with Lymphoid Cell Malignancy

The Behavior of Autologous Indium- 114m-

Labeled Lymphocytes in Patients with Lymphoid Cell Malignancy David Hamilton, Richard A. Cowan, Harbans L. Sharma, Mark Drayson, Pamela M. Nuttall, John Wagstaff, David P. Deakin, and Derek Crowther Regional Department ofMedical Physics and Bioengineering, CRC Department of Medical Oncology, Department ofRadiotherapy, Christie Hospital and Holt Radium Institute; Manchester; Department ofMedical Biophysics, Department oflmmunology, University of Manchester, England

It has been shown that radioactive material can be localizedto lymphocyte trafficareas using radiolabeled autologous lymphocytes and that 11@―ln deposited in such a way in rats produces

a lymphopoenia by establishing a selective internal irradiationof circulatinglymphocytes. The study reported here was undertaken to investigate the feasibilityof using this technique in patients with lymphoid cell malignancy. Up to 22.7 MBq was administered to seven patients with active non-Hodgkin's lymphoma involving the spleen and the behavior of the radioactive

material was followedover subsequent months. Estimates of the activityin peripheral blood, bone marrow, excrete samples, and of the variationin the whole-body distributionwere obtained. The administered radioactive material cleared rapidlyfrom the blood, 85% being removed within the first 30 mm. There was an almost immediate uptake of most of this by the spleen and liver with 4@J U

Cl) 0

15-

n

,. -

Bone Marrow Content

ci ri

-C

Bone marrow aspirates were obtained from five patients,

a-)

one of whom provided repeat samples severalweeks apart,

4@)

and were assayed in the same way as the blood samples. The total activity contained in this compartment was estimated

C

assumingthat activitywas distributedevenlythroughout the bone marrow and that this comprises [email protected]%of body weight

(9).

CD 0.

C-

ci

(n I 0-

-:@nfl ..JL@

E

@. I_4ri

-C

5.

0

Excretion The elimination of activitywas followedfrom administra tion to the end of each patient study to assess the total excretion of the radionuclide. Twenty-four-hour urine and feces collections were obtained in all patients. This informa

tion was supplementedin Patient 5 by data from a shadow shield whole-body monitor using an energy setting of 0.724 MeV ±20% and in Patients 6 and 7 by data from the longitudinal profile scans.

Volume29 • Number4 • April1988

0

0 0

2

4

6

Hours Post Adm@nistration FIGURE 1 The variation of activity in the peripheral whole blood and

ftscell-associated componentforPatient6 overthefirst6 hr postadministration.

487

Subsequently, and for as long as measurements were

relationship

continued,

study; the height of the spleen peak relative to that of the liver peak decreased. This is shown in Figure 2. Count ratios were obtained from these data by inte

a figure of 2 ±1% was maintained.

The secondary peaks had only a small effect on the general shape ofthe clearance curves which were essen tially exponential. Three phases could be identified and

the average half-times of these were 2.4 ±1.1 mm, 22 ±9 hr and 50 ± 33 days. These accounted

11 ±4, and 3 ±1% of administered

for 42 ± 15,

activity, respec

tively. The early behavior of the cell-associated activity in the peripheral blood, as a percentage ofthe total activity in that compartment, is shown for a typical patient in Figure 1. It decreased from a peak at administration and showed increases which were coincident with the secondary

peaks of the total activity curve. The later

behavior exhibited a decrease to a minimum of27% at 65 hr followed by an increase to 67%. A similar pattern was discernible in all patients with the low point in the curves occurring at a mean time of 50 ±18 hr.

