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Marrow autotransplantation accelerates haematological recovery in patients with malignant melanoma treated with high-dose melphalan.

In a Phase I study, melphalan 140 mg/m2 was administered to 8 patients with disseminated malignant melanoma. Marrow was removed from the patients immediately before melphalan administration and returned i.v. 8 h later. Studies on marrow culture and melphalan pharmacokinetics predicted that this was a safe time to administer non-cryopreserved marrow. Four patients received lower doses of i.v. melphalan without autologous marrow. In the group receiving autologous marrow the time for recovery of peripheral-blood granulocytes to 800/mm2 or greater was significantly less (P = 0.01) than in those not receiving marrow. In 7 patients the tumour showed evidence of response to the drug and there was 1 complete remission. This treatment deserves investigation in patients with tumours more sensitive to drugs than melanoma.

A PROBLEM in human cancer chemotherapy is that for some cytotoxic drugs the safe dose is limited by damage to the marrow, yet for compounds such as alkylating agents an increased dose would be expected to increase the tumour-cell kill. Melphalan, for instance, shows a loglinear dose-response curve against human malignant melanoma xenografts growing in immune-deprived mice over the dose ranige 5-25 mg/kg (Selby, 1978, personal comm.), reflecting the clinical experience that, although conventional oral doses (.10 mg on 5 days monthly) of melphalan produce regressions in only about 9% of melanoma patients (Luce, 1975), the higher concentrations achieved in isolated limb perfusions are much more effective (Wreaver et al., 1975). Where marrow toxicity is the dose-limiting factor, this problem can to some extent be overcome by the use of reverse-barrier nursing, broad-spectrum antibiotics and platelet concentrates to support the patient during the period of marrow hypoplasia, and we used this approach in a small series of patients with faradvanced tumours (2 with malignant melanoma, 2 with testicular teratoma) who were given i.v. melphalan in doses ranging from 60 to 125 mg/Mr2. Although 2 patients (one from each group) achieved significant tumour regressions, this regimen was too toxic, since white-cell counts took nearly 4 weeks to become reestablished above 1000/mm3. However, serious toxicity was limited to myelosuppression, which raised the possibility of marrow "rescue" with non-cryopreserved autologous marrow harvested immediately before treatment.
Combining marrow autografting with chemotherapy is not new. Attempts have been made to do this, using cryopreserved marrow, in Burkitt's lymphoma (Ziegler et al., 1977) and in a wide variety of other tumours (Westbury et al., 1959;Clifford et al., 1961;Humble et al., 1975;Tobias et at., 1977). Furthermore, Ariel & Pack (1962) treated 31 melanoma patients with 100 mg i.v. melphalan plus non-cryopreserved autologous bone marrow and noted objective responses in li of the tumours, although unfortunately the haematological effect of the marrow transfusion was not clearly documented.
In Ziegler's study with Burkitt's lymphoma the results were encouraging, but it would be fair to say that in most other studies the results have been equivocal, both from the standpoint of demonstrating that autologous marrow infusion accelerates marrow recovery from drug damage and from that of showing that higher doses of drug produce a significantly greater antitumour effect. However, the impression remains that the autografts were of some value, and the antitumour effects were sometimes greater than expected.
There are considerable practical problems in the cryopreservation of large voltumes of marrow, and Tobias et al. (1977) found very variable stem cell viabilities at the time of reinfusion of marrow preserved in this way. We have therefore approached this problem with 2 main aims first to choose a drug which has such a short life in the patient that cryopreservation of the marrow is unnecessary, so that the whole proceduremarrow harvest, drug administration and return of marrow to the patient can be accomplished in a few hours and, second, to establish unequivocally whether or not nmarrow autotransplantation aids marrow recovery. Because of the short half-life of i.v. melphalan it seemed possible that the whole procedure of marrow harvest, drug administration and marrow reinfusion could be performed on the same day without recourse to cryopreservation. Patients with advanced malignant melanoma were chosen for the study, because conventional doses of drugs rarely produce regression rates greater than 20%o and complete remissions are very rare, so it seemed proper to attempt more radical chemotherapy in these patients.
In order to establish the optimal timing for removal of the marrow, melphalan administration, and reinfusion of the marrow, it was necessary first to measure melphalan levels in plasma and urine after a single i.v. injection, since the persistence of toxic levels of the drug at the time of reinfusion could destroy the reimplanted marrow. Secondly it was necessary to measure the viability of aspirated human marrow kept without cryopreservation. Hitlherto there have been no studies on the levels of melphalan in body fluids after the high doses given in the present study. However, a recently published study (Tattersall et al., 1978) in w%Nhich conventional doses were given (i.v. injection-20-23 mg/M2) produced evidenice that the plasma half-life wras short (,1 h) though there was evidence for prolonged persistence at low levels in a terminal elimination phase. Likewise urinary output, measured both in terms of excretion of radioactivity after administration of the 14C_ labelled drug, and by mass spectrometrystable isotope dilution, diminished rapidly after 2 h. The latter technique was used in the present determinations of plasma and urine levels of melphalan. Only the alterations from the experiment procedure described previously (Tattersall et al., 1978)  Assay of melphalan in urine.-Six patients were studied. Urine was collected by catheterization, and samples removed at hourly intervals and the volume determined. Samples were stored frozen (-30°C). To an aliquot was added the appropriate volume of a solution of melphalan-d2 (1-00 mg in 1 ml 0-1N HCI).
Because of the sharp fall in the levels with time, the volumes of urine taken and the volumes of standard solution added were appropriately varied to give convenient ratios in the mass spectra for m/e 230:m/e 232 [M-CH(NH2)CO2CH31+ for the methyl esters of melphalan and its d2 analogue. Thus for the samples taken up to 4 h. 50 ,lI of standard was added to 0 5 ml of urine, from 4 to 6 h 20 ,ul in 1 ml and subsequently 10 ,ul in I mnl. Crude inelphalan was recovered from Amberlite XAD-2 resin as described previously. and was purified by thin-layer chromatography on Silica gel (Merck Kieselgel GF254) using chloroform methanol: water, 28:10:1-8, as developing solvent. The UVabsorbing bands corresponding in RF value to melphalan were removed, eluted wxith methanol and the eluate conventionally methylated with ethereal diazomethane. From the mass spectrum of the resulting mixture of the methyl esters, the ratio of melphalan to its d2 analogue w-as determined as previously described.
The levels are expressed in terms of cumulative excretion of drug ( Fig. 1).
Assay of melphalan in plasma-.Five patients were studied. The procedure followed was that used for urine, except that in all cases 10 pd of standard w,as added to 1 ml of plasma. Blood samples w%ere collected at 5 and 30 min, and 1, 2, and 3 h. The plasma levels are show%in in Fig. 2. Five patients had initial (5 min) melphalan levels measured, 4 had levels measured at 30 min and 1 h, and 3 had levels measured at 2 h.
For the calibration line using normal plasma, 1 ml aliquots were treated with 10 pu of standard and either 0, 1, 2, 5 or 10 ,ul of melphalan (1.00 mg/ml). The observed ratios for m/e 230: m/e 232 in the mass spectra obtained after work-up were corrected (Tattersall et al., 1978) mainly for recontribution to m/e 232 from the 37CI-containing ion from the unlabelled melphalan, and the corrected values plotted against melphalan concentration ( Fig. 3). RESULTS

