatment of the nonmalignant hematological disorders, e.g., storage defects, immune deficiencies, hemoglobinopathies, and congenital or acquired
The
cytopenias.
Newer purging methodologies consisting of gene deletion and substitution carried out via electroporation followed by transfection may well obviate concerns of graft failure in the nonmalignant marrow disorders. This approach in somewhat modified form will also apply to certain of the marrow malignancies, with gene manipulation following upon negative selection, e.g., unwanted cell removal will precede DNA selection. A major concern relative to gene splicing is the potential for late-occurring diseases directly attributable to viral transfection; and in this regard, metabolically inert, nonmutagenic material, such as methotrexate, appears to be a benign vector substitute. Efforts to add to the donor pool (less than one-third of available siblings are HLA identical) by using haplo-identical or matched unrelated donors, paralleled studies designed to test the feasibility of autologous marrow—and, subsequently, peripheral blood to serve the same purpose. Similar studies were also designed for use in enhancing marrow recovery in megadose therapy-treated patients with a variety of solid tumors. The net effect was an exponential leap in purging research. Today, somewhat more than two-thirds of all marrow transplants are accompanied by some type of purging maneuver. Autologous bone marrow transplantation (ABMT), unlike allogeneic transplantation (alloBMT), very likely requires removal of every last malignant cell prior to reinfusion. In alloBMT, elimination of residual (host) tumor cells are very likely addressed by donor induced GvL. In ABMT, such is not the case, and purging, therefore, is designed to reduce the tumor burden without significant loss in the totipotent cell pool. The purging techniques, which do not lend themselves well to large burden cytoreduction, i.e., greater than a 5% tumor mass, fall into four categories: pharmacologie, biophysical, physical, and immunologie, as well
299
GROSS combinations of the same. Among the more common pharmacologie modalities, all of which produce in are the activated cyclophosphamides (mafosfamide, 4-HC, etc.), glucocorticoids, cisplatins, doxorubicin, arabinofuranosylcytosine, deoxycytidine, etopiside, vincristine, and alkyl-lysophospholipids. This negative selection approach rarely yields a greater than a 4 log depletion of the targeted cells; and specificity is somewhat less than precise. Stem cell losses, especially following the use of combination drug purging, are considerable; and time to marrow recovery is invariably prolonged (rarely less than 38-42 days) and, on very rare occasions, infinite. Dependence on granulocyte-macrophage or erythroid colony-forming units (GM-CFU, E-CFU) in culture as a predictor of response time is unreliable with pharmacologie purging. Far more certain is reliance on number of CFU-L. The best examples of the effective application of such agents are the studies carried out with mafosfamide or 4-HC, the former for AML and ALL, the latter solely for AML. The reasons for the differences in disease response between two such essentially similar agents are unclear. Although the volume of studies does not approach that of mafosfamide or 4-HC, the least toxic pharmacologie agents appear to be the alkyl-lysophospholipids, which .have enjoyed success in acute lymphocytic leukemia (ALL) purging. Radioisotopes and photolytic agents are the best examples of biophysical purging technologies. Elutriation is the single best example of a purely physical modality. Because of the technical difficulties in controlling emission, radionuclide purging continues to be a promising, albeit still laboratory-based, process. Photoradiation procedures, on the other hand, have been successfully used in clinical trials. There are two main groups of photolytic agents: merocyanine 540 (MC-540) and dihematoporphyrin ether (DHE). Light activation is a function of the particular photolytic agent, and,in turn, tumor cell cytotoxicity is dependent upon the mode of light activation. Common to both groups of agents, however, is preferential localization and subsequent photoexcitation in tumor cells following exposure to ionizing irradiation. Successful application of these techniques has been carried out in AMBT for Hodgkin's disease, non-Hodgkin's disease, acute myeloid leukemia (AML), ALL, and neuroblastoma. Further trials are clearly appropriate. Elutriation is effective predominantly in density separation of populations of like cells. In this regard its application is limited to T-cell separation as a means of GvHD control, where its efficacy has been well demonstrated in large-scale studies carried out in a few highly organized programs. The margin of success is dependent on a narrow line that addresses pan-T-cell removal for control of GvHD without untoward engraftment failure. Current