Eur J Haematol 1987:38:437-441

Key words: labelling index - survival - non-Hodgkin’s lymphoma

Survival following combination chemotherapy in advanced high grade non-Hodgkin’s lymphomas: Relation to proliferative activity of the lymphoma cells

Lars Brandt & Hgkan Olsson

Department of Oncology, University Hospital, Lund, Sweden

In 18 untreated adult patients (median age 62.5 yr) with advanced non-Hodgkin’s lymphoma of unfavourable histology, thymidine labelling indices (LIs) of the lympho- ma cells were assessed. The patients were treated with combination chemotherapy and have been followed for 29-60 (median 52) months or until death. The survival curve had a steep fall during the first 2 yr. Between 2-5 yr after treatment there was a flattening of the curve and survival seemed to be similar to the survival expected for a Swedish population matched for age and sex. 11 patients died with 2 yr and 7 patients have survived for a longer period. Age, histopathologic classification and clinical stages were comparable in short-term and long-term survivors and treatment was not more aggressive for the long-term survivors. The LIs were significantly higher (median 8.2) in short-term survivors than in the long-term survivors (median 1.4). Long-term survival following combination chemotherapy of advanced NHL of unfavourable histology seems to be achieved mainly in patients with a low proliferative activity of the lymphoma cells. It is suggested that in NHL a high proliferative activity may facilitate the generation of new mutants and that some of these are spontaneously resistant to various chemotherapeutic drugs.

Accepted f o r publicution Junuury 26, 1987

Combination chemotherapy may induce long- lasting remissions in many patients with ad- vanced non-Hodgkin’s lymphomas (NHL) of histologically unfavourable type (1, 2) and the introduction of new multidrug regimens has increased the probability of long-term survival (3, 4). It is largely unknown why some patients with advanced aggressive lymphomas may achieve long-term survival whereas others with a clinically and histologically similar dis-

ease treated in the same way will die from progressive disease within a relatively short period. Using various methods it has been documented that the proliferative activity of NHL cells has great prognostic importance (5, 6, 7 , 8, 9). In the present study the rate of cell proliferation at diagnosis of advanced his- tologically aggressive lymphomas has been related to the survival following combination chemotherapy.

438 BRANDT & OLSSON

Material and met hods Patients. 18 previously untreated patients aged 33-80 (median 62.5) yr with NHL of histologically unfa- vourable type and clinical stage 11-IV have been studied. All patients had nodal disease. Clinical data are given in Table 1. All patients have been followed for a minimum of 29 months or until death. The median time of observa- tion for patients alive at the time of this report was 52 months. Follow-up was performed at our hospital or in close collaboration with other hospitals in our region.

Determination of labelling index (LI). Material from the lymphomas was obtained through aspiration using an empty 10 ml syringe with a needle of 0.7 mm diameter. Usually, 3-4 aspirations were performed from the same tumour. The aspirate was ejected into 1 ml of heparinized autochtonous plasma. Contaminant phagocytizing cells were removed by incubating the cell suspension with iron filings at + 37" C under rotation for 30 min and removing the iron and phagocytes with a magnet. The viability of the lymphoid cells following this preparation is 90 * 8 070

(SD) according to the trypan blue exclusion test. Tritiated thymidine (specific activity 24 Ci/mmol,

Radiochemical Center, Amersham, U.K.) was added to a concentration of 1 pCi/ml and after 60 min at + 37" C cytocentrifuge preparations were prepared. The cytocentrifuge slides were coated with auto- radiographic emulsion K2 (Ilford Ltd, Ilford, U.K.), exposed for 8 d, developed and stained with MGG at

pH 5.8. The percentage of labelled lymphoid cells (LI) was determined through examination of 500 cells.

Staging procedures. Clinical staging was performed according to the Ann Arbor classification (10). The staging procedures have been described previously ( 5 ) and did not include staging laparotomy.

Treatment. The initial chemotherapy regimens were CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), CHOP-M (CHOP + methotrexate), MEV (methotrexate, cyclophosphamide, vincristine) or COMLA (cyclophosphamide, vincristine, methotrex- ate, leucovorin, cytosine arabinoside). For those patients not responding to MEV the regimen was changed to CHOP. Patients not responding to CHOP or CHOP-M were treated with COMLA.

Survival. The method of Kaplan & Meier (1 1) was used to obtain a survival curve for the patients. A curve demonstrating the expected survival in a Swedish popu- lation with a sex and age distribution corresponding to the patient group was estimated according to Haku- linen & Abeywickrama (12).

Resu I ts The survival for each patient is given in Table 1 and the survival curve for the whole material in

TABLE 1 Clinical data and LIs f o r I 1 patients (no. 1-11) who died within 2 yr and 7 patients (no. 12-18) who have survived this period

Age, Sex Patient no. Histology Stage Initial treatment LI Survival

(months)

1 2 3 4 5 6 7 8 9

10 11

12 13 14 15 16 17 18

65 M 72 M 49 M 59 F 52 M 80 M 56 M 53 M 53 M 70 M 69 M

68 F 33 F 66 M 73 M 46 F 73 M 57 F

LB LB LB IB IB CB CB

CB-CC Diff. CC Large cell

Hi Hi

LB IB IB CB

CB-CC Diff. CB-CC Diff.

Hi

IV 111 IV 111 IV 111 IV I1

IV IV 111

I11 111 I11 IV IV IV IV

CHOP-M COMLA CHOP-M CHOP MEV MEV CHOP-M CHOP CHOP-M MEV CHOP

CHOP-M CHOP MEV CHOP MEV MEV CHOP

19 20

8 23

1 17 8

18 11 24 11

29 + 52 + 60 + 49 + 53 60 + 34 +

8.2 7.4

19.8 13.8 18.4 2.4

21.4 4.4

11.2 5.0 8.0

7.2 4.2 0.8 1.8 0.4 1.4 0.2

LB = Lymphohlastic; IB = Immunoblastic; CB = Centroblastic; CC = Centrocytic; Hi = True histiocytic; Diff. = Diffuse.

