SHORT REPORT

Alpha interferon gene deletions in post-transplant lymphoma

ANGELA WOO D, BRIAN ANGUS,* PATRICK KEST EVAN,† JOHN DARK,† GIANCARLO NOTARIANNI, SUE MILLER,MARTIN HOWARD,‡ STEVE PRO CTOR AND PETE MIDDLETO N University Department of Haematology, Medical School,and *Department of Pathology, Royal Victoria Infirmary, and †Department of Haematology, Department of Cardiology,Freeman Hospital, Newcastle upon Tyne, and ‡Department of Haematology, York District Hospital, York

Received 11 November 1996; accepted for publication 11 June 1997

Summary. Post-transplant lymphoproliferative disorder(PTLD) is a well-recognized complication of organ transplantand has been associated with high mortality using con-ventional chemotherapy. We have investigated 11 cases ofPTLD for alterations to the interferon alpha (IFNA) and p16genes on chromosome 9p using archival material. 4/9 (44%)cases had deletions of the IFNA genes, in contrast to 1/59

(1.7%) cases of intermediate/high-grade de novo NHL drawnfrom the same geographical region. PTLD may thereforerepresent a distinct NHL subgroup exhibiting distinct genepathology.

Keywords: post-transplant lymphoma, interferon alpha,p16.

Post-transplant lymphoproliferative disorder (PTLD) is awell-recognized complication of organ transplantation, andalthough a heterogenous group of disorders, most are B cellin phenotype and EBV associated. Using present conven-tional therapeutic regimens the long-term disease-freesurvival is <30%. The use of IFNa in post-transplantlymphomas is currently being assessed with early indicationsthat EBV-positive tumours respond well to this cytokine(Liebowitz et al, 1996).

Deletions of the chromosome region 9p21, particularlythat region involving the tumour suppressor genes p15 andp16, have been reported in up to 15% of diffuse large celllymphomas, with no deletions seen in intermediate/low-grade histologies, indicating that deletions are associatedwith aggressive disease. The interferon alpha (IFNA) genecluster maps close to the p15 and p16 genes and isfrequently co-deleted in a number of different neoplasmsincluding high-grade NHL and ALL.

We have assessed gene pathology (IFNA, p16 and c-MYC)and EBV status of 11 cases of PTLD for whom outcome wasknown. Archival material in the form of paraffin-embeddedsections (eight cases) or material supported on storedunstained microscope slides was the only material available.Nine cases were diagnosed as high-grade NHL using the Kielclassification and two as intermediate-grade NHL. Clinicaldetails are given in Table I. Patients 2, 5, 9 and 11 were stillalive at time of writing.

Although the data set has omissions which result fromworking with such archival material, some clear character-istics emerge (Table I). 4/9 (44%) of cases of PTLD showedloss of IFNA genes (in two cases a suitably reliable DNApreparation could not be obtained). This contrasts with ourfinding of one deletion in 59 cases (1.7%) of a previouslycharacterized panel of intermediate and high-grade de novoNHL drawn from the same geographical region (North ofEngland) (Levasseur et al, 1995). Those cases of PTLD withIFNA gene loss were also EBV positive. Where determined,the p16 genes were also deleted along with the IFNA genes.(The PCR method for the p16 gene was found to be lessrobust than the PCR for IFNA genes and performed poorly onarchival material.) One case (case 10) had a point mutationin the c-MYC gene intron1/exon1 control region (c-MYCgene point mutations have been reported in immuno-deficiency-related lymphomas (Bhatia et al, 1994)). Thesefindings suggest that PTLD may represent a distinctpathological entity with a high incidence of 9p deletion.

The incidence of post-transplant lymphoma appeared toincrease with increased immunosuppression. In the centrefrom which these cases were taken, lung transplantrecipients receive anti-thymocyte globulin (ATG) in additionto the standard regimen and the incidence of lymphoma ishigher in this group than in the heart transplant group (theoverall incidence of PTLD in this centre is ,2%). The casesfound to have abnormalities of p16, IFNA or c-MYC genesall received either ATG or OKT3 in addition to standardtherapy of cyclosporin, azathiaprine and prednisolone.Patients were initially treated with a combination of reduced

British Journal of Haematology, 1997, 98, 1002–1003

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Correspondence: Dr Angela Wood, Medical Molecular BiologyGroup, Floor 4, Catherine Cookson Building, The Medical School,Framlington Place, Newcastle upon Tyne NE2 4HH.

immunosupression and high-dose aciclovir. Chemotherapyusing CHOP-based regimens or radiotherapy was given asclinically indicated. As expected, mortality was high;however, none of the surviving patients were found to haveloss of 9p or mutations of c-MYC, suggesting that suchabnormalities may be associated with more aggressivedisease, as in the de novo NHL group. Given the number ofcases of PTLD showing deletion of IFNA genes, it is temptingto speculate that loss of these genes may have a role indisease aetiology and could influence the sensitivity of thetumour population to IFNa therapy. If confirmed, thecharacteristics of PTLD in its response to some therapiesmay therefore reflect the gene pathology of a distinct diseaseentity and not just a virally-driven disease state.