The activity in the peripheral blood which was not cell-associated was only a small proportion of the total administered activity and showed a variation over the three phases ofthe total peripheral blood clearance. The mean values, as a percentage ofthe administered activ ity, forphases 1, 2, and 3 were 6.7 ±2.8%, 4.4 ±2.6%,

and 1.1 ±0.7%, respectively.

between the two peaks altered during the

grating the counts under the peaks and comparing them

to the counts integrated over the rest of the body. They indicated that the activity accumulation in the spleen reached a maximum

at —2days and decreased approx

imately mono-exponentially, thereafter, with a mean half-time of 46 ±16 days. Such a pattern was not observed for the liver. Organ Counting

The results obtained from the organ counting follow ing the postmortem investigation of Patient 4 on Day 37 showed that the spleen, which occupied a volume

1493 cm3, contained 25.4 ±0.2% of administered activity. Both a normal (volume 6 cm3) and an abnor

mal (volume 20 cm3) splenic hilar lymph node were shown to contain 4J U -C ci.@ C@.

a-)

4J U,

C

E -a

10 7 0

20

40

60

0

20

40

60

Time Post Administration (days) FIGURE 5 Semi-log plot of the variation of absolute activity in the spleen and liverwith time, calculated from the gamma camera data for patients 5 (), 6 (0) and 7 (0). Also shown is the spleen activity obtained from the organ counting for Patient 4 (is).

Volume 29 • Number 4 • April 1988

489

7 6 >.

-4-, ‘-4

>

5

-4-,

C-,

4

-a C— 4-, U)

3

C E

2 I

FIGURE 6 Histogram showing percentage of administered activityestimated to be in the bone marrow compartment

0

for

I

I

each patient. The error bars shown are ±2 s.d. and are calculated from the counting statistics.

2

713

1

Time Post Administration 4 5 Patient No.

3

(weeks) 6

7

and 1.00% ofadmimstered activity. The results for each Several authors have considered the dosimetry of patient are shown in Figure 7. The mean excretion for human lymphocytes labeled with [‘ ‘ ‘Inloxine(12—14), all patients was 0.35 ±0. 14% via the urine and 0.42 ± @-80% of which is localized in the nucleus (15). It is 0.33% via the feces. reasonable to assume that [‘ l4mIn]oxine behaves in the same way and the microdose-rate will then be similar DOSIMETRY because the Auger electron emissions are similar in Three types of radiation dosimetry are applicable to both energy and intensity (16). this technique.

The micro-dosimetry

of the low energy

Auger electrons and their effect on the radiolabeled lymphocytes, the local dosimetry of the blood cells passing through the radiation field and the macro dosimetry of the critical organs. Micro-Dosimetry

The low-energy Auger electron emissions affect only the radiolabeled lymphocytes, compromising their abil

ity to migrate and function. >@ -4-J

Assuming a nuclear diameter 4 sm then, for a label ing concentration ofO.6 MBq (16.2 j@Ci)/l08 cells, the

radiation dose absorbed by a lymphocyte nucleus in the first hour would be 226 mGy (22.6 rad) for ‘ ‘ ‘In and 139 mGy (13.9 rad) for @@4mIn. This microdosimetry has been calculated assuming a particular nuclear diameter and it must be emphasized that any variation in this dimension will dramatically alter the figures. However, since a dose of 1.5—2.0 Gy

2

> 4@) U

@0

a-) a-)

4@)

(I) C

.f—I

FIGURE 7 Histogram showing the mean per

centage of administered activity ex

E @0

creted per day via the feces (hashed)

and urine. The error bars shown are ±2 s.d. and are calculated

variation in total excreted with time.

490

from the

activity

Hamilton, Cowan, Sharma etal

// ,/1 // 7/

77

7// // //

I

/ 7-,

2

3

Patient

6

4

7

no. The Journal of Nuclear Medicine

(150—200rad) to lymphocytes in vitro is lethal (17), death of the cells within a few days can be confidently predicted.

Local Dosimetry This is by far the most difficult aspect of the dosim

etry to describe and to estimate. Calculations of radia tion dose received by the blood components during their transit through the spleen can only be carried out

if the time of transit and route through the radiation field are known (18) and this information is not avail able from this study. Macro-dosimetry Estimations of radiation

absorbed

dose have been

made for the spleen, liver, bone marrow, and whole body ofa standard man. It has been assumed that 45%

of administered activity initially accumulates in the spleen, that this decreases mono-exponentially

with a

half-time of 34 days, that 40% accumulates in the liver and that 5% accumulates in the bone marrow. The dosimetry is summarized in Table 2. DISCUSSION Several methods have been applied to achieving se lective depletion of lymphocytes. These include the techniques ofthoracic duct drainage (19), antibody lysis

(20—21),and the very recent targeting of Ricin (22). Most, however, have relied on the administration of ionizing radiation, making use ofthe radiosensitivity

of

lymphocytes resulting from their susceptibility to inter phase death.

