REPORTS ON SEMINARS
(1) Seminar on “Bone Marrow Transplantation,” November 7-8, 1986, Tokyo, Japan.
Dr. Yasusada Miura of the Japanese Foundation for Cancer Therapy initiated the presentations with an examination of stem cell function in BMT patients. He discussed the kinetics of appearance of various colony forming units (CFUS) with specific examples of using 4-hydroperoxy-cyclophosfamide (4-HC) as an in vitro purge. 4-HC had a profound inhibitory effect on CFU-GEMM, BFU-E and CFU-GM. In addition, the colonies formed were larger (fewer) units. He concluded that 4-HC preserves the most immature progenitors, and that 4-HC will be a useful tool for studying human pluripotent stem cells.
Dr. Hirotoshi Shibata of Osaka discussed the use of colony stimulating factor for BMT. CSF human urine stimulates granulocytes and was used in 51 patients. Toxicity was mild fever only, for this group of ABMT/AlloBMT. CFS was employed at doses of 2x106 units/day to 8x106 units/day for 46 available patients, starting on Day 1-3 in most patients. He believes that CSFHU speeds marrow recovery by a significant degree (16.7 vs. 24 days to leukocyte recovery) but has not yet been shown to stimulate leukemic cells. Problems with the purity of this natural (not recombinant) product and the heterogeneity of patients make these data difficult to analyze. However, a double-blind, controlled, prospective Phase III study is being planned.
Dr. William Peters of Duke University, described his perspective on bone marrow purging especially for ABMT, in solid tumor patients. Failures occur if the preparative regimen is ineffective or if the marrow is contaminated by tumor. In terms of cytoreduction (preparation), dose intensity is crucial, and non-overlapping toxicity is useful. The Duke University Program for Breast Cancer is CPA + CDDP + BCNU + ABMT in 18 patients (9 prior adjuvant therapy) and 8/18 CR and 5/18 PR. Patients generally relapse in sites of prior (bulky) involvement. Toxicities have been significant including hemorrhage, veno-occlusive disease toxic hepatitis and late opportunistic infection. Requirements for purging include selective killing of tumor (vs. normal) cells and being practically useful (easily added and removed in vitro). Breast and SCLC patients (but not melanoma) theoretically require ABMT purging. Duke University is studying 127 MoAbs directed against 7 antigens (multiple epitopes). Individual MoAbs detect breast cancer cells variably but the situation is improved with multiple Abs. Now they can detect 1:10,000 contamination with tumor cells (5x105 cells in FACS). Using antibodies or immunotoxins has also demonstrated some benefits.
Dr. Yoshiaki Moriyama of the Niigata School of Medicine presented his work with hyperthermic purging of tumor cells (AML) for ABMT. Human AML cells were heated in H2O bath for 15-60 minutes, and then stem cell survival determined. At 42°C, tumor lines were more sensitive than normal cells and up to 2 logs of AML cells were killed. An increase of 1°C increased cell kill by tenfold. Normal cells showed least sensitivity for immature mixed stem cell colonies.
Dr. Karen Antman of the Dana-Farber Cancer Institute described a large integrated program of multiple alkylating agents for solid tumors (“STAMP”). Hypotheses employed include chemotherapy steep dose/response relationships, tumor heterogeneity (drug resistance), lack of cross resistance and synergy. Increases of 5 to 50 fold in doses of alkylators can be clinically achieved with ABMT. Cellular transport is mediated by many different mechanisms, and thus, resistance can result from different perturbation. In an initial trial of 61 patients treated with CPA + BCNU + CDDP, toxicity was significant with 15% toxic deaths, and 16 patients had herpetic disease. Consequently, now all patients receive Acyclovir prophylaxis. Deaths were due to infection (2), bleeding (1), and VOD (3). The greater the prior chemotherapy, the worse was the toxicity, and the median time to progression overall was 4.7 months. Responses were very rapid (by approximately day 7) and predicted for overall response. More recent studies of ThioTEPA + CPA also look promising. Patients receive CPA 6 gm as a 4 day infusion + TTPA + L-PAM. Variable PK and PD has been noted for 500 and 750 mg/m2 of TTPA. Responses have been noted in breast cancer and melanoma patients to date in this Phase I-II trial. Currently SCLC patient are treated with “STAMP I” after induction. In sarcomas, IFF + CBDCA are used. In breast cancer “STAMP III” is being tested. A proposed study is for breast patients to receive CAF induction and then randomly receive STAMP III or the same drugs at 1/10 the dose (without marrow transplant).
