Clin. Radiol. (1977) 28, 23-28

E L E C T R O N SPIN R E S O N A N C E M E A S U R E M E N T S O F B L O O D C A E R U L O P L A S M I N A N D IRON T R A N S F E R R I N L E V E L S IN P A T I E N T S WITH N O N - H O D G K I N ' S L Y M P H O M A

MARGARET FOSTER, AUDREY DAWSON, T. POCKINGTON and LYDIA FELL

From the Departments of Medical Physics, Haematology and Chemical Pathology, University of Aberdeen, Aberdeen AB9 2ZD, Scotland

Caeruloplasmin and iron transferrin level were measured in blood of patients with non-Hodgkin's lymphoma in different stages of disease activity and compared with erythrocyte sedimentation rate and NAP level in the same samples. It was found that both caeruloplasmin level and sedimentation rate showed a slight increase in mean level in patients with active disease as compared with those in remission, particularly in the group of patients with poorly or undifferentiated diffuse disease. No difference was observed in levels of iron transferrin or NAP. Both caeruloplasmin and sedimentation rate showed occasional abnormal values in patients in remission but in most cases where both were elevated the patients subsequently entered a more active phase of the disease.

INTRODUCTION

The value of serial measurements of caeruloplasmin and iron transferrin in whole blood by electron spin resonance spectroscopy (e.s.r.) has been demon- strated in the management of patients with Hodgkin's disease (Foster et al., 1977). Although the variation in serum copper or caeruloplasmin level (usually measured by other time-consuming and often less accurate techniques, particularly for caeruloplasmin) in active Hodgkin's disease is well known, as is the disruption of iron metabolism, there is some debate in the literature regarding these findings in the non-Hodgkin's lymphomas. For example, Mortazavi et al (1972) examined serum copper in various types of lymphoma and found that there was an increased level as disease activity increased and a reduction on successful treatment of this active phase. However, Hrgovcic and his co-workers (1973a) found that in the non-Hodgkin's lymphomas, particularly in reti- culum cell sarcoma, the serum copper level is less helpful in determining the patient's state or response to therapy although after a more detailed study (1973b) they did find that this parameter did have some clinical relevance.

Since the technique of e.s.r, is simple and rapid to perform and also gives information of caeruloplasmin and iron transferrin levels in the same whole blood sample, it was decided to investigate a group of patients with non-Hodgkin's lymphomas to see if the results were comparable with those found by us for Hodgkin's disease, and also to test the value of these parameters as compared with the standard parameters of erythrocyte sedimentation rate and neutrophil

alkaline phosphatase (NAP) level in indicating an increase in disease activity.

TECHNIQUES AND PATIENTS

The techniques used in this study were identical to those used in investigating similar parameters in Hodgkin's disease. Caeruloptasmin and iron trans- ferrin were measured by e.s.r., a signal height being recorded rather than an absolute level of either protein, sedimentation rate was measured by the Westergren method and NAP level by the technique of Dacie and Lewis (1969). Haemoglobin level, packed cell volume, white cell count and platelet count were also recorded. The patients were examined clinically by a physician at a follow-up lymphoma clinic and on the basis of this examination, plus additional information such as X-ray films and gallium - 6 7 scan where necessary, were assigned to groups of disease activity. These groups were similar in most respects to those used previously in classifying Hodgkin's disease patients, level (1) being clinically inactive disease, level (2) intermediate activity either due to early reactivity of the disease clinically or during recovery from an active phase, and level (3) being clinically active disease. The final group, level (4), differed slightly from that used in the Hodgkin's disease classification. In this latter all cases showing an abnormality in some parameter of the blood but being assessed as clinically inactive and showing no other reason for the ab- normality were allocated to level (4). However, the various blood parameters showed such a wide spread in the non-Hodgkin's lymphoma group that this was

23

24 C L I N I C A L R A D I O L O G Y

Table 1 - Distribution of samples from non-Hodgkin's lymphoma patients

(Nine patients at some time failed to attend for clinical assessment after blood sampling and hence were not allocated an activity level)