PROLIFERATION AND SURVIVAL IN NHL 439

- 20-

15-

5 10-

5 -

0.1 1 0-I

Survival L 2 y 0 1 2 3 4 5

A

0

8

r

A

0

A

a A t

> 2 y .

YEARS CE-CC diff. A I B

0 CC large cell 0 LEI

A CB + True hist

Figure 1. Survival of 18 patients with advanced diffuse NHL of unfavourable histology (lower curve) and estimated survival of an age- and sex-matched Swedish population (upper curve).

Figure 2. Labelling indices of lymphoma cells in 11 patients who died within 2 yr after diagnosis and in 7 patients who have survived for more than 2 yr.

Figure 1. The shape of the curve appears to be biphasic with a sharp fall during the first 2 yr after diagnosis. Thereafter there is a flattening of the curve.

The median age of the patients who died with- in 2 yr was 59 yr (range 49-80) and for those surviving more than 2 yr it was 66 yr (range 33- 73). Thus, there was no overrepresentation of young patients among the long-term survivors.

The distribution of various histopathological subgroups was comparable in the 11 short-term survivors and the 7 long-term survivors. LB lym- phomas were diagnosed in 3/11 vs. 1/7, IB in 2/11 vs. 2/7, diffuse follicular centre cell lymphomas (CB, CB-CC or CC) in 4/11 vs. 3/7 and true histiocytic lymphoma in 2/11 vs. 117 (Table 1).

The distribution of clinical stages was also similar in short- and long-term survivors. Among those who died within 2 yr stage 111-IV disease

was diagnosed in 10/11 and among the long-term survivors 7/7 had clinical stage 111-IV (Table 1).

The initial treatment of the patients who died within 2 yr was not less active than the treatment of the long-term survivors. Thus 7 out of 11 short-term survivors were initially treated with CHOP or CHOP-M. The corresponding figures for long-term survivors were 4 out of 7. Treat- ment with MEV was given to 3/11 short-term survivors and to 3/7 long-term survivors. COM- LA was initially given to 1 patient with short survival and to none of the long-term survivors.

The median LI of the lymphomas associated with short survival, I 2 yr, was 8.2 and for lymphomas with long-term survival 1.4 (Figure 2). The difference is significant (p = 0.002, Mann-Whitney U-test). Because CB-CC and CC lymphomas may be considered to be of inter- mediate rather than high malignancy grade (13),

440 BRANDT & OLSSON

a comparison of LIs was also performed exclud- ing these lymphomas. The difference between LIs in short-term survivors (median 8.0) and in long-term survivors (median 1.8) is still signifi- cant (p = 0.01).

Discussion Several studies of the effect of combination che- motherapy in advanced NHL of unfavourable histology show a strikingly similar shape of the survival curve; there is a rapidly declining part of the curve during the first 2 yr. Thereafter there is a flattening of the curve representing patients with long-term survival (1, 14, 15, 16). The sur- vival curve for the present group of patients also had this characteristic shape. Although based on a relatively small number of patients, the ob- served survival is therefore probably representa- tive for advanced NHL with unfavourable histol- ogy and initially treated with the combinations of drugs mentioned. This assumption is supported by the very similar shape of the survival curves for 67 patients with generalized NHL of un- favourable histology initially treated with CHOP and for 74 patients treated with MEV reported by Hagberg et a1 (15).

Apparently, the differences in age, clinical stage and histopathologic classifications between the patients who died within 2 yr and the long- term survivors were negligible and the initial treatment was not less active for the patients with a short survival than for those who survived for more than 2 yr. In contrast to these similarities between short-term and long-term survivors, the LIs of the lymphoma cells were significantly higher in the patients who died within 2 yr than in the long-term survivors. Thus long-term sur- vival following combination chemotherapy in advanced NHL of unfavourable histology seems to be achieved in a subgroup of patients charac- terized by a low proliferative activity of the tumour cells. Similar results were recently re- ported by Bauer et al (17) using flow cytometry for the assessment of the proliferative activity in diffuse large cell lymphomas.

In malignant tumours, mutants resistant to

chemotherapeutic drug may arise early and spon- taneously (18) and a high proliferative activity may increase the risk of such mutations (19). In untreated NHL with high LIs, larger numbers of clonal chromosome aberrations have been found than in lymphomas with lower LIs (20). These results suggest that a high proliferative activity in NHL is associated with frequent spontaneous changes in the genetic material. It is possible that some of these changes may cause resistance to various chemotherapeutic drugs and that some lymphoma cells therefore continue to proliferate despite treatment. Lymphomas with low LIs may be less prone to develop new mutants and the number of cells with spontaneous drug resistance may be relatively low at diagnosis. Combination chemotherapy should be more effective in such lymphomas. Moreover, if some drug resistant cells are present also in these lymphomas, a low proliferative activity is compatible with a late relapse of symptoms.

In advanced, histologically unfavourable NHL the proportion of long-term survivors may be longer than in the present series due to a more aggressive multidrug treatment (3, 4). Neverthe- less, a considerable number of the patients will die within a short period of time due to treat- ment failure. According to the present results new therapeutic efforts are especially important for NHL patients with a high proliferative activity of the lymphoma cells.

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Correspondence to: Dr. L. Brandt Department of Oncology University Hospital S-221 85 Lund Sweden