Dr. Mine Harada of Kanazawa University then reviewed his extensive experience with ABMT in various solid tumor categories. Many different preparative regimens were studied with highly variable results. Treatment failures occurred because of relapse (5), not CR (2), and fatal toxicity (7). The results of multicenter Japanese studies were presented. Eleven centers cooperated, and 110 patients received ABMT (22 NHL, 29 AL, 12 SCLC, 11 neuroblastoma predominating). Results were generally favorable for NHL, and some AML and solid tumor patients are long-term survivors (including 2 NPC, 1 CNS, 1 neuroblastoma, 1 Ewing’s).
Dr. Geoffrey Herzig of Washington University (St. Louis) reviewed his ABMT experience in NHL and solid tumor patients. He outlined his technique of Phase I development-escalation scheme, choice of agents and choice of patients. He developed a menu of possible choices for 2 and 3 drug combinations for 7 tumor types. He described his experience with high dose CPA + TBI (1,000 cGy) and compared autologous with allogeneic transplant (thus examining tumor contamination of marrow). For HD and NHL, some 20-35% of patients survive in CR for a median duration of 15 months. Having achieved a prior CR seemed to be of most prognostic significance. Especially promising data for VP16 + CPA + BCNU in HD were presented (13/17 CR with CCR of 3 > 12 months). He concluded with a probing series of developmental questions for the future, including opportunities for clinical trials to be considered.
Dr. Tohru Masaoka of the Center for Adults Diseases in Osaka presented data on allogeneic BMT for leukemic patients over a 10-year period (1975-85). For 356 patients, 202 deaths were noted, with interstitial pneumonitis, relapse and sepsis predominating. Overall, 70% of ALL patients in the first CR survived free of disease. Prognostic factors for ALL appeared to be GVHD, BMT status and sex. For ANLL, about 60% were cured in first remission, and similar prognostic factors were demonstrated. A mild degree of GVHD was best for patient survival. Data for CML were also displayed, with CMV titer, age and TBI dosage of prognostic import. He also indicated that decontamination with ozone or garlic reduced fungal overgrowth.
Dr. Keith Sullivan of the University of Washington (Seattle) presented his data for allogeneic BMT for leukemia and lymphoma patients. Longterm, very durable remissions have been demonstrated (especially post 3 years) for ALL and ANLL. For ANLL, 50% are cured in the first CR and 25-35% in early relapse. These results are inferior for 2nd and 3rd relapse/remission. Rather similar data exist for ALL patients. Prognostically, the younger patients fare better than older ones for allogeneic transplants. Syngeneic transplants are effective in CML (90% survival), and accelerated-blast phase CML (20-30%). In general, the sooner the transplant (time from diagnosis), the better is the outcome. For HD and NHL, 100 patients were treated (76 with resistant, refractory disease), and 60 had allogeneic grafting (27 autologous and 13 syngeneic). With CPA + TBI, 20% of resistant, refractory NHL survive. However, for earlier patients, better results were evident. He described the Seattle attempts to increase the pool of HLA matching, since only about 30% of patients will have appropriate family HLA + MLC donors. The rejection rate for HLA well-matched transplants was tiny (0.05) and for 2 loci mismatched (0.20) only slightly worse. To achieve less GVHD, a variety of techniques to deplete T cells have been proposed. Optimal GVHD control was achieved with short course MTX plus Cyclosporine for 180 days. Effective therapy for chronic GVHD includes early alternating Prednisone/Cyclosporine, as does Prednisone + Azathioprine. The dilemma of GVHD and long term survival was also clearly delineated.
Dr. Shunichi Kato of Tokai University, presented a collection of studies on pediatric patients. Some 233 patients with all sorts of diseases were treated with BMT. Especially good results were noted in patients with 1st relapse ALL and CML. The techniques of pediatric transplant at Tokai University were described and data on leukemia, aplastic anemia, solid tumors and SCID presented. Surprisingly, there was a generally low incidence of infection in the early period (0-30 days); overall 3/30 died of infection. Transfusion products were from CMV(-)donors. IG administration and fractionated radiation seemed to be beneficial. Finally, an excellent plan for evaluating long term toxicity and quality of life considerations was presented.
The first day concluded with a presentation by Dr. F. Leonard Johnson of the University of Chicago on the BMT experience in the U.S. pediatric patient population. He reviewed the historical development of BMT in children in both non-malignant and malignant disorders. Approximately 80% of SCID with completely matched donors survive, but other patients do less well. Manipulating cyclosporine can both limit GVHD and maximize engraftment. For relapsed ALL, the use of transplant offers the best chance of cure (46% vs. 14% for chemotherapy). However, for ANLL, the value of BMT is not so clearly demonstrable because of late relapses and deaths. Also, aggressive chemotherapy seems to be as effective as BMT. For preleukemia and solid tumors, data suggest that real benefits exist. He then reviewed the major problems which exist today, including rejection, GVHD, infection, mismatching, and long-term side effects. His review was monumental and critical, and provided a forum for a detailed discussion.