No. o f No. o f Samples during cases samples treatment

All Female 16 67 7 Male 16 76 39 Total 32 143 46

Table 2 - Distribution of non-Hodgkin's lymphoma cases by histological type

No. o f cases Histological type

4 13

Nodular: well-differentiated lymphocytic Nodular: mixed cell, hystiocytic,

poorly differentiated lymphocytic Diffuse: well-differentiated lymphocytic Diffuse: poorly differentiated lymphocytic

undifferentiated, mixed cell, histiocytic

Activity (1) Female 9 23 1 Male 10 22 6 Total 19 45 7

Activity (2) Female 8 13 2 Male 8 26 19 Total 16 39 21

Activity (3) Female 9 18 4 Male 7 23 11 Total 16 41 15

Activity (4) Female 4 4 0 Male 3 5 3 Total 7 9 3

impractical and at least two blood parameters had to be abnormal and the clinical assessment inactive before non-Hodgkin's lymphoma patients were ad- mitted to level (4). Distribution of cases and samples in the various activity levels is shown in Table 1.

The test group of non-Hodgkin's lymphoma cases was unselected but did not include any female taking oral contraceptive steroids. It consisted of 32 patients, 16 males and 16 females, ranging in age from 30 to 85 years who were attending the Joint Lymphoma Clinic at Aberdeen Royal Infirmary. Originally these cases were separated into two major groups, reticulum cell sarcoma and lymphosarcoma. However, it was possible to review the histology in most of the patients and divide them according to Rappaport 's classification (1956). With only 32 cases in the study group it was not possible to split the information into so many sub-groups and achieve meaningful sample sizes in each group so a pre- liminary analysis was made by examining the block of data as a unit. Subsequently the analysis was ex- tended to see if any particular histological condition showed any trend away from the general pattern. However, only 28 of the 32 cases could be given a detailed histological classification as some of the

/

initial diagnoses had been made elsewhere using another classification and the histology had not been checked in Aberdeen (Table 2).

RESULTS

Fig. 1 shows the difference in spread of peak-to- peak height of the caeruloplasmin signal between fully active non-Hodgkin's lymphoma patients and those in complete remission, and Table 2 shows the mean figure with standard deviation for this and all other blood parameters in the two states. The spread is wide in both sets of samples, the patients in remission showing caeruloplasmin signals up to 2.15, whereas those with active disease range from over 2.8 to below 1.0. The mean value for patients with active disease is higher than that for inactive patients but the overlap is much greater than that found in patients with Hodgkin's disease. Analysis of the data by age, sex and administration of cytotoxic therapy at the time of sampling showed none of these factors to influence the spread of results.

A further splitting of data was made from patients in activity categories (1) and (3) into caeruloplasmin signal spread by histological type. Unfortunately only one value was available from a patient with active disease who had a well-differentiated, nodular type of disease which makes impossible any comment of differences in the nodular group. More information was available for patients with the diffuse disease type and a fairly clear picture is indicated of an increase in caeruloplasmin level with increased activity being found mainly in those patients with poorly or undifferentiated cell types, as seen in Table 4. One patient with mixed cell, diffuse disease and another with histiocytic diffuse disease are omitted as they did not fit well into either histological group. Omission of these data did not significantly alter the results for either group.

Fig. 1 shows the spread in signal size from iron transferrin in patients with fully active non-Hodgkin's lymphoma and those in complete remission. Both the

E L E C T R O N SPIN R E S O N A N C E M E A S U R E M E N T S O F B L O O D C A E R U L O P L A S M I N 25

mean values and the spread picture show that there is no general variation in this parameter of the blood and analysis of the data by sex and age showed no trend for either of these groupings. However, when the data were split into the histological types, as done for the caeruloplasmin signal, there was a very slight trend indicating that in the poorly and un- differentiated group of patients with diffuse disease the iron transferrin level drops with increasing activity, whereas in the well-differentiated diffuse group it remains within normal levels (Table 4).

Once again analysis of the data for patients with nodular disease was not possible due to the small numbers available. Also the picture for diffuse disease is complicated by one very odd value of 5.22 for a patient with active disease in the undifferentiated group. This single point has been omitted from the averages of this group, and of the active group in general as the patient had obstructive jaundice (from

involved nodes in the porta hepatis) which may affect the iron balance of the blood. At no other time, even on other allocations to activity level (3), did he have an iron transferrin level below 1.0 or above 2.0.