AND DISCUSSION (I)
Urinary excretion of unchanged melphalan was virtually complete in 6 h ( Fig. 1), supporting the choice of this time as safe for autologous marrow reinfusion. There was a wide range of melphalan recovery as a proportion of the total dose administered. This ranged from 96% (240 mg recovered/250 mg administered) for VA to 32% (70 mg recovered/220 mg administered for AK. We do not know why, but possible explanations are variation in the degree of melphalan binding to plasma proteins between patients or variations in the degree of hydrolysis of the melphalan. It cannot be due to metabolism of the drug, since this does not occur. Plasma data ( Fig. 2) were less complete. One ,ug of melphalan was easily quantified in 1 ml of normal plasma (Dr M. Jarman) containing 10 /tg of d2 standard. Thus in the mass spectrum of the mixture of methyl obtained therefrom, the signal intensity (see Fig. 3) for melphalan (m/e 230) was 12% of that for the d2 analogue (m/e 232). The relative intensity for m/e 230 when no melphalan was added was 2%. Thus I ,ug/ml is in principle easily detected and measured by the method described here. However, some of the plasma samples from treated patients, particularly those taken for the later times, failed to provide such contaminantfree spectra, and in such cases only upper limits could be established. In Fig. 2  for each patient. The 5min levels varied widely, with a range 5-3-115 ,ug/ml, but in no patient could melphalan be detected in the plasma after 2 h. These limited data supported the conclusion that 6 h and after was safe for marrow reinfusion, since plasma levels would be sufficiently low to minimize toxic effects on the reimplanted cells.