A profound lymphopoenia can be induced by whole body irradiation

involving sub-lethal

whole-body

cx

posure to X or gamma radiation (23-24) or by repeated doses of whole-body irradiation with external shielding of non-lymphoid organs to achieve a “total lymphoid irradiation― (25).

Improvements by anatomic confinement of the ra diation so that lymphocytes are selectively irradiated and damage to non-lymphatic tissues is avoided, have been attempted

using several approaches.

Lymphocytic

depletion can be produced by extracorporeal irradiation of blood. This technique exploits both the radiosensi tivity and the migratory properties of lymphocytes to deplete the whole recirculating pool and relies on most 2RadiatiOn TABLE AdministeredActivity Absorbed Doses per (kilt of

for the CriticalOrgans ManmGy/MBq of a Standard Rad/mCiSpleen 2,800Liver

760

600Bonemarrow

160

100Wholebody

Volume29 • Number4 • April1988

is the production of a permanent field of radiation confined to lymphoid tissue by the use ofa phosphorus 32 strip attached to the surface of the spleen (1). This

technique has been modified by the injection of colloi dal tungsten-185 trioxide directly into the spleen and certain lymph nodes (28).

The study presented here was undertaken to investi gate the feasibility of using autologous lymphocytes labeled with ‘ l4mInas vectors to concentrate radioactive

material in lymphoid tissue by exploiting both the radiosensitivity and the migratory lymphocytes.

properties

of the

Of primary concern was that the viability of the lymphocytes should be maintained throughout the har vesting, labeling, and administration in order that their migratory properties be preserved. Animal studies, in vestigating the purity of the radiopharmaceutical dem onstrated normal lymphocyte migratory patterns.

Confirmation that the lymphocytes were viable at administration was afforded by a number of observa tions. Each sample of radiolabeled cells for administra tion excluded trypan blue. Although establishing their integrity this did not prove that the migratory properties ofthe cells were intact and this had to be deduced from other observations. The damage to the migratory pattern of lymphocytes is characterized by a long-term retention in the lung (3) and a subsequent selective uptake in the liver (10). The shape of the activity-time

curves in the early dynamic

study and the lack of lung activity on both the profile scans and the gamma camera images represent migra

tion patterns associated with viable rather than dam aged lymphocytes. The secondary peaks on the periph eral blood clearance curves indicate re-circulation

of

the radiolabeled lymphocytes (3,10-11) which are con firmed to be labeled cells returning to the vascular compartment

by the coincident

increases in the pro

portion of the activity which was cell-associated. The three phases identified for the clearance of activ ity from the peripheral vascular compartment can be explained by assuming that: the first phase describes

the rapid migration ofcirculating radiolabeled lympho cytes out ofthe vasculature; the second phase describes the gradual depletion in the numbers of radiolabeled lymphocytes available for re-circulation as they die from the effects of the ionizing radiation damage; and the third phase describes the equilibrium between activ ity leaking into the blood and activity being excreted from theblood. Initially, there was a lower percentage of cell-associ

30

1

other cells in the blood being sufficiently radioresistant to escape irreversible damage (26—27). A second method which exploits both the radiosen sitivity and the re-circulation properties of lymphocytes