Fig. 2 shows that there is some upward spread in sedimentation rate in non-Hodgkin's lymphoma cases with active disease, although the majority are within the normal range, and abnormally high values can be met among patients with inactive disease. Analysis by histological classification as in Table 4 shows the same pattern already seen for caeruloplasmin level, that in patients with diffuse poorly or un- differentiated tumour there is an increase in sedi- mentation rate with increasing disease activity, and that this is not usually the case if the tumour is well-differentiated lymphocytic, with one exceptional value at 63 mm/h.

Fig. 2 also demonstrates that there is little difference in scatter of NAP values between patients

8

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CAERULOPLASMIN

ACTIVE DISEASE

Mean =1.80-+0"525 S.D. n :41

IRON TRANSFERRIN

ACTIVE DISEASE

Mean : 1.40-+ 0"893 S'D- n= 28

INACTIVE

Mean : 1"49 -+ 0-314S.D. • n=45

Signal

INACTIVE

Mean= 1.21-+ 0"373 n=38

SD.

Size

Fig. 1 - Spread of values for size of caeruloplasmin and iron transferrin signals in active and inactive non-Hodgkin's lyrnphoma.

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ELECTRON SPIN RESONANCE MEASUREMENTS OF BLOOD CAERULOPLASMIN 27

ERYTHROCYTE SEDIMENTATION RATE N.A.P. LEVEL II

[ I NORMAL RANGE I t NORMAL [ ~ u RANGE

ACTIVE DISEASE ACTIVE DISEASE 8

[~~:] Mean 27-7-+ 26"80S 'D. Mean 53.1 + 36"39 S'D. 6 n = 39 n= 22

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INACTIVE DISEASE INACTIVE DISEASE 10

Mean 14.7 +- 13"31 S-D. Mean 45.5 + 34.08 S.D.

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Fig. 2 - Spread o f values of erythrocyte sedimentat ion rate and NAP level in patients with non-Hodgkin 's l y m p h o m a in active state or full remission,

with active or inactive disease. In both cases the majority of values lie below the figure of 100, which marks the normal limit, and indeed only one active sample was above this, whilst three inactive samples were outside the normal range. Analysis of these data by histological type shows no pattern comparable to that found with any other parameter, indeed for both well and poorly differentiated diffuse disease the mean NAP level in active patients was below that for patients with inactive disease.

No other parameter of the blood measured in this study showed a significant difference between patients with clinically active or inactive disease.

Table 5 gives mean values for the four main blood

parameters under examination for activity levels (2) and (4), The values for level (2) reflect the small differences between fully active and inactive states for non-Hodgkin's lymphoma. The category (4) values for iron transferrin signal size and NAP level also show the lack of difference found when this group of diseases is examined as a whole, but the caeruloplasmin signal size and erythrocyte sedi- mentation rate are both elevated well above the mean value for fully active patients. Category (4) includes only those patients who are considered to be inactive clinically but who show some abnormality in the blood. The nine samples allocated to this category are listed in Table 6 where it can be seen that both the

28 CLINICAL RADIOLOGY

caeruloplasmin level and the sedimentation rate are elevated in all cases. Among the seven patients involved, two were allocated to category (4) on two successive visits. Of the seven, five showed an increase in activity to fully active disease on the next visit after being allocated to level (4), one had no follow-up and the final one showed no increase in disease activity. Of the five patients showing increase in disease activity two had nodular mixed-cell disease, one had well-differentiated lymphocyt ic and another undifferentiated cell, diffuse disease and the final case had not had the histology confirmed.

All iron transferrin levels and both NAP levels were within normal range ,but all caeruloplasmin levels and all but one sedimentation rate were outside normal limits. It would seem, therefore, that there is a strong correlation between simultaneous increase in both sedimentation rate and caeruloplasmin level and an increase in disease activity, even in patients who do not normally show an elevation in either of these parameters during the active phase of the disease, e.g. those with well-differentiated disease. However, where only one of these parameters is elevated there is usually no subsequent increase in disease activity.

