The Therapeutic Effect of Total Body Irradiation Followed by Infusion of Autologous or Isologous Marrow in Humans

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Chairman, Coml ttee on Research, Cincinnat FROM: Edward 8.Sil b erste in, M.0, 5lBJECT: The Therapeutic Effect of Total Body Irradiatlon Followed by Infusion of Autologous o r Isologous Marrow i n Humans jntroduction and Purpose o f Study: Host forms o f widespread metastatic cancer i n man are not amenable to conventional chemotherapy w i t h a few notable exceptions such as carcinoma of the breast and prostate. Curative radiotherapy requires dosts o f radiation generally i n excess of 3500 rads, and such large amounts of irradiation cannot be given over wide areas o f the body. Therefore whole body radiotherapy has been employed f o r several years (1-5) as palliation for metastatic carcinoma and lynphoma. The rationale behind our belief In the efficacy o f this mde o f treatment I s the fact that the mamnalian Do (the dose o f radiation required to kill 63% ofa mamnalian cell population) lies between 100-180 rads for all cell types stud1 ed (6). Thus, even though 1arge masses o f tunor i n VIVOmay not always be comparable t o these in vitro dose-response assays, some proportion of tumor cells irradiated should always be killed. should frequently relieve symptoms secondary t o tunor pressure within or on an organ and, by lowering the rider of tunor cells present, ps l bly prolong This In turn

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Results o f these studies will be analyzed by means o f the life table technique, 1.c. percent surviving each year o f those a t risk for that year as conpared t o the survlval fi5 ~ r e so f several papers which have examfned the natural history of various carcinomas with llver or osseous metastases (7-8). To further evaluate the efficacy o f low dose-wtde field radiotherapy, a protocol (appended) has been'devised randomi zing patients with adenocarcinoma of' the colon aetastatic t o the liver into grouDs receiving 5-fluomuracil 15 mg. per kg. (9) or 300 rads cobalt gama-lrradlatlon to a w i d e fic ? d from xiphoid to feet. For patients receiving rad fat fon doses i n excess of 150 rads some form o f protection is required to 8vdd life-threatening leukopenia and thronbocytopenla. Pmvlsion o f "sterile" environmnts (10-11). platelet (12) and leukocyte (13) transfusions, and radioprotective chemicals (14) have been employed; we are using marrow auto-transplantation without storage to avoid these canpl ications. The patient has marrow m ved from 0800 t o 1000 hours I n the operating mom, I s irradiation a t about 1300-1400 hours and receives his narrow (stored a t 4' C.) Intravenously from 1430 t o 1600 hours. The method of radiationdose calculation can be found elsewhere (15). The m r m transplantation

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Hater lal s Roqul red: 1 ~ awnds rtop Vacutriner tube, labeled with patlent's nane and nunbcr, plus I fom for c8C 1 md top Vacutrinar tube, lsbeled wlth patient ' s nane and nder for the blood bmk, plus a blood bank form 200 cc. hepadn ro lu tl on containing 50 units heparln per cc. This I S made by putting one cc. o f preservative-free hcparln, 10,000 unlts per CC., In 199 CC. o f normal saline. h cxtrrl vial of this heprrfn Is brought t o the operatlng mm. 7.3-7.4 and added t o a Fenwal pack. 150 12 CC. ster lle syringes t o be ob kl n td by the oper rtl ng room. Signamtor punp wlth tubing attachrnts. Sterilized triple filter system wlth three arsh CI lter opcnlngs of 297, 149, 74 mi cra d lw ter respectlvely . o 2 Fenwal Blood Pack Unlts No. TA-10, lo00 cc. 1 box I terl 1e mi croscope .c 1 Ides t

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+ 3 Matt blood rdminlrtration sets with fl lter opening masurlng 150 by 2 10 cc. syringes w i t h 18 gauge needles Formalin tube for Pathology De pr rb nt study of marrow aspirate. Blood bank rtquest tom the patlent I s pmnedlutrd with 0.6 mllligrana atropine and 100 mg. bconal Intramscularly, a t 6 a.m. The patlent pes t o the operating room r t appmxlmstcly 7 a.m. where, after inductlon wlth Pentothal, endotracheal lntubatlon Is per f amd, and the patient i s given general anesthesia with Nalothane and placed I n the prone posltlon. After appropriate antiseptic preparation o f the patient's iliac crest area, bone marrow asplratlon mqy At this the, prior to aspiration, blood for peripheral white cell Count and dlfferentlal I t obtained and 5 cc. o f blood are sent t o the blcc? bank far typing and crossmatchfng o f 2 unlts o f whole t:tGd. As an alternatl be obtained 1 . , ~ mthe transplant and reinfuscd -..AS, so as t o ellmi nate artefrctual rarPi'> .

