Correspondence

LOW-GRADE GASTRIC MALT LYMPHOMA AS A SECOND MALIGNANCY IN CHRONIC

LYMPHOCYTIC LEUKAEMIA

The association between chronic lymphocytic leukaemia(CLL) and second primary malignancies has been wellknown for decades. Several epidemiological studies havecon®rmed a signi®cantly increased risk of developing secondcancers in CLL patients compared with age- and sex-matched controls with an overall incidence of 8´7±10´2%(Travis et al, 1992; Mauro et al, 1999); both disease-relatedimmunode®ciency and individual predisposing factors havebeen suggested to be involved in the pathogenesis of thesesecond neoplasias. Skin cancers appear to be the mostfrequent type of tumours, with gastrointestinal, lung, kidney,bladder, brain and breast tumours also being rathercommon. A correlation of this risk with previous therapyand age has not been proved (Mauro et al, 1999). Clonaltransformation of CLL in the form of large-cell non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Richter'ssyndrome) can be expected in about 2±3% of patients(Robertson et al, 1993), although this risk appears to behigher in younger patients (5´9%) and not in¯uenced byprevious therapy (Mauro et al, 1999). Some rare cases of denovo large-cell NHL in CLL patients have also been described.We report a case of gastric low-grade MALT NHL complicat-ing B-CLL.

A 72-year-old woman was diagnosed as having B-CLL in1981; the diagnostic criteria recommended by the NationalCancer Institute (Cheson et al, 1988) were applied. Thepatient had always had the features of smouldering CLLdescribed by Montserrat et al (1988) and therefore neverrequired any treatment. In October 1997, she presented withabdominal epigastric pain and non-speci®c dyspeptic symp-toms; after gastroscopic biopsy of ulcerated lesions in theantrum and prepyloric region, a histological diagnosis of B-cell low-grade gastric MALT NHL was made (atypicallymphoid in®ltrates in the lamina propria and lympho-epithelial lesions) and rod-shaped structures consistent witha Helicobacter pylori infection were observed. A [13C]-ureabreath test was positive and a karyotype performed on aparaf®n-embedded gastric tissue section was normal. Gastricin®ltration by CLL was ruled out by immunohistochemicalstudies of the lymphoid cells (CD5±, CD19�, CD3±, CD23±,CD10±). An abdominal computerized tomography (CT) scanshowed no abnormalities and a bone marrow biopsy did notreveal any signs of NHL involvement. Anti-H. pylorieradication therapy with a 1-week course of amoxycillin(1 g b.d.), clarithromycin (500 mg b.d.) and omeprazol(20 mg b.d.) was prescribed. Assessment of a satisfactorytherapeutic response included a negative [13C]-urea breathtest and a gastric biopsy with isolated mild atrophic changes1 and 6 months after the end of treatment respectively.

Eighteen months later, further follow-up including a 6-monthly [13C]-urea breath test and multiple gastroscopicbiopsies has con®rmed remission of the gastric lymphoma.

It is interesting to note that the co-existence of twoclonally different B-cell lymphoid neoplasias in the samepatient (such as B-CLL and multiple myeloma; Quaglino et al,1982) is a rare but possible event; actually, to our knowl-edge, the association between B-cell CLL and gastric low-grade MALT lymphoma had not been previously reported. Inour case, the patient had not received any treatment and herCLL had not shown any signs of active disease fromdiagnosis, which is consistent with prior evidence forabsence of any correlation between the risk of secondcancers and any of those parameters. A differential diagnosiswith gastric involvement by CLL (an uncommon event) byimmunohistochemical and cytogenetic studies is important;this could be potentially expected in aggressive CLL cases(advanced clinical stages, extensive nodal and/or extranodalin®ltration, unfavourable cytogenetic patterns). Furtherstudies will be required to clarify whether the incidence ofgