T shrinkage which Burkitt s lymphoma undergoes

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1 MORPHOLOGY OF BURKITT S LYMPHOMA DURING REGRESSION INDUCED BY CYCLOPHOSPHAMIDE B.. OSUNKOYA, MB, BS, PHD, V. A. NGU, MS, FRCS,? AND FRANCES C. MOTTRAM, BSc* The histologic and cytologic eatures o Burkitt s lymphoma during the irst ew days ollowing the administration o intravenous cyclophosphamide are described. The 3 cases studied showed almost complete disappearance o tumor cells within 72 hours. Surviving tumor cells were enlarged, and some attained a giant size ( gigantoblasts ). There were dierences in the ultimate response o the 3 cases to treatment. One case showed complete regression and total disappearance o tumor cells while in another case, ater an initial good response accompanied by massive lymphocytic iniltration at the tumor site, numerous tumor cells reappeared. All cases showed marked histiocytic prolieration and karyophagocytosis. It is considered that the presence o gigantoblasts may lead to diiculty in histologic diagnosis o biopsies taken rom patients who have been receiving cyclophosphamide. HE RAPID AND DRAMATIC, THOUGH VARIABLE, T shrinkage which Burkitt s lymphoma undergoes during cytotoxic therapy is a wellknown clinical eature o the disease.2-6 Little is known however about changes which take place within the tumor during regression. The sites o completely regressed tumor nodules have the appearance o ocal scars at autopsy, and it has been suggested2*3 that such sites are rarely, i ever, the site o tumor recurrences. The present study is concerned with morphological changes which take place in Burkitt s lymphoma tissue during the irst ew days o regression ollowing cytotoxic chemotherapy. MATERIAL AND METHOD Tumor regression was studied in 3 Nigerian patients suering rom Burkitt s lymphoma, all o whom were treated with intravenous cyclophosphamide. Brie clinical summaries o the cases are shown in Table 1. From University College Hospital, Ibadan, Nigeria. Supported in part by the U.S. National Institutes o Health, and the British Empire Cancer Campaign. Lecturer in Department o Pathology. t Proessor in Department o Surgery. * Visiting Biologist in Department o Pathology. The authors wish to thank the Medical Illustration Unit, U.C.H., Ibadan, or preparation o photomicrographs. Received or publication August 28, Biopsies: Pretreatment surgical biopsy was taken rom each case. Needle aspirates o tumor were obtained at 24, 48, 72, and 96 hours ater the irst dose o drug was given. On the ith day, another surgical biopsy was taken. A inal needle aspirate was obtained rom the tumor site 13 days ater commencement o therapy. Processing o biopsies: Processing o specimens was undertaken immediately ater biopsy. Each surgical biopsy was divided into 3 portions. One portion was ixed in 1% ormol-saline or histologic sections. The second portion was used in preparing touch (imprint) smears. The third portion was minced in a pool o TC 199, and a drop o the dispersed cell suspension was examined under the phase contrast microscope. The remaining cell suspension was centriugated, and smears were made rom the cell deposit. The smears were prepared rom needle aspirates and a drop examined by phase microscopy. Smears were stained by May-Griinwald-Giemsa and Unna-Pappenheim (methyl green-pyronine) methods. Formalin-ixed surgical biopsies were embedded in parain and sections were stained with hematoxylin and eosin. RESULTS Histology: The 3 cases showed dierences in the degree o disappearance o tumor cells.