Analysis of further cases of PTLD for p16, p15 and IFNAgene loss is required to confirm these initial findings and toinvestigate any relationship between gene pathology,response to therapy, especially to IFNa, and clinical outcome.Access to fresh or frozen biopsy material would be essentialin this respect given the technical limitations of working withfixed material.

The speculative link between gene pathology andmechanism of action of IFNa therapy is an interestinghypothesis worth pursuing in this distinct disease state.

REFERENCES

Bhatia, K., Spangler, G., Gaidano, G., Hamdy, N., Dalla-Favera, R. &Magrath, I. (1994) Mutations in the coding region of c-myc occur

frequently in aquired immunodeficiency syndrome-associatedlymphomas. Blood, 84, 883–888.

Bradley, J.F., Rothberg, P.G., Ladanyi, M. & Chaganti, R.S.K. (1993)Hypermutation of the MYC gene in diffuse large cell lymphomaswith translocations involving band 8q24. Genes, Chromosomes andCancer, 7, 128–130.

Henco, K., Brosius, J., Fujisawa, A., Fujisawa, J-I., Haynes, J.R.,Hochstadt, J., Kovacic, T., Pasek, M., Shambock, A., Schmid, J.,Todokoro, K., Walchli, M., Nagata, S. & Weissmann, C. (1985)Structural relationship of human interferon alpha genes andpseudogenes. Journal of Molecular Biology, 185, 227–260.

Kamb, A., Gruis, N.A., Weaver-Feldhaus, J., Liu, Q., Harshmen, K.,Tavtigan, S.V., Stockert, E., Day, R.S., III, Johnson, B.E. & Skolnick,M.H. (1994) A cell cycle regulator potentially involved in genesisof many tumor types. Science, 264, 436–440.

Levasseur, M., Middleton, P.G., Angus, B., Proctor, S.J., Norden, J. &Howard, M.R. (1995) c-MYC gene abnormalities in high gradecentroblastic-centrocytic non-Hodgkins lymphoma. Leukemia andLymphoma, 18, 131–136.

Liebowitz, D., Anastasi, J., Hagos, F., LeBeau, M.M. & Olopade, O.I.(1996) Post transplant lymphoproliferative disorders (PTLD):clinicopathologic characterization and response to immuno-modulatory therapy with interferon-alpha. (Abstract). Annals ofOncology, 7, (Suppl. 3), 28.

Lo, Y-M.D., Mezal, W.Z. & Fleming, K.A. (1989) In vitro amplificationof hepatitis B virus sequences from liver tumor DNA and fromparaffin wax embedded tissues using the polymerase chainreaction. Journal of Clinical Pathology, 42, 840–846.

Pringle, J.H., Barker, S. & Warford, A. (1992) Demonstration ofEpstein-Barr virus in tissue sections by in situ hybridisation forviral RNA. Journal of Pathology, 167, (Suppl.), 133A.

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Table I. The results of analyses obtained on the 11 cases described.

Months todiagnosisfrom Site of initial Immuno- Cause of Survival time

Patient Transplant transplant lymphoma Histology supression Chemo death (months) IFNA p16

1 Heart 23 Mediastinum IM C/A/P OKT3 No Cardiac 10 Del. N/A2 Heart 4 Lung IM C/A/P No 88+ N N/A3 Heart 72 Mediastinum IM C/A/P ATG Yes Sepsis 15 Del. Del.4 Heart 70 Gut DLCL C/A/P Yes Lymphoma 3 N N/A5 Heart 4 Disseminated IM C/A/P ATG Yes 30+ N N/A6 Heart 72 Lung IM C/A/P No Cardiac 9 N N/A7 Lung 6 Lung, spleen IM C/A/P ATG N/A Lymphoma PM diagnosis Del. Del.8 Lung 4 Lung CB C/A/P ATG Yes Sepsis 7 Del. N/A9 Lung 3 Lung DLCL C/A/P ATG Yes 48+ N N/A

10* Lung 9 Lung IM C/A/P ATG R/T Lymphoma 2 N/A N/A11 Renal 24 Tonsil CB C/A R/T 36+ N/A N/A

IM, immunoblastic; CB, centroblastic; DLCL, diffuse large cell lymphoma; C/A/P, cyclosporin, azathioprine; prednisolone; ATG, anti-thymocyteglobulin; R/T, radiotherapy; Del., homozygous deletion; N, normal; N/A, no result.

Methods: DNA was extracted from archival material by mechanical removal from the supporting microscope slide or block, de-waxed usingxylene extraction, ethanol extraction, proteinase K digestion and ethanol precipitation essentially as described (Lo et al, 1989). IFNA gene statuswas determined by PCR using primers directed to a conserved region of the IFNA gene family (1161–1450 bp of the IFNA consensus sequence(Henco et al, 1985)), with internal control of primers directed to the b-globin gene (Lo et al, 1989). p16 gene status was determined by the samemethod using primers producing a 142 bp region of exon 2 (Kamb et al, 1994). c-MYC mutation studies were performed for the exon 1/intron 1boundary region and the exon 2 region as described (Bradley et al, 1993; Bhatia et al, 1994). EBV status was determined by in situ hybridizationfor the EBV EBER I and L mRNAs (Novo Castra EBV probe; Pringle et al, 1992).

*c-myc mutation intron 1/exon 1 boundary.