DISCUSSION

The results reported in this survey indicate that neither the iron transferrin level of the blood nor the NAP level reflects the activity o f disease in patients with non-Hodgkin's lymphoma and appear to have no clinical usefulness in the monitoring of these diseases. However, both caeruloplasmin level and erythrocyte sedimentation rate show some elevation with an increase in activity of disease for some histological types and in particular they both appear to show considerable increase before the onset of a period of activity even though one or both parameters may return to normal level during the active phase itself. However, either parameter can show abnormal levels, unrelated to any obvious factor, in patients in remission and only in those patients where both parameters became abnormal was there a strong correlation with impending increase in disease activity. It would appear, therefore, that the serial measurement of these parameters in the blood of such patients could be of some clinical significance in patient management but only if both are measured at each visit o f the patient.

Although in their general review ( 1 9 7 3 a ) o f serum copper levels in lymphomas and leukaemias, Hrgovcic and co-workers suggested that in non-Hodgkin's lymphoma the clinical significance was less marked than in Hodgkin's disease, in a more detailed survey published the same year (1973b) they found that

increases in serum copper level with disease activity were general in their non-Hodgkin's lymphoma group, although most marked in those patients with un- differentiated disease. With this latter finding we are in agreement although even here there is considerable scatter of results. However, we did not find the increase which those workers reported in the other types of lymphoma, and even in the group where there was elevation, this one parameter, taken alone, was not sufficiently reliable to constitute a good test for disease activity.

One of the most interesting findings to have been made in this survey of both Hodgkin's and non- Hodgkin's lymphoma is that there is very marked difference between the diseases in the parameters of the blood when the patients enter a more active phase of their disease. Although all these diseases are classed together under the single heading of malignant lymphoma it is possible that these different blood responses are a reflection of the deeper significance of the difference between Hodgkin's disease and other lymphomas.

Acknowledgements. - The authors would like to thank Professor J. R. Mallard of the Department of Medical Physics and Professor S. C. Frazer of the Department of Chemical Pathology, University of Aberdeen, for encouragement, advice and facilities which have helped with this study. We also acknowledge the help and interest of the late Dr E. F. Ridley, Consultant Radiotherapist, Malignant Disease Unit, Aberdeen Royal Infirmary. This work was supported by the Cancer Research Campaign grant No. SP1273.

REFERENCES

Dacie, J. V. & Lewis, S. M. (1969). Practical Haematology, 4th edn, pp, 91-93. Churchill.

Fairley, G. H. & Stansfeld, A. G. (1974). Malignant disorders of the lymph nodes. In Blood and its Disorders ed. Hardisty, R. M. & Weatherall, D. J. Blackwell.

Foster, M., Fell, L., Poeklington, T., Akinsete, F., Dawson, A., Hutchison, J. M. S. & Mallard, J. R. (1977). Electron spin resonance as a useful technique in the management of Hodgkin's disease. Clinical Radiology, 28, 15-22.

Hrgoveic, M., Tessmer, C. F., Brown, B. W., Wilbur, J. R., Mumford, D. M., Thomas, F. B., Shullenberger, C. C. & Taylor, G. (1973a). Serum copper studies in the lymphomas and actue leukemias. In Progress in Clinical Cancer, ed. Ariel, I. M. Vol. 5, Grune and Stratton Inc.

Hrgovcic, M., Tessmer, C. F., Thomas, F. B., Ong, P. S., Gamble, J. F. & Shullenberger, C, C. (1973b). Serum copper observations in patients with malignant lymphoma. Cancer, 32, 1512-1524.

Mortazavi, S. H., BanbHashemi, A., Mozafari, M. & Raffi, A. (1972). Value of serum copper measurement in lym- phomas and several other malignancies. Cancer, 29, 1193-1198.

Rappaport, H., Winter, W. J. & Hicks, E. B. (1956). Follicular lymphoma: a re-evaluation of its position in the scheme of malignant lymphoma, based on a survey of 253 cases. Cancer, 9, 792-821.