On the second day of the symposium, issues of BMT for non-malignant conditions and supportive care issues were raised. Dr. Akihisa Kanamaru of the Hyogo College of Medicine began with a review of the Japanese experience with aplastic anemia. A total of 77 cases was presented (median age = 16) of variable etiology (90% idiopathic). A total of 77 cases of variable etiology (90% ideopathic) were presented (median age=16). A total of 9 different conditioning regimens were employed, and both MTX and cyclosporine were used for GVHD. The overall rejection rate was about 25%, and there were no differences between engraftment rates for males or females. No statistically significant differences were detectable with respect to MTX vs. cyclosporine, <6 or 6 months from diagnosis to BMT, nor pretransplant transfusion history. Overall, 52/72 cases survive post-transplant.
Dr. Sullivan presented the aplastic anemia experience at Seattle. He described the supportive care aspects of care, including infection control, caloric support and antiemetics. Overall, BMT data on 455 patients with a 17% rejection and 40% GVHD (II+) rate were described. TBI (standard dose fractions) causes intolerable complications (IP), but no single preparative program has demonstrable superiority. An important prognostic feature is the dose of marrow cells administered, but donor sex, negative MLC and buffy coat infusion are of less clear import. For unclear reasons, rejection has been decreasing since 1980, and so GVHD has increased in importance. Crucial features for GVHD control included laminar flow rooms (LAF), HLA matching, immuno-suppression, and in vitro marrow treatment. In contrast, in leukemics, LAF has no substantial impact (poorer gut decontamination). The cause of this effect is unknown, but perhaps enteric flora share antigenic features with gut epithelium (hence, better decontamination yields less GVHD). Immunosuppression is very important, and MTX or MTX + cyclosporine A is necessary. The combination of cyclosporine + MTX dramatically decreased the incidence and severity of GVHD acutely, but not chronically.
Dr. Jun-ichi Yata of the Tokyo Medical University described the Japanese experience with BMT in SCID. Factors affecting BMT in SCID include the same set of factors noted above. When major HLA and MLR incompatibility occurs, only about 5% of patients survived, and little GVHD was noted. Soy bean agglutinin and anti-T cell antibodies have been applied. Successful engraftment was achieved in 7/16 Japanese patients. The relative merits of T-cell depletion techniques were discussed. Unique aspects of SCID include the fact that no conditioning is needed, HLA = donors are acceptable, and granulocyte and platelet systems are preserved. However, conditioning programs are necessary for all non-SCID conditions. He then postulated that large granular lymphocytes might inhibit T-cells in certain immunodeficient patients.
A discussion focusing on GVHD was led by Dr. Yoshihisa Kodera of the Nagoya First Red Cross Hospital. A total of 46 allogeneic BMT patients (37 leukemic, 9 aplasia) was treated in Nagoya, and 248 patients were collected from other centers. The prophylaxis consisted of MTX or CY-A. The incidence of acute and chronic GVHD in leukemia adults was 57% and 44%. The number of patients in any particular category was quite small, not permitting extensive analysis. One-third of patients developed chronic GVHD without antecedent acute GVHD. The spectrum of clinical pathology was as expected. Among 22 chronic GVHD patients, 13 had good response to therapy (only 5 died of uncontrolled GVHD). As expected, mild GVHD is best for leukemic patients. A comprehensive overall review of the Japanese experience was presented.
Dr. Shigetaka Asano of the University of Tokyo discussed the challenges of interstitial pneumonitis (IP). Approximately 25% of BMT deaths (59/202) are attributable to IP. Several risk factors for IP have been postulated, including infections (CMV, etc.), pulmonary alveolar damage (TBI) and delayed immune reconstitution. Prevention and therapy of CMV-IP included CMV immune plasma, selective blood product use, antiviral therapy and accelerated immune reconstitution. Delayed constitution seems to be crucial, and efforts with IFN and IL-2 have been made. The use of HG-CSF isolated by Dr. Asano seems a promising option for the future. He has described the nucleotide sequence of G-CSF, which seems to be unique compared to other CSF’s studied.
In summary, this symposium was one of the most successful in recent years. The reasons are largely unexplainable, but the excellence of the presentations certainly contributed. All the investigators offered excellently prepared remarks - thorough, critical and lucid. Additionally, there was an open and intense exchange of information which both groups found useful.
Both U.S. and Japanese investigators face similar, formidable problems. The costs of BMT (financial, effort, patient resources, etc.) are so high that careful strategic planning is needed. The orderly development and application of BMT and supportive care techniques will demand considerable attention for the foreseeable future. Bone marrow transplantation is less widely employed in Japan than in the United States at this time, but will certainly increase in the next decade. This topic will likely be reexamined 4-5 years hence.
