A 36-year-old Caucasian Portuguese male was admitted to the hospital in October 2010, with fever (38 C), pallor, weakness, and jaundice. His medical history revealed chronic alcohol abuse. The physical examination showed hepatosplenomegaly and there were no palpable superficial lymph nodes. Blood counts demonstrated pancytopenia: white blood cells 1.80 x 109/L, neutrophils 0.69 x 109/L, lymphocytes 0.64 x 109/L (no evidence for morphologically abnormal cells), platelets 46 x 109/L, and hemoglobin 6.6 g/dl. Biochemistry analysis revealed markedly increased serum lactate dehydrogenase levels (LDH) 2815 IU/L (135-225 IU/L) and abnormal liver tests: total bilirubin (TB) 1.5 mg/dl (<1.2 mg/dL), aspartate transaminase (AST) 124 IU/L (10-37 IU/L), alanine transaminase (ALT) 62 IU/L (10-31 IU/L) and gama-glutamil transferase (GGT) 107 IU/L (10-49 IU/L); coagulation tests were normal. The abdominal computerized tomography (CT) scan confirmed the hepatosplenomegaly (liver and spleen longitudinal axis of 209 cm and 158 cm, resp.) and did not show other abnormalities (Figure 1(a)).
The bone marrow (BM) aspirate had more than 80% of morphologically immature cells, with a pale or slightly basophilic cytoplasm sometimes with fine azurophilic granules and a nucleus with an immature chromatin, with one or two distinct nucleoli, and the first hypothesis for the diagnosis was that of an acute leukemia (Figure 2).
Flow cytometry (FCM) analysis of the BM aspirate cells using the EuroFlow lymphoid screening tube (LST) and antibody panel for NK cell chronic lymphoproliferative diseases (NK-CLPD), complemented with other cell surface markers, showed that the neoplastic cells were positive for CD45 (high), CD2, CD26, CD38, CD94 (high), and HLA-DR (high, heterogeneous) antigens, and negative for surface CD3, TCR, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD56, CD57, and CD161, as well as for cytoplasmic CD3; in addition, they express intracellular granzyme B and perforin (Figure 3). B cell (CD19, CD20, and CD79a), myeloid (CD13, CD14, CD15, CD64, CD65, and myeloperoxidase), immature (CD34, TdT), and dendritic (CD123) cell associated markers were all negative (data not shown). In the PB there were 10% of phenotypically abnormal CD45+high, CD2+, CD16+low, CD94+high, and HLA-DR+ NK cells, 19% of which expressed dimly and heterogeneously the CD56 molecule, at levels that were much lower than those observed in normal PB NK cells (data not shown). Flow cytometry propidium iodide based NK cell DNA studies revealed a diploid cell DNA content (DNA index 1.02) and a high proliferative rate (G0/G1 phases: 67.4%; S phase: 31.8%; G2/M phases: 0.8%). Polymerase Chain Reaction (PCR) TCRG gene rearrangements studies were consistent with a germ-line configuration.
The BM biopsy revealed a hypercellular marrow infiltrated by CD45+, cytoplasmic CD3epsilon+, EBER+ and CD56, CD34, CD20, CD68, myeloperoxidase, and lysozyme-negative atypical lymphoid cells, interpreted individually given rise to the possibility of an immature EBV+ T cell leukemia (Figure 4).
The BM karyotype with chromosome banding analysis showed complex aberrancies: 46,add(X)(q27),-Y,i(7q), +8,add(17)(p13),add(19)(q13) (Figure 5).
Infection markers were negative for human immunodeficiency virus types 1 and 2, viral hepatitis B and C, and human T cell lymphotropic virus types 1 and 2. The whole blood EBV load, detected by quantitative PCR for viral DNA, was of 57 x 105 copies/ml.
As no neurologic symptoms were present, central nervous system involvement was not evaluated.
In view of these findings and according to the WHO classification of neoplasms of the hematopoietic and lymphoid tissues, the final diagnosis was an EBV+ ANKL, Ann Arbor stage IVB, high risk NKIPI (NK/T cell lymphoma international prognostic index), ECOG status 4.
The patient was treated with a combination of gemcitabine (1 g/m2 days 1, 8, and 15), cisplatin (100 mg/m2 day 15), and methylprednisolone (1 g days 1-5) (GEM-P) repeated every 28 days.
The first BM reevaluation, performed on day 23 after the first chemotherapy course, revealed 0.5% of phenotypically abnormal (CD2+, CD56-, CD94+, and HLA-DR+) NK cells and a normal karyotype (46, XY). In addition, there was a marked decrease in the EBV load in the PB to 4 x 105 (day 14) persisting at 6.6 x 105 (day 21) copies/ml. Also, the BM aspirate showed 4.1 x 105 EBV copies/ml. These changes correlated with an improvement in clinical status and blood analysis: WBC 5.14 x 109/L, neutrophils 2.78 x 109/L, lymphocytes 0.92 x 109/L, platelets 45 x 109/L, hemoglobin 8.9 g/dl (not dependent on blood or platelet transfusions), TB 1.10 mg/dl, AST 37 IU/L, ALT 39 IU/L, GGT 71 IU/L, and LDH 425 IU/L. A second evaluation of the BM aspirate after the second GEM-P showed only 0.09% of neoplastic NK cells. At that time, the BM biopsy provided evidence for a partial hematopoietic recovery, although intrasinusoidal niches of neoplastic NK cells were still observed; in addition, the BM karyotype was again abnormal, with different chromosomal aberrancies: 77,add(X)(q27),-Y,i(7)(q10),inc[5]/46,XY[15]. Similarly, an increase of the EBV load was observed (42 x 105 copies/ml). Considering the refractoriness to GEM-P, it was decided to change the chemotherapy.
Nearly one month later, when the alternative schema was being discussed, the patient was admitted to urgency reporting loss of vision. At that time, the ophthalmologic and neurological examination revealed an almost total bilateral decline in visual acuity, markedly decreased pupil reflexes, right retinal detachment, and impaired right eye abduction compatible of palsy of the 6th right cranial nerve. Blood analyses were as follows: WBC 4.78 x 109/L, neutrophils 2.14 x 109/L, lymphocytes 1.69 x 109/L, and platelets 155 x 109/L, depending on regular blood transfusions, TB 0.88 mg/dl, AST 34 U/L, ALT 29 U/L, and LDH 429 IU/L. Abnormal NK cells had increased in blood (1.9% by FCM) and there was a marked increment on the EBV load (89 x 105 copies/ml) and on the LDH (1587 IU/L). Head CT revealed a thickening of the right optic nerve, compatible with neoplastic infiltration, without evidence for other abnormalities in brain tissue and structures. Five days later he also developed dysphonia, dysphagia, impaired abduction of the left eye, and left eyelid ptosis and the head MRI showed a thickening of the left lateral rectus muscle (Figure 1(b)). At that time, there was clinical evidence of palsy of multiple cranial nerves (bilateral palsy of the 2nd and 8th left and right cranial pares, left palsy of the 3rd and 5th left cranial pares, and right palsy of the 6th and 7th right cranial pares). The CSF had 262 cells/mul with 88% of atypical mononuclear cells, being consistent with leukemia meningitis. Immunophenotypic characterization was not performed, as on the day of the lumbar puncture flow cytometry was not available at our institution; EBV DNA analysis was not possible due to insufficient sample volume. At this point, no further chemotherapy was administered, and the strategy from here on was best supportive care. Death occurred a few days later, 107 days after the diagnosis.