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Bone marrow aspirations are per fo md using the Blemn needles for the sternum, and Kernick xeedles far the Illac crests. No more than 3 cc. we obtained with the needle bevel i n one positlon. The bevel Is rotated through four quadrants for each aspiration. Aspiration I s performed with a syringe which has been filled with one cc. o f the heparin solution containing 50 units per CC. An Abbott Venopak adapter connects a sterile bottle contalnlng 200 cc. o f heparin solution, 50 units per cc., toa twoway stopcock adapter i n which each syringe is placed tu flll It with one CC. o f heparin. This stopcock is also turned SO that the heparin so lu tl on i s a closed system bebeen asplrations. The syringe with aspirated marrow i s inverted several times todis - tribute the heparin and handed t o an assistant who injects it into the Fenwal pack contsining TC 199 through a two-way stopcock adapter t o which the plastic tubing o f the Fenwal pack has been attached. The emptied syringe is left In place but the stopcock handle i s turned each time so as to close off the Ferual pack system. After each aspiration another assistant applies pressure t o the area fmm whlch marmw has been obtained t o decrease hematoma formation. The areas t o be aspirated wil : be posterior and anterior iliac crests and the sternum. After 500 cc. of marrow are collected, the bag i s inverted 5-10 times and then a sanple t s obtained for nucleated cell - evloy the Sigmamotor punp and infusion set, alcohol bottle of3 CC. of 0.9% saline for injection, scissors and the triple fl lter system 'noted above. Three Abbott blood infusion sets are present for emer-

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After the marrow has been collected and stored i n the blood bank a t 4" C. for several hours while the patient receives whole body irradiation, i t I s then taken to the patient's ward where the Fenwal pack holding the mrm* Is inverted several tims t o insure homogeneous distribution of Cells and then fitted to the Slgmamtor infusion system. The Signamotor pomp is turned on and the marrow passes through the triple filter system downstream from the punp a t a rate o f 60 to 90 drops (4-6 cc.) per minute. Aliquots are collected inmediately after the punp i s turned on for the determination of cell viability, nucleated cell count and differential as well as blood culture. If there is evidence of malfunction of the f i l t e r system, as shown by bubbles appearing i n the infusion tubing after the marrow 1 s filtered, the punp i s turned off and the intravenous drip of normal saline through which the marrow has been infused ''piggyback" is restarted. The Fenwal pack containing the marm is then connected t o a standard Abbott blood infusion set which i s attached t o the bottle containing this saline solution and the infusion begun again. After the infusion has been completed, sterile normal saline is flushed through the filter set which has been disconnected from the pump. The marrow infusion 1 s regularly followed by chills and fever unless 10 grains of aspirin are given orally or rectally during the procedure. Blood cultures a r e routinely obtained and have always been sterile. a filtered specimen using Kernict's t2:k:: ., oat a in the nucleated CL;; count per single hemocytometer square by counting eight s q t t a e - t - ' dividing the total by elght. Then we p"'' # i s number by (IC,-to get

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3count per mn. ) x (40, dilutior, factor i n counting) x (1000, number o f an i n 1 cn ) x (fraction of viable cells) x ( 5 0 0 cc. volume of marrow) x (1.4, the dilution correction f r o m 100 cc. TC 199 and 100 cc. of heparin i n the Fenwal pack) x (fraction o f marruw made up of erythroid and ndeloid PrecurSon, including stab cells). This simplifies t o (cell count per hemocytometer square) x (2.8) x (lo8) x (fraction o f viable cells) x (fraction of marrow cells). Intravenous Infusion of bone marrow has been known to repopulate the marrow space (18-22) of animals and there is now ample experience w i t h hunan subjects as well (23). The marrow transplant technique developed i n our laboratory involves'a fully closed system, a significant advance over the technique published recently by the Seattle group (24). We have chosen n o t t o employ stored autologous narrow for several reasons. Storage of large amounts of marrow is difficult technically a l - though within our capability . However, we wish to return uni rradiatcd mrrow to i t s necessary microenvironment (25) as soon as possible to avoid any delay I n stem cell proliferation and maturation i n vivo. &lays giving marmu until the cell counts have fallen t o clinically dangerous levels i n the fourth week, one must still allow 5to 10 days for the precursors o f circulating leukocytes (26) and probably the same time for platelets (27) to d,:ferentlate. By this time the patient may be seriously 111 or recovering spontaneously. With infusion of marrow a t this late time one cannot then t e l l if the marrow transplant has been efficacious, whereas i f the Mrm is given on the day o f radiotheraov the 5wor ? c;tt:p3i,? during the expected i