(2) Seminar on “Neoadjuvant and Adjuvant Therapy,” February 3-4, 1987, Makaha, Hawaii.
After opening remarks by Drs. Michael Friedman and Makoto Ogawa, the program was begun.
Session l: Head and Neck Cancer
Dr. Waun K, Hong of M.D. Anderson Hospital and Tumor Institute began with a review of the background for therapeutic approaches to advanced (Stage III or IV) oropharyngeal cancer. Overall, 40-50% have local and 20-30% distant recurrences (and 10-40% have a 2nd primary tumor). For those with metastatic disease, response rates of 5 to 40% are seen with 5%-10% CR rate.
Neoadjuvant therapy with CT preceding definitive RT or surgery is being explored. The advantages to this approach are obvious, but the drawbacks are also potentially important (such as enhanced local toxicity, immunosuppression, nutritional deficits, etc). Combinations of CDDP, bleomycin, MTX, and 5-FU have been evaluated. Overall, CDDP + bleomycin induction resulted in 10-20% CR and 30-40% PR. The addition of a 3rd agent (such as a vinca) did not result in major improvement. Aggressive combinations of CDDP + 5FU seem to be most effective with CR at 30%. The importance of clinical CR rates is now being appreciated. About 1/2 of clinical CR have a pathological CR. Moreover, the MDS is 58 months for complete response, compared to 11 months for non-responders.
The importance of clinical CR rates is now being appreciated. About one-half of clinical CRs have a pathological CR. Moreover, the MDS is 58 months for complete response, compared to 11 months for non-responders.
The toxicity and tolerability of CT preceding RT are now better defined. Full course RT is possible, but must be given with care. The question remains as to whether induction chemotherapy can substitute for surgery in CR patients. Laryngeal sparing surgery may then be possible. Moreover, patients who respond well to CT tend to respond well to RT also.
Unfortunately, to date, there are no randomized studies to document the benefits of neoadjuvant treatment. However, the CR rate was consistently quite low (10% or less). For laryngeal cancer, the use of CT to preserve voice is being evaluated. A VA-cooperative trial of 340 patients will evaluate CDDP + 5FU pre-operatively and randomized allocation to RT or surgery. Begun in l/85, currently 175 patients have been accrued, and a CR rate of 35% observed. A similar trial of 3 courses of CDDP + 5FU prior to RT is being studied. Finally, an NCI intergroup study is ongoing with the following design: 
A new 3-drug induction of CDDP + 5FU + bleomycin is being evaluated. New directions include intensification programs, ABMT, and sequenced CR + RT + BRMS.
Dr. Y. Nomura of the Kyushu Cancer Center presented data from Japan for these patients treated with CDDP + pepleomycin. Interest in preoperative IA CT has been increasing. Preclinical data suggest that CDDP preceding PEP is superior to other sequences. Clinically, 50 mg/m2 CDDP is given prior to 5 days of PEP. To date, 55 patients (beginning in 5/81) received neoadjuvant CDDP + PEP. Post-induction therapy included RT, CT or surgery.
For maxillary sinus tumors, a complex approach of 3 doses of CDDP + PEP followed by 5FU + RT was employed. After CT + RT 9/22 had pathologic CR. For NPC patients preoperative CT attempted to reduce systemic metastases, 3/12 achieved a CR with CT and impressive antitumor responses were observed.
Overall, CDDP + PEP seems to provide reasonable CT, and opportunities for combined modality trials exist.
Session 2. Breast Cancer
Dr. Robert B. Livingston of the University of Washington (Seattle) focused rather specifically on how dose/response considerations might be crucial for adjuvant breast cancer studies. Three considerations include peak plasma levels, area under the curve and intensity per unit time. Data suggest that higher intensity of therapy results in better clinical findings; and probably full dose chemotherapy results in better findings. For advanced disease patients, higher dose ADM is more beneficial than low-dose, and similar data exist for CDDP as well. Hrunuyk and Bush have examples of higher dose resulting in higher response and survival. For ultra high-dose CT (ABMT), 3/20 CR and 13/20 PR were noted for single agent alkylating agents. It might, therefore, be worthwhile to examine ABMT in patients achieving CR. Such consolidation and salvage programs should be further explored.
Dr. Nomura then presented the relevant Japanese data from 3 breast cancer trials. These studies examined hormonal and CT, for example, for stage II patients:
MMC + FT206
MMC + FT206 + TAM
These therapies resulted in a 88-92% 3-year DFS. For stage II and IIIa patients in a second study, MMC + FT206 were again studied. This perioperative chemotherapy was well tolerated, and for stage IIIa ER+, TAM seems to add benefit. A third study uses multiple alkylating agents with or without TAM and is ongoing. These studies have been included in Richard Peto’s overview studies and will be published separately in the near future. Considering quality of life, hormonal therapy is generally superior.