Marrow viability at 4°C
Colony-forming ability in vitro (CFU-C) was used as a measure of the functional viability of the marrow after aspiration. Aliquots of whole marrow, in preservativefree heparin were kept at 4°C for up to 24 h before the assay was set up. At the time of each assay, a sample of marrow was allowed to sediment by gravity at room temperature, the buffy coat removed and the cells washed 3 times. Colony formation was assayed with leucocyte feeder layers as the sources of colony-stimulating activity (Pike & Robinson, 1970) with the addition of lysed rat erythrocytes to improve colony growth (Gordon et al., unpub.). After 10-day incubation at 37°C in a humidified atmosphere of 10% CO2, colonies containing more than 50 cells were counted under a dissecting microscope.
The results in Fig. 4 show that there was no significant loss of colony-forming ability when the marrow was held at 4°C for up to 10 h. Longer periods of storage led to a gradual decrease in marrow viability measured in this way.
Since melphalan was undetectable in plasma after 2 h and in urine after 6 h and non-cryopreserved, heparinized marrow will grow control numbers of granulocytic colonies after 8 h storage at 4°C, 8 h was chosen as the Patients.-Eight patients, details of whom are given in Table I, were treated with 140 mg/m2 melphalan i.v. followed by a marrow autograft. All had widely disseminated malignant melanoma which was sufficiently ad-vanced to require palliation but were otherwise in good physical condition, and all were under the age of 65. Patients were excluded from the study if they had impaired renal function, marrow infiltration, or if the bone scan or radiological skeletal survey showed bone involvement. Procedure.-Under a light general anaesthetic a urinary catheter and central venous pressure line were introduced. The patients were heparinized wiith a single dose of 1,500 u/M2 to facilitate marrowr aspiration. They were placed supine and marrowA and blood were aspirated from the anterior iliac crests through heparinized Salah marrowA aspiration needles into 20 ml sterile heparinized syringes. After 200-300 ml of blood/marrow had been aspirated the patients were placed face down and the posterior iliac crests were sampled. Finally the sternum was used as a source of marrow. Between 2 and 4 ml of marrow was aspirated from each site. Multiple skin punctures were unnecessary, as the needles could be moved around under the skin, and usually only 2 skin punctures were needed on each side. Marrow was expressed from the syringes into a sterile plastic bag until 2-5 x 108/kg nucleated marrow cells had been harvested (200-500 ml of blood and marrow).
When the marrow harvest wNas complete the mixture of heparinized blood and marrow was placed in a 4°C refrigerator and the patient returned to the ward w-here 140 mg/ m2 melphalan was given i.v. The drug is supplied with its own diluent, and was made up immediately before injection, and it was injected as a bolus into the side arm of a fast running drip. (It can be very painful if a large vein is not used and the injection is not made slowly).
The melphalan was followed by 20 mg frusemide i.v. and 2 1 of fluid were given i.v. over the next 3 h in order to prevent a high urinary melphalan concentration. Antiemetics were given as required.
Hypotensive episodes occurred in the first 2 patients within 12 h of this procedure, probably due to a combination of the anaesthetic and the loss of blood aspirated wAith the marrow, and 2 units of blood were therefore given rapidly in the theatre to subsequent patients, w-hose pulse, blood pressure and central venous pressure were recorded every 15 min and urine output hourly until the marrow was returned. After the adoption of this routine, there wtere no further hypotensive episodes.
Eight hours after the melphalan had been given the blood and marrow were reinfused i.v. The marrow was unfiltered apart from its passage through the 300 ,um filter in a standard blood-giving set. It wvas returned to the, patient as fast as possible.

Management of patients after treatment.
Patients were barrier-nursed in single rooms during the period of neutropenia, and nonabsorbed antibiotics were given orally to suppress potential gut pathogens (Framycetiin, colistin and nystatin-Storring d al., 1977). Febrile episodes were treated promptly w!ith broad-spectrum i.v. antibiotics, and platelet concentrates wN-ere given when indicated. RESULTS (11) Complications of treatment All patients were nauseated, and vomited within 4 h of treatment, aind of the anti-emetics used 5 mg haloperidol i.v. appeared the most effective. Otherwise there were no immediate complications, apart from the hypotensive episodes mentioned above, and pain or discomfort from the marrow aspiration sites were not appreciable. After 5-6 days there was a marked loss of appetite, associated in most cases with nausea and depression, which persisted throughout the period of marrow hypoplasia. Alopecia developed during the 3rd week. More serious complications were febrile episodes, occurring in all patients during the 2nd week and from 2 of whom organisms were isolated in blood cultures (Staph. aureus and E. coli respectively) and transient elevation of blood urea in 2 patients (peak blood urea levels 18 0 and 24 4 mM).
Fever resolved when the granulocyte count rose above 400/mm3, so that the ungrafted patients had longer periods of pyrexia than the grafted ones.
Both patients whose blood urea levels were raised after high-dose melphalan had high urinary concentrations of the drug during the first 2 h after its administration, and once the induction of a diuresis with frusemide and i.v. saline became routine practice there were no further cases.
The effect of treatment upon the tumour One patient (Case 8, Table I) achieved complete remission. She had a large axillary nodal recuirrence with extension / I into breast tissue and multiple liver metastases. She remained in complete remission for 4 months after treatment, and then developed an axillary recurrence which was treated with radiotherapy. Seven months after treatment there was still no evidence of recurrence of the liver metastases. A second patient (Case 3) had disease limited to the retroperitoneal tissues and her abdominal ultrasonogram cleared completely after high-dose melpha-Ian, although minimal disease persisted on lymphography. Of the remainder, 2 show-ed more than 500/ regression of measurable skin deposits, as did a third whose liver metastases progressed nevertheless. One patient had no response at all, and the other 2 had transient regressions of skin deposits. None of these partial regressions was sustained for longer than 3 months.