2 56 CANCER March 197 VOl. 25 TABLE 1. Clinical Data o the 3 Cases o Burkitt s Lymphoma Studied Dose o Case no. Hosp. no. Sex Age Site o tumor cyclophosphamide Schedule Response FOIIOW-UP 1 F. N. F 9 Rt. maxilla 6 mg/kg iv Over 24 hours Complete Recurrence ater (& ovaries) regression 3 months, but regressed again ater treatment. Alive and well 1 yr. 4 months. 2 J. S. Jl 9 Lt. orbit & 4 mg/kg iv Single dose Very slight Tumor increased maxilla transient in 1 month. 1 mg/kg iv Single dose regression Patient lost to on 7th day ollow-up. 3 T.. M 1 Rt. maxilla 6 mg/kg iv Over 24 hours Marked Regression main regression tained when last seen (2 months ater treatment). In case 1, there was complete disappearance o tumor cells within 5 days. The tumor was replaced by large numbers o histiocytes, scattered small lymphocytes, and small clumps o necrotic cells (Figs. 1,Z). In case 2, however, reduction in the number o tumor cells was slight. Many tumor cells were present in the ith-day section. Histiocytic prolieration was evident. Case 3 showed marked but incomplete re- FIG. 1. Typical histologic appearance o Burkitt s lymphoma beore treatment. A starry sky eect is produced by large pale histiocytes scattered between masses o closely-packed hyperchromatic primitive lymphoid ( blast ) cells (H and E, ~4).

3 No. 3 REGRESSION OF BURKITT S LYMPHOMA * Osunkoya el al. 57 FIG. 2. Burkitt s lymphoma 5 days ater intravenous cyclophosphamide (case 1). There was good response to treatment as indicated by complete disappearance o blast cells. Tumor consists o prolierating oamy histiocytes some o which show phagocytosis o pyknotic cells. A ew small lymphocytes are also present (H and E, ~4). duction in the number o tumor cells. Histiocytic prolieration was also quite marked. Cytology: The daily needle aspirates rom the tumors yielded adequate material or sequential study o cytologic changes during the irst ew days o treatment. These changes are depicted in Table 2. The 3 cases showed similar changes during the irst 48 hours. There was evidence o necrosis o some tumor cells within the irst 24 hours. At 48 hours, appreciably ewer tumor cells were surviving, and almost all o these showed increase in cell-size and depletion o cytoplasmic lipid granules. Ater 48 hours, the 3 cases showed dierent cytologic eatures. In case 1, there was complete disappearance o typical tumor cells ater 72 hours. The ew surviving cells were large. Lymphocytes were present in small numbers but there was no granulocytic iniltration. The picture remained more or less the same, with gradual total disappearance o tumor cells; by the thirteenth day, no tumor cells were seen. In case 2, no typical tumor cells were seen, but many giant primitive cells appeared at 72 hours. The cells were o enormous size (3-5 p in diameter) and had a large olded nucleus with multiple prominent large nucleoli. Like typical Burkitt s lymphoma cells, they had cytoplasmic lipid granules and showed intense cytoplasmic pyroninophilia, but, unlike typical Burkitt s lymphoma cells, they were actively motile (Figs. 3, 4). Some were seen in mitosis. Other striking eatures noted at 72 hours were granulocytic and massive lymphocytic iniltration. Even more striking was the reappearance 24 hours later o many typical tumor cells which persisted till the thirteenth day. The giant primitive cells disappeared at 5 days. Cytologic eatures observed in case 3 were intermediate between those described in cases 1 and 2. There was persistence o small numbers o normal tumor cells throughout the period o study. Occasional giant tumor cells were seen at 72 hours. Granulocytic and lymphocytic iniltration were also noted, although the latter was much less in degree than in case 2.