Session 3: Lung Cancer
Dr. Mark R. Green, of the University of California, San Diego Cancer Center, reviewed approaches to neoadjuvant trials in NSCL cancer (Stage III). The use of CAP therapy with or without RT in 167 patients did not result in statistically significant benefits (60% one year survivors). Currently, there are 3 studies examining this issue in the USA. The 3 programs examine VP16 + CDDP, MACC, or FOMi-CAP chemotherapy. One important experiment he described is the use of 5FU + CDDP simultaneously with radiation. The study of Trybula described 64 patients receiving 5FU 800 mg/m2/d x 5 days CI and CDDP 60 mg/m2 q 14 days, along with 2Gy/dx5 RT. Of 31 patients who received a subsequent thoracotomy, 7 had a pathologic CR. Overall, although the recurrence rate is 61%, 20% of patients have pathologic clearing of local tumor. The LCSG has generated data for conventional adjuvant (post-operative) CT. The use of intrapleural BCG did not prove to be beneficial. CAP CT is beneficial in adeno and large cell patients. Moreover, local control is achieved with post-operative RT. For incompletely resected NSCL cancer, CAP + RT seems to offer some DFS benefits but little impact on overall survival.
Dr. M. Ogawa of the Cancer Institute Hospital indicated that although gastric cancer is still the number 1 cause of malignant death in Japan, its incidence is decreasing, and lung cancer is number 2 and increasing. There is proportionally more adeno and squamous cell lung cancer (40 and 37% overall). Roughly half of patients are operated upon but only 20% have curative resection. The Japanese have explored regional administration of MMC as an adjuvant therapy. In a selected small series, 30-50% survived 5 years. The use of nonspecific immunostimulants has not proven to be of definite value. From 1975 to 1985, nine studies were reported. Only three were randomized, and with small numbers of patients some promising results with BRM’s were reported.
Session 4: Gastric cancer
The discussion of gastric cancer was initiated by Dr. Bernard Levin of M.D. Anderson Hospital and Tumor Institute. He outlined general prognostic features and the activity of CT combinations. An extensive discussion of the GITSG adjuvant study of 5FU + MeCCNU vs. control then followed. Of approximately 160 patients, the CT group seemed to enjoy benefits in terms of DF and OS. These data are discordant since two other studies are negative (ECOG and VASOG). Comparisons between U.S. and Japanese data were suggested as a fruitful area for discussion. In Japan, predominantly earlier stage patients are being diagnosed and treated. Future directions include the use of perioperative therapy, IP CT, new drug combinations such as FAMTX, and radiation therapy.
Dr. T. Nakajima of the Cancer Institute Hospital presented the Japanese gastric cancer data. The central role of resection is unquestionable. Over 40 years, 8,437 patients were treated, and 5,708 (59%) had a curative resection. There has been a general decrease in the incidence of locally advanced disease. He reviewed eight sequential trials for these patients. Common features include the use of MMC in CT programs and a surgery only (control) group. There are a variety of programs with preliminary evidence of benefit. A large, complex, randomized study of Japanese and Chilean patients has been initiated. Three therapies are possible for Stage I-III patients and two programs for Stage IV individuals. It is planned to accrue 700 patients. To date, no significant benefits for any therapy arm have been noted.
Session 5: Colorectal cancer
Data for colorectal cancer therapy in Japan were presented by Dr. T. Tominaga of Komagome Hospital. He described the Kajitani Study of MMC + FT207 vs. no therapy for colonic or rectal cancer. Benefits were noted for the CT group for the more than 1,000 patients studied. A second study was performed in 1982-83 with over 5,000 patients. To date, only 2 year DFS are available, and no definite trends are apparent. The most recent study examines the value of regional CT (portal vein vs. systemic vs. control) for colon and IA vs. IV vs. control for rectal cancer. More than 1,000 patients per study are entered. A specific trial for rectal cancer examining preoperative radiation is also being conducted. Attention is being directed to pathologically assessed cellular changes induced by RT.
Session 6: Soft Tissue and Bone Sarcoma
Dr. K. Furuse of Tottori University began the discussion on bony and soft part sarcomata. A trial of the Orthopedic Oncology Group examined 117 patients with osteosarcoma. They received either ADM(80) or ADM + MTX(37). Overall, 53/117 survived and 64/117 died; the 5-year disease-free rate was 39%. The multidrug group fared better than ADM alone. Two other non-randomized studies were performed. Both demonstrated superior results for multidrug programs consisting of ADM + MTX = CDDP, etc. The Japanese have also independently evaluated the T- 10 protocol of MSKI (Rosen) with good results. For soft-tissue sarcomas, the data are less extensive. Those receiving adjuvant chemotherapy had a variety of agents given via several routes. Overall, about 56% = 10 survived 5 years. Finally, a set of guidelines and recommendations for adjuvant therapy in Japan was discussed.