4

ated activity in the peripheral blood than reported for studies using ‘ ‘ ‘In(3). This was considered

a conse

491

quence of the different production techniques used for the two radiopharmaceuticals. During the study, there was a fall in this percentage,

emissions. It is expected that images of lymph nodes could be obtained in the higher activity studies planned,

to a minimum at @@-50 hr, which could be explained by

From animal data (2), the activity administered in this study was too low to be expected to elicit a thera peutic response and certainly no toxicity was experi enced by the patients. However, the consistent results of uptake and excretion obtained for the seven patients enabled a preliminary dosimetric assessment to be

a gradual depletion in the numbers of radiolabeled lymphocytes available for re-circulation. Thereafter, there was a rise which could be explained by macro phages, containing radioactive material from destroyed lymphocytes, starting to enter the peripheral blood. The single peak maintained in all the longitudinal

profile scans at the level ofthe spleen and liver indicated that there was no large redistribution ofactivity to other parts of the body. From the data of the seven patients studied, on average @@@-45% of administered activity ac cumulated initially in the spleen and 40% in the liver.

All patients had considerable splenomegally with vary ing degrees of liver involvement and the observed pat tern ofuptake was interpreted as representing a selective localization

of radioactivity

at the predominant

site of

disease. After the first 2 days the distribution of radioactivity changed only slowly. Macrophages are highly radio resistant (29) and are not rapidly redistributed to other tissues (30). The slowly changing distribution thus sug gests the trapping of radioactive material by macro

phages at the site of lymphocyte death along the lym phocyte migration pathway. There was a definite reduction of radioactivity in the spleen with time, however. The mean half-time of the splenic elimination data from the gamma camera in vestigation correlated well with that from the profile investigation and with phase three of the blood clear

ance investigation. Thus there was some leakage of activity from the spleen; either because of macrophage redistribution or because of the release of activity from

macrophages which had been destroyed. This definite pattern of activity loss was not observed for the liver. The accumulation of a large amount of radioactivity in the bone marrow could cause undue damage to the hemopoeitic stem cells, and may be the dose limiting

factor. For this reason the highest recorded case of a 5% accumulation in the bone marrow compartment was used in the dosimetry calculations. However, total exclusion of activity from the bone marrow is possibly undesirable, as this would provide a sanctuary site for malignant cells. The relative importance of these two aspects will be a consideration in the studies using higher administration activities now underway which will establish whether this technique can be used to adequately treat lymphoma within the bone marrow.

It is believed that the activity not accounted for was distributed throughout

made. The highly selective uptake, stable in vivo distribu tion and low excretion reported here suggest that ad ministration of autologous radiolabeled lymphocytes

offers a convenient and effective method of introducing radioactive material into lymphoid tissue. The physical characteristics of [‘ I4mIn]oxine will result in an intense local field ofradiation which should make the technique suitable for therapeutic purposes. Because of the distribution of the radionuclide the technique should have potential in the treatment of both chronic lymphocytic leukemia and generalized

lymphomas. Higher activity studies are currently un derway to investigate the clinical therapeutic

potential

of this technique. CONCLUSIONS Autologous ‘ I4mlfllabeled lymphocytes exhibit a rapid clearance from the peripheral blood and localize preferentially in the spleen and, to a lesser extent, in

the liver and bone marrow. From a maximum concen tration at 48 hr there is a slow excretion ofactivity from the spleen resulting in a prolonged splemc radiation field which gives rise to a large absorbed dose within the organ and also acts as a source for the selective internal irradiation of the re-circulating lymphocyte population. We therefore conclude that it should be feasible to deposit sufficient activity in the spleen to achieve a lymphocytopoenia and it remains to be shown

whether adequate activity may also be deposited in diseased lymph nodes and bone marrow

to have a

significant anti-tumor effect. We propose that this tech nique may have therapeutic potential in the treatment of lymphoid malignancy. Clinical studies are currently underway at higher activities. ACKNOWLEDGMENTS The authors thank Mrs. Ann Marie Smith for preparation of the radiopharmaceutical and Dr. Wesely Shiu who partici pated in the early stages of the work.

the lymphatic tissue ofthe body

and as such would not be detected by external moni toring techniques at the activities used, because of the combined effects of its low activity per unit volume of surrounding tissue and the low intensity of gamma-ray

492

as mentioned previously.

Hamilton, Cowan, Sharma etal

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