investigations also involve the use of elutriation in association with immunemediated CD8 purging to enhance GvHD control and further lessen graft failure. The earliest immunological purging modalities consisted of combination monoclonal antibodies (MoAbs) as targeting agents and either plant ricins or complement as the killing sources. As in pharmacologie and radionuclide or photolytic purging, the net effect is in situ cell lysis. More recent immunologie techniques include the use of immunorosetting and immunomagnetic technology (MoAbs with magnetic microspheres). With the latter, the "magnetically attracted" material is recoverable for analysis or subsequent utilization. Unlike any other purging procedure, immunomagnetic purging has the capability of addressing both positive and/or negative selection. With the advent and steady accumulation of effective and highly specific MoAbs, the opportunity to use them in association with either ricin, C, or microspheres is limited only by the target type, itself. Accordingly, MoAbs et al have been successfully used in purging AML (CD14,15), ALL (CD9,10,19,20), neuroblastoma, and retinoblastoma (neural-derived MoAbs), lung and breast cancer (site-specific MoAbs), and myeloma (B-linkeage MoAbs). Moreover, it is impossible to select out the single "best" immunologie procedure, for, in different hands, the same procedure may well produce different results. In general, institutions carrying out in-depth studies usually produce the most reliable data. Accordingly, those groups carrying out long term MoAb + C purging efforts in the AMLs have as high a rate of success as those institutions involved in extensive use of 4-HC for a similar cohort of disorders. The same applies to the various B-cell lineage luekemias as well as neural crest tumors and cancers. Another newer generation genetic approach is the use of amplification probes to identify a single gene rearrangement or a combination of genetic alterations, as in disordered gene sequences or whole translocations. Oligonucleotides tend not to be ingested by normal cells, and thus insertion (and acceptance) of this nonsensical DNA material into abnormal cells invariably results in selected cell death. Larger clinical trials as
situ destruction,
are
underway.
300
EDITORIAL
Strictly interpreted positive selective applies solely to the use of stem cells for marrow reinfusion. The two techniques in this regard include the use of (i) magnetic removal using CD34+ MoAbs and microspheres followed by chymopapain release of the targeted cells, and (ii) column immunoabsorption that utilizes biotinylated anti-CD34 MoAbs and passage over avidin-coated beads. As of this date, the latter procedure has been effectively applied to both ape and human trials with successful engraftment of trilineage hematopoiesis. The ability to upgrade the MoAb-microsphere approach has been hampered by the lack of effective large-scale chymopapain splitting of antibody-coated cells. In addressing the role of both ABMT and purging, two factors deserve special mention, viz., the lack of a GvHD (GvL) response to ABMT in the hematological malignancies and the inability of pharmacologie purging to remove drug-resistant cells. In the former, GvL lack had been a serious issue, which now appears to have been resolved concomitant with the use of low-dose cyclosporin A as a means of initiating both GvL and increasing the extent of malignant cell autocytotoxicity. The same concerns apply to removal of multidrug-resistant (MDR ) cells. The single, most effective purging approach, one that is not subject to the need to overcome P-glycoprotein expression, is the use of MoAbs with complement or ricin (for in situ destruction) or with microspheres (for entrapment). Mention has not made of the use of either cord or peripheral blood in the context of purging. It is nonetheless clear that patients with disease involving the marrow have disease in the circulation as well. Peripheral blood is not a substitute for marrow purging. Its application is ideally suited to situations of compromised marrow. Cord blood, on the other hand, with modestly immunoincompetent lymphocytes, may prove to be yet an most-studied
+
for control of GvHD. in all of these efforts is the need to limit studies to those institutions involved in extensive Implicit for investigation; in the final analysis, the fundamental issue is how best to successfully exploit purging in autologous marrow reinfusion and as a means of controlling the severity of GvHD. Purging cannot be looked upon as a separate issue; and until such time as the different purging procedures are sorted out and reach investigational maturity, they cannot be marketed for either general use or for head to head studies.
alternate
means
Samuel Gross, M.D. Professor and Chief Pédiatrie
Hematology I Oncology
Director, Pédiatrie Bone Marrow
301
Transplantation Program College of Medicine University of Florida