Dr. Laurence H. Baker of Harper Hospital described a series of studies from both ECOG and SWOG. The combination of ADM + DTIC seems to be worthwhile, and ifosfamide is an important new agent. Of prognostic significance is the fact that Grade 1 and minimal necrosis tumors behaved more favorably. Demonstrating a benefit for adjuvant chemotherapy is difficult. Consequently, a randomized study of 145 patients receiving either ADM or observation was initiated.
For bony sarcoma the use of adjuvant ADM + other agents is well established. Consequently, two major studies are planned. A discussion of joint plans and potential problems ensued.
Session 7: Statistical Analysis
A discussion of statistical issues and related methodology was presented by Dr. A. Sakuma of the Tokyo Medical and Dental University. He reviewed use of statistics in the literature in current Japanese medical journals. The problems of multiple comparison and methods of handling multiple endpoint comparisons were discussed. Examples included tests of predetermined sequential analyses, timepoints used, repeated chi-square ordered categorical data, and confidence band of regressions.
Finally, Drs. Ogawa and Friedman led a discussion on general aspects of adjuvant chemotherapy trials.
Session 8: General Discussion
(3) Seminar on “Biochemical Pharmacology of Treatment Failure,” March 26-27, 1987, Shoreham Hotel, Washington, D.C.
Dr. Kenneth Cowan of NCI initiated the discussion of the “Biochemical Pharmacology of Treatment Resistance” with a description of the Multi Drug Resistant (MDR) phenotype. This system of cross resistance for anthracylines, actinomycins, podophylotoxins and vincas can result in decreased drug accumulation, increased P170 glycoprotein, increase in some cytosolic proteins, and gene amplification. The MDR gene may reside on chromosome 7 and is over expressed in many human tumors. Transport resistance does not equate with drug resistance. For example, verapimil can reverse certain transport defects, but does not completely reverse drug resistance. Other differences also exist in MDR cells. A model system used examines carcinogen impact on cells. A toxin enters the cell and can be processed by Phase I (activating) or Phase II (conjugating) metabolism. There is an associated decrease in toxin accumulation in the MDR cell. Phase I enzymes are decreased while Phase II enzymes (such as GSH-S-Transferase) are increased (an acidic or placental form of the enzyme).
The clinical relevance of these findings may be significant. For example, 10/12 specimens studied have increased anionic GSH-T and P170. MDR may be naturally present in 90% of human colon cancer and less than 10% of normal colon tissue. The association between acquired and inherent resistance will be a fruitful area for further investigation.
Dr. Takashi Tsuruo of the Japan Foundation for Cancer Research next discussed his work with a MoAb against the MDR gene (Adriamycin resistant). P170 glycoprotein is presumed the target of this Ab. MoAb “l6” or “17” can inhibit growth of resistant cell lines. Several possible uses for this MoAb exist, including the identification of resistant patients and as antitumor therapy. He has successfully transfected murine cells with MDR gene (4.5 Kb fragment). As described by Pastan and Ling, the structure of P glycoprotein can be phosphorylated by phorbol ester or diacyl glyceride. The phosphorylation pattern is different between verapimil and phorbol ester activation.
MDR 1 gene amplification is not found in wild type P388 cells but is in resistant P388 and is more in P170. Considerable collaborative work is planned for this topic area.
Dr. Robert Ozols of NCI discussed the relevance of drug resistance to clinical human ovarian cancer. He stressed that dose intensity is a crucial consideration. High dose CDDP (200 mg/m2) plus cytoxan (1 gm/m2) in 37 patients was employed. For Stage III disease there seems to be an improvement in survivorship. The toxicity is severe, especially neuropathy, which is dose-limiting. Methods of decreasing toxicity are being examined. WR 2721 and diethyl-thio-carbamate (DDTC) are pharmacologic protectors for some toxicity. CBDCA also has activity in refractory patients but usually in CDDP sensitive cells. DDTC given after CDDP can ameliorate normal tissue toxicity but cannot reverse DNA-adducts. A clinical trial in refractory patients will be initiated soon at NCI.