4 58 CANCER March 197 Vol. 25 TABLE 2. Types o Cells Present in Tumor Beore and Ater Cytotoxic Chemotherapy Posttreatment Pre- Cell types treatment 24 hrs. 48 hrs. 72 hrs. 96 hrs. 5 days 13 days Case 1 Normal size Small lymphocytes Polymorphs Eosi nophi Is Histiocytes Pyknotic cells Large size Giant size Case 2 Normal size Large size Giant size Small lymphocytes Polymorphs Eosinophils Histiocytes Pyknotic cells Case 3 Normal size Large size Giant size Small lymphocytes Polymorphs Eosinophils Histiocytes Pyknotic cells None. Occasional. Few. Present in good numbers. Present in large numbers. Apart rom histiocytic prolieration at the tumor site, other eatures shared in common by the 3 cases were persistence o pyroninophilia in surviving tumor cells, marked karyophagocytosis by histiocytes, and presence o numerous extracellular granular lipid material. DISCUSSION The clinical response o any malignant tumor to cytotoxic chemotherapy depends on a combination o several actors. Such actors include eective drug concentration at tumor site, sensitivity o tumor cells to the drug, and the rate o tumor growth (replication o tumor cells). The part which additional actors, such as host deense mechanisms and possible malignant transormation o normal cells, also contribute to ultimate clinical ie- sponse is diicult to assess, but all are pertinent to the outcome o chemotherapy in individual patients. The 3 cases studied shared in common the eatures o initial rapid and almost complete disappearance o tumor cells rom the tumor site with concomitant prolieration o histiocytes. The rapid elimination o the tumor cells occurred within 72 hours and was accompanied by slight enlargement o most surviving cells 48 hours ater commencement o therapy. The dierences subsequently observed in the cytologic eatures o the 3 cases highlight 2 points. First, the established clinical impression o variability in response o individual patients to cytotoxic chemotherapy is relected in the 2 extremes depicted by cases 1 and 2. The ormer showed complete and durable disappearance o tumor cells, whereas the latter showed apparent persis-

5 No. 3 REGRESSION OF BURKITT S LYMPHOMA Oszinkoya et al. 59 tence o many tumor cells. Second, the role, i any, which massive lymphocytic iniltration may play in the reappearance o tumor cells is not clear. The absence o such massive lymphocytic iniltration in case 1 which showed complete regression would suggest that there may be some interrelationship between lymphocytic iniltration during regression and ultimate clinical response in individual cases. Although contrary to accepted concepts o grat rejection, the observed phenomenon may be due to a peculiar immunologic reaction resulting in the blastogenic transormation o iniltrating lymphocytes at the tumor site. However, the relatively poor response o case 2 may be due simply to the administration o a lower dose o drug. The appearance at the tumor site o giant primitive cells ( gigantoblasts ), particularly in case 2, would appear to be an exaggeration o the increase in cell size which cyclophosphamide tends to induce on the tumor cells. Similar cells have been described in the bone marrow o acute leukemia patients receiving rubidomycin.1 Although it is most likely that these cells are derived rom the malignant tumor cells, their exact origin, role, and signiicance is debatable. O practical importance is the diiculty which the presence o such cells may cause in the histopathologic diagnosis o biopsy or necropsy specimens rom Burkitt s lymphoma patients who have already received some chemotherapy. The presence o the giant cells may cause conusion with Hodgkin s lymphoma and rhabdomyosarcoma, or lead to the diagnosis o atypical Burkitt s lymphoma. FIG. 3 (top). Freshly dispersed Burkitt s lymphoma cells beore treatment. One small lymphocyte (center) is shown surrounded by 8 nonmotile blast cells, case 2 (Phase contrast, ~45). FIG. 4 (bottom). Giant tumor cell ( gigantoblast ) in needle aspirate rom Burkitt s lymphoma 72 hours ater cyclophosphamide therapy (case 2). Note large pale nucleus, prominent nucleoli, and several lipid granules. Photomicrograph was taken while cell was moving slowly rom let to right and pushing red blood cells in ront o it (Phase contrast, ~45). REFERENCES 1. Bernard, J.: Acute leukemia treatment. Cancer Res , Burkitt, D. P.: Chemotherapy o jaw tumours. In Treatment o Burkitt s Tumour, J. H. Burchenal and D. Burkitt, eds., Berlin, Springer-Verlag, 1967; pp Burkitt, D. P., Hutt, M. S. R., and Wright, D. H.: The Arican lymphoma: preliminary observations on response to therapy. Cancer 18:399-41, Cliord, P., Singh, S., Stjemsward, J., and Klein, K.: Long-term survival o patients with Burkitt s lymphoma: an assessment o treatment and other actors which may relate to survival. Cancer Res. 27: , Ngu, V. A.: Clinical experience in the therapy o Burkitt tumour. In Treatment o Burkitt s tumour, J. H. Burchenal and D. Burkitt, eds., Berlin, Springer- Verlag, 1967; pp