The effect of treatment on blood count
The mean granulocyte and platelet counts in the 8 grafted patients who received 140 mg/M2 of melphalan are shown in Fig. 5, and the actual granulocyte   Table II. These are compared with those in the 4 patients receiving melphalan alone in doses ranging from 60 to 125 mg/M2 i.v. Note that all the grafted patients received higher doses of melphalan than the ungrafted ones, and yet there was faster recovery of the granulocyte count in the grafted patients, an effect that can only be due to the marrow graft.
After melphalan treatment in both groups of patients, the total leucocyte count remained normal for the first 5 days, and then fell rapidly to a few hundred/ mm3. In both groups of patients the total granulocyte counts had fallen to less than 500/mm3 by Day 10, with the exception of one grafted patient in whom the lowest recorded granulocyte count was 950/mm3 on Day 6. By Day 16, 3/4 of the nongrafted patients had no detectable peripheral blood granulocytes, whereas of the grafted patients 2 had counts of more than 2000/mm3, and 5 had counts of more than 1000/mm3. By Day 20 all the grafted patients had granulocyte counts of 950/ mm3 or more. None of the non-grafted patients had a count of more than 400 at Day 22. The number of days taken to establish a granulocyte count of 800/mm3 or greater was measured for each patient. The 2 groups were significantly different by the Mann-Whitney U test (P=0 01). There was no significant acceleration of platelet recovery in the grafted patients. The reason for this is not known, but it is well known that in patients receiving marrow allografts from sibling donors a similar tardiness in platelet recovery is seen compared with the rate of granulocyte recovery. Possibly it is more difficult for megakaryocytic stem cells to become established and to mature when transplanted. However, this is less of a problem, since supportive treatment with platelet transfusions is readily available and easily given.

DISCUSSION (II)
This study shows that autologous, un-6 preserved marrow infused 8 h after highdose i.v. melphalan accelerates marrow recovery, and this is reflected most markedly in the recovery of peripheralblood granulocytes, where the time taken for recovery to a mean count of 800/mm3 in the grafted patient was 7 days, compared with 18 days in the non-grafted ones.
The effect upon the tumours was disappointing, apart from one patient in whom there was a complete remission. From this standpoint we have done little to answer an outstanding question in cancer chemotherapy-does increasing the dose of drugs increase the response of tumours? Experience with chemosensitive transplanted animal tumours has shown that this is so, but as Tattersall & Tobias (1976) have pointed out, there is little evidence in man that increasing the dose of most c]inically useful antitumour drugs does more than increase toxicity to the patient. Clearly, in this context, the choice of both tumour and drug is likely to be important and, although we have found that malignant melanoma is relatively unresponsive to high-dose melphalan, other tumours more sensitive to conventional doses of alkylating agents, such as ovarian cancer, lymphomas, neuroblastoms and breast cancer, may prove to be more responsive.
The choice of drug for very-high-dose chemotherapy is also bound to be limited by the effect upon normal tissues other than the marrow, even if means are available to protect or rescue the marrow from drug damage. For example, it is unrealistic to think of increasing the dose of the many classes of drugs by more than about twice the upper limit of conventional dosage, since vinca alkaloids would produce prohibitive neurotoxicity, anthracycline antibiotics cardiotoxicity, nitrosoureas hepatoxicity and fluorouracil gastrointestinal toxicity. Even the well known exception, methotrexate, which can be given in enormous doses with folinic acid rescue, has not been shown convincingly to have an increased therapeutic effect when used in this way. In practice, we are left with alkylating agents and even some of these, such as cyclophosphamide and ifosphamide, produce urothelial toxicity which prohibits their use in very large doses.
Finally, melphalan itself could not be used in doses much greater than those employed in this study since damage to the gastrointestinal tract would supervene. Studies in sheep  have shown that melphalan gut toxicity can be reduced by the use of small "priming" doses of cyclophosphamide given before the melphalan, and we have shown a similar sparing effect upon human marrow (Hedley et al., 1978), but at present there is no evidence that this effect can be produced in human gut and, even if it could, it is not known by how much the melphalan dose could be increased nor whether such an increase would be therapeutically valuable.
Our future policy will be cautiously to explore the use of this treatment for tumours known to be more sensitive than malignant melanoma to alkylating agents and in whom resistance to conventionally employed agents has arisen.