Epstein-Barr Virus-Positivity in Tumor has no Correlation with the Clinical Outcomes of Patients with Angioimmunoblastic T-cell Lymphoma

Patients

Over a period of 8 years (from August 1998 to July 2005), 70 AILT cases from 8 medical centers were identified based on the histological and immunohistochemical criteria with using the REAL and/or WHO classifications. We enrolled 27 patients who had additional tissue slides for EBV in situ hybridization (ISH). Ten patients' BM slides were also analyzed in this study. Eight patients had their cryopreserved BM mononuclear cells for performing EBV DNA PCR.

All the patients were staged according to the Ann Arbor staging system. The complete staging procedures included physical examination, chest X-ray, complete blood cell counts, blood biochemistry, computed tomography of the thorax, abdomen and pelvis, and BM aspiration and biopsy. The clinical and laboratory records were reviewed for all the patients.

Treatment

The chemotherapy regimens included CHOP (cyclophosphamide, Hydroxydaunomycin, vincristin and prednisone), CHOP-E (etoposide) or ESHAP (etoposide, solumedrol, cytarabine and cisplatin).

ISH for EBV in the tumor and BM biopsy12)

We performed EBV ISH on 27 tumor specimens and 10 BM biopsy specimens. The paraffin-embedded sections of the biopsy specimens were deparaffinized with xylene, and this was followed by treatment with proteinase K. The sections were then hybridized with fluorescein isothiocynanate-conjugeted EBV oligonucleotides (Novocastra, Newcastle, U.K.) that were complementary to the nuclear RNA protein of the EBER1 and EBER2 genes. Positive labeling was identified only when the cells showed nuclear staining with EBV oligonucleotide. As negative controls, we used EBV negative lymphoid tissues and a hybridization mixture without the EBV oligonucleotides. As the internal positive control, the tumor and BM slides of patient with NK-cell leukemia were used.

Sample DNA preparation and EBV PCR11)

The BM mononuclear cells from 8 patients were studied by performing EBV PCR. EBV DNA was extracted from the patients' BM aspiration samples (frozen monocytes) with using the QIAmp DNA Blood Mini Kit (Qiagen, USA). Approximately 100ng of DNA was used for PCR reaction. The PCR primer sequences were 5'-GACGAGGGGCCAGGTACAGG-3' and 3'-GCAGCCAATGCAACTTGGACGTTTTAGG-5', respectively. The PCR reaction was performed using the primer set (Maxim biotech, Inc, USA). The PCR mixture contained 250 µL of the pre-mixed primers and 750 µL of optimized PCR buffer. The PCR was preformed in a total volume of 50 µL, which contained 10.0 L of DNA, 39.8 L of the PCR mixture and 0.2 µL of Taq DNA polymerase. The amplification reaction was performed for 37 cycles under the following conditions: initially, 96℃ for 1min for activation (one cycle), followed by a denaturation phase at 94℃ for 1min, an annealing phase at 5 8℃ for 1min and an extension phase at 72℃ for 1min (35 cycles). Another 10 min extension phase at 72℃ was performed at the end of each round of PCR. Ten microliters of the PCR products were subjected to electrophoresis (100V, 25min) on a 2% agarose gel, and the products were stained with ethidium bromide. The presence or absence of specific PCR targets was observed under UV illumination.

Statistical analysis

Overall survival was measured from the time of diagnosis to death or to the last follow-up. The survival curves were calculated using the Kaplan-Meier method and they were then compared using the log-rank test. Other clinical factors were compared using chi-square tests or t-tests. Differences were considered statistically significant if the p value was <0.05. All statistical analyses were performed using the SPSS for Windows 13.0 statistical package (SPSS Inc., Chicago, IL).

Clinical features

One patient among the 27 AILT patients refused any further staging work-up. The clinicopathologic data of the 27 patients are summarized in Table 1. This series included 19 men and 8 women with ages from 17 to 75 (median age: 57 years-old). All the patients except one had stage III or IV disease. Anemia was detected at diagnosis in 10 patients; two of them had autoimmune hemolytic anemia. Twenty five patients received chemotherapy, including 23 patients who received CHOP, 1 patient who received CHOP-E and 1 patient who received ESHAP, while 2 patients received only supportive care.

Table 1

Clinicopathologic findings of the 27 patients with angioimmunoblastic T-cell lymphoma

EBV ISH in the tumor and its clinical impact

Among the tumor specimens obtained at the time of the initial diagnosis from the 27 AILT patients, 10 were positive for EBV ISH. The correlations of the clinical status, such as the international prognostic index (IPI) score, age, stage and other laboratory findings, with the EBV status of tumor are summarized in Table 2. Only CD20 negativity in the tumor was significant correlated with EBV-positivity in the tumor (p=0.035).

Table 2

Correlation between tumor EBV positivity on ISH and the clinico-laboratory findings

The median overall survival and progression-free survival of the 25 treated patients were 48.9 months (95% CI: 18.6~79.2 months) and 32.3 months (95% CI: 11.5~53.1 months) (Figure 1A). There were no significant correlations between EBV-positivity in tumor and the overall or progression-free survival.

Figure 1

Kaplan-Meier curves for overall (solid line) and progression free survival (dotted line) for the 25 treated patients with angioimmunoblastic T-cell lymphoma (A). Comparison of the overall survival (B) and progression-free survival (C) between the patients with bone marrow involvement (solid line) and those without BM involvement (dotted line)

BM involvement by Hematoxylin-eosin staining, ISH or PCR

In 13 of the 26 AILT patients, gross tumor involvement was recognized in the initial BM sample by hematoxylin-eosin staining (H-E stain). None of the 10 patients who had additional BM slides available revealed EBV-positivity on BM ISH. Among these 10 patients, 3 patients showed BM involvement by H-E staining, but this was not identified on EBV ISH (Figure 2) or PCR. Yet one patient among the 5 patients without BM involvement showed a positive band on PCR (Figure 3). BM involvement by H-E staining was not related with the clinical status as defined by the IPI, age, stage and laboratory findings (Table 3). The overall survival and progression-free survival for the treated patients among the 13 patients with BM involvement according to H-E staining, were longer than those for the patients without BM involvement (p=0.054 and p=0.005, respectively; Figure 1B, 1C).

Figure 2

In situ hybridization for EBV in the bone marrow biopsies. Positive staining for EBV in a case with NK/T-cell leukemia (A), and negative staining in a patient with angioimmunoblastic T-cell lymphoma (B)

Figure 3

PCR analysis showed the EBV-specific DNA sequences. The EBV DNA was detected in the BM mononuclear cells of one patient (white arrow in line 8). Lines 1~8 are for the BM samples of the angioimmunoblastic T-cell lymphoma cases, lines 9~10 are for the BM samples of NK/T-cell lymphoma cases, and line 11 is for the positive control in the EBV PCR kit (240 base pairs)

Table 3

Correlation between BM involvement and the clinicolaboratory findings

Clinical factors affecting survivals

On univariate analysis, older age, male gender, pulmonary parenchymal involvement, the presence of edema and negative BM were adverse factors for progression-free survival and overall survival. Male gender (p=0.041), pulmonary parenchymal involvement (p=0.039) and negative BM (p=0.042) all significantly shortened the progression-free survival on multivariate analysis. Among the significant factors for overall survival, male gender (p=0.078), pulmonary parenchymal involvement (p=0.044) and negative BM (p=0.080) also had modest significance on multivariate analysis.