Another interesting compound is tetraplatinum, and Phase I trials at NCI are planned for the near future. For screening purposes, a series of cell lines was prepared. Mostly, these are from resistant patients. Unexpectedly, there is some cross resistance between Adriamycin and L-PAM. His laboratory is studying several methods of enhancing drug effects. Of note is that in human ovarian cancer, the MDR phenotype may not be the crucial resistance mechanism. GSH may be a more relevant consideration. GSH can detoxify electrophilic agents, and destroy free radicals (of greater importance for anthracyclines). In ovarian cells resistant to L-PAM or Adriamycin, there is increased GSH. Decreasing GSH by using BSO increased alkylator cytotoxicity, but BSO may increase normal tissue injury as well (such as in kidney). There are now parallel studies to try to exploit and overcome some mechanisms of resistance.
Dr. Akiyama presented data on new screening systems employing MDR considerations. The KB-CHR cell line he uses is resistant to colchicine, vinca, anthracycline, etc., and isoprenoids can overcome this P170 resistance while not blocking calcium channel activity. Chloroquine can also act in this way. These amphophilic cationic drugs are of interest.
Cephathine is a compound currently being screened, a plant alkaloid that has been used clinically. It can inhibit rapid drug afflux. Reserpine also seems to decrease drug afflux. This area of work will hopefully define other agents of clinical utility.
Dr. Youcef Rustum of Roswell Park Memorial Institute next examined the pharmacologic basis of Ara-C effectiveness in patients with AML. Newly diagnosed AML patients receive Ara-C infusion plus Adriamycin which results in an 80% CR rate, but only 20% have a durable response. Ara-C is the prb drug for Ara CTP (the active form) and is inactivated by a deaminase (resulting in Ara-U). There is little correlation between plasma levels of Ara-C (PK) and the cellular concentration (cytotoxicity), The intracellular kinase activity determines AraCTP retention and cytotoxicity. The average Ara-C level is 55 ng/ml at conventional dosage and 75,000 ng/ml at very high dose. There are three pharmacologic phenotypes; the rarest is that 5-10% who have no Ara-C intake (lack of CTP kinase). The other two groups are responsive to Ara-C induction (but retention determines CR duration). High dose Ara-C is required for refractory patients, and those had transport deficiency. Two gm/m2 Ara-C may be as effective as any higher dosage (higher may give only more toxicity). Some patients who have good drug retention may need only qd dosage, while the low retainers should be treated tid or qid.
He also described Ara-CDP depalmitin (CD 84027) as a prototype nucleotide with modification being made at RPMI. Initial research indicates that some candidate molecules yield more Ara-CTP retention, and these will be evaluated.
Dr. Hideo Suzuki of the University of Tokyo discussed potential reversal of Ara-C resistance by Cadequomycin and related compounds. Both in vitro and in vivo data show potentiation with decreases in deamination (DCMP deaminase).
Dr. Carmen Allegra of NCI presented the NCI data on biochemical modulation of 5FU with folinic acid (FA). 5FU affects DNA (5-FdUTP) and RNA (5-FUTP). Thymidine synthetase (TS) is the primary target enzyme (dUMP —> dTMP) requiring reduced folate co-factors. A protocol at NCI examined refractory breast cancer patients and attempted to quantify FA - 5FU interactions. Clinically, FA 0.5 gm IV x 5d with 5FU 375 mg/m2 x 5 d (2 hours later) was used. Currently, 33 patients are evaluable and about 90% had some prior 5FU. Overall, 9/33 had a clinical response (7 had soft tissue responses). GI toxicity was not as bad as myelotoxicity. Dr. Allegra tried to assay tissue T.S. (total, bound and free). When 5FU was given alone, less T.S. was bound than when 5FU + FA was administered.
Dr. Makato Inaba of the Japan Foundation for Cancer Research described his studies of 5FU, and suggested that decreased uptake and decreased uridine kinase activity were factors of importance. Differences in 5FU, FUR and FUDR were noted in several cell lines (P388, CEM, KU2 and A549). Possible mechanisms of resistance include:
Dr. Eddie Reed of NCI presented a series of experiments evaluating CDDP interaction in human tissues. He has studied DNA treated with polyclonal MoAb for an ELISA assay of DNA-adduct formation. The most common CDDP modification (90%) is in bidentate intrastrand dGpG and dApG linkages. He then investigated the relation of this assay to clinical results. Patients who demonstrated no adduct formation had no clinical response. About 50% of those patients with CDDP adducts had an objective response.
Examination of possible artifactual confusion ruled out WBC or lysosomal contamination. In his evaluation of ovarian cancer patients, there was a strong statistical correlation between adduct formation in WBC and response. These adducts persist for long periods of time, especially in brain, spleen, and kidney. In liver and lung, the adduct levels were lower. There is a dose response relationship; higher doses of CDDP correlated with a higher adduct level.