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viability of 95-99: after freezing and thawing as one can with our Our patients receive daily white blood cell counts and differential white counts, platelet and ret icu lo cyte counts, and a check of the hematocrit and hemoglobln levels. They are sent home two days poct - irradiation and the degree o f cytopenia observed pos t-ir radi at i on in our patient has not warranted rehospitalization. Thus the total time consuned fc the procedure is3 1/2 hospital days with or without marrow transplantation, divided as Day 1: Admi ss ionto hospi tal. Preparatory di agnostic procedures including a marrow aspiration under local anesthesia. Twenty-four hour control urine collection. "Sham" irradiation for radiation dosimetry and t o make control psychologic observations. Day 2: Marrow aspiration under general anesthesia if radi atl on dose exceeds 150 rads t o whole body. Irradiation with 6oCo gam rays. Day 3 and 4: Post - irradiation observations on patient ' s psychologic reaction, blood and urine. Discharge 1500 hours day 4. We have demnstrated t h a t marrow i;msplantation can prevent the severe thmrfbocytopenia and leukopenia which 200 rads will cause (16). who received 150 rads, platelet transfusions and parenteral anti bio tlc s were required t o Prevent severe bleeding and obviate s??:~I:T::J (:le Patlent was penic). We are attempting t o modify the syndrome of nausea and romiting which For one patient u d - p,ism ever cultured from the blood while severely leuko-

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rccompany whole body irradiation with increasing frequency as the dose exceeds 100 rads (28). A comnerclally available synthetic dl kt, Vlvonex, approved by the F.D.A., decreases tht gastrointestinal flora by a factor of 1OOO (291 and may be helpful i n t h i s regard. Phenothiazines are now used t o re1 ieve post - Irradiation nausea and emesis. Assessment o f the effects of large field low dose irradiation Is under way for the following parameters: 1. Psychologic performance decrement. 3. Biochemical indicators of radiation damage. 4. Radiation-induced a1 terations o f the function of circulating peripheral blood cells. Radiation-induced alteration in imnunologic response to a set of phages, covered under a separate protocol o f Hess, Li twin,et.al. tptho d-----t o be Used i n Prcjcuring Consent: These forms, containing i n lay language an explanation o f the pmcedures described above have been forwarded t o your Comnittee. The patient is tc ld t'tat the radiotherapy t o be given i s oat a cure but that It may relieve pain (sham radiation does not have this placebo effect i n our cancer patients1 and that its efficacy i n prolonging life ISunder study. Patients w i t h metastatic carcinoma may have marrow aspirated and frozen.

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tat ions and Therapeutic Efforts, Defense Atomjc Support Agency Report No. Incidence and Duration o f Remission, Cancer &:523-530, 1969. Johnson, R.E. Total Sody Irradiation with Fractionated Dosage Schedules, Front. Radiation Ther. Onc. 3:152-162, 1968. Total Body Radiation, Nitrogen Mustard and Triethylene Melamine on the Hematopoietic System o f Terminal Cancer Patients, Science =:161-163, 1963. Miller, L.S., Fletcher, G.H. and Gerstner, H.G. Systemic and Clinical Effects Induced i n 263 Cancer Patients 5y Whole Body X - tr radiation with Nominal Air Doses o f 15 t o 200 R. School of Aviation Medicine, U.S.A.F., Little, 3.8.. Cellular Effects of Ionizing Radiation, New Eng. J . Med. Rapport, A.H. and Burleson, R.L. Survival o f Patients Treated with Systemic Fluorouracil, Surg., Gyne. and Obstet. =:773-777, 1970. Colonic Resection for Carcinoma with Simltaneous Liver Metastases, Surg., Eastern Cooperative Oncology Group, Five - Flu om uracil in Cancer: An Improved Regimn, Ann. Int. Med. 2:897-900, 1970. Patient i n a Protected Environment, Am. J. Med. Sci. 256:112-121, 1968. Penland, W.Z. and Perry, S. Portable Laminar-Air-Flow Isola tor, Lancet fusions: Fun c tim of Transfused Granulocytes from Donors with Chronic Myelocytic Leukemia. Blood %:432-442, 1970. Prolonged Survival o f a Leukemic StoraQe of Platelet Concentrates

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Modlfying Biological Effects of Radiation, Arch, Environ. Health a : 8 8 - Changes In Eunans Following Total -Body Irradiation, Defense Atomic Support Agency Report No. 1844, February 1960 through April 30, 1966. Kernick, N.B. "Autologous Bone Marrow i n the Treatment of Severe Iatrogenic tlyelosuppression" i n D i a nosis and Treatment of Acute Radiation Injury, Uorld Heal t h Organi la* Geneva, Lorenz, E., Congdon, C.C. and Uphoff, 0. Modification of Acute Irradiation Injury i n Mice and Guinea Pigs by Bone Marraw Injections, Radiology 2: Porter, K.A. Effect of Homologous Bone Marrow Injections In X-irradiated Lethally Irradiated …