Dr. Kousuke Okada of Hiroshima University described various means of affecting Topoisomerase activity in order to examine drug resistance. Using a camptothecin analogue, he studied Topoisomerase I inhibition. Since VP16 affects Topoisomerase II in a clinically meaningful way, these type I agents may also be of interest. A major screening activity was carried out in Japan using calf thymus Topoisomerase I. Several DNA forms (supercoiled + and - as well as relaxed) were studied. An inhibitor of interest, “Topostin” can inhibit even captothecin resistant cells. A series of collateral sensitivity experiments are also being performed.
Dr. Gregory Curt of NCI presented data on MTX resistance mechanisms of clinical relevance. MTX Glu5 is an avid inhibitor of DHFR and even TS. Unbound MTX is very important in the cell in order to prevent drug afflux. There are five general mechanisms of MTX resistance. One of particular interest is increased DHFR (gene copy number) and double minute chromosomes in SCLC. In screening 9 SCLC cell line thought to be somewhat MTX resistant, at micromole concentrations of MTX, there was acceptable transport and binding to DHFR. Differences were noted in polyglutamation (Glu 3,4,5) in 4/9; 2/9 had low TS levels and 1/9 had more DHFR.
In normal human bone marrow, more Glu metabolites were noted after 24 hours of 10 micromole exposure to MTX. In human choriocarcinoma, there is high sensitivity to MTX. Two chorioearcinoma cell lines are the most efficient polyglutamate formers. New antifols will be examined to optimize TS binding, GAR transformylase and AICAR transformylase.
Dr. Enrico Mihich of Roswell Park Memorial Institute summarized several issues of biochemical pharmacology. Multifactoral items of factors include pharmacokinetics, transport, activation and retention, competition with metabolite pools, adaptation or repair, drug perturbation cascade, and metabolic requirement at time of drug control. The specific example of 5FU was discussed at length. Thymidine plus 5FU yields thymine which spares 5FU (and favors 5FU proceeding to RNA inhibition). However, this combination was toxic and has been clinically abandoned. 5FU blood levels are generally higher with thymidine.
The feasibility of 5FU + FA has been explored clinically. The more FA the better is the TS bindin. A randomized study in colorectal cancer was begun at RPMI, with initial evidence of 5FU + FA being more effective than MTX + 5FU and 5FU alone. Biochemical parameters correlated with these findings.
Dr. Robert Shoemaker of the NCI then described the NCI in vitro screening techniques. There is an overall coordination of sampling. The task is enormous (1,000,000 cultures per month). For example, in P388/A PR there were 12/242 compounds which showed collateral sensitivity. In the MCF model, there was a similar 9% of 324 collaterally sensitive compounds. Unfortunately, there was no overlap in the compounds (i.e., none were collaterally sensitive in both screens). This area will be an important one for exploration.
Dr. Makoto Inaba of the Japan Foundation for Cancer Research presented data on the use of the nude mouse model system. He has employed this animal system to obtain a rational dose for predicting clinical effects and toxicity. He has examined human gastric and lung tumor lines, and the in vitro predictions of response correlated reasonably well with observed clinical results. Thus, this technique may be useful for testing new compounds at the end of Phase I testing.
The preclinical studies in the United States of new compounds of interest were presented by Dr. Charles Grieshaber of NCI. He extensively reviewed all agents in the decision network system at this time.
Dr. Shigeru Tsukagoshi of the Japan Foundation for Cancer Research reviewed new drugs of interest in Japan. One 5FU analogue is in Phase I testing. A camptothecin analogue (CPT II) is also in testing, and an ICRF-159 derivative is being tested. Phase I trials are planned with platinum analogues, heterocyclics, anthacycline and nonspecific immune modulators.
Dr. Daniel Hoth of NCI described the enormous review of all reported trials for Phase II studies of NCI drugs over the past 15 years. Some 46 new agents were screened and 2,500 publications reviewed. Of 46 drugs, 24 were considered active (that is, a response rate > 10% in selected diseases). Identified were 7/18 active in leukemia, 17/23 in lymphoma, but only l/42 colon, 6/34 breast, 3/37 lung cancer.
Finally, Dr. Makoto Ogawa of the Japan Foundation for Cancer Research reviewed the Japanese Phase II data. 2545 is a CDDP analogue 10 times more soluble and more active against some murine tumors; of interest, thrombocytopenia was dose-limiting with milder leukopenia. Also, new anthracycline, azathiopurine and quino-carmycin data were reviewed.
The meeting proved to be a productive exchange of information, and opportunities for research were defined.
SEMlNAR AGENDA AND PARTICIPANTS
(1) SEMINAR ON BONE MARROW TRANSPLANTATION
Century Hyatt Hotel, Shinjuku, Tokyo
November 7-8, 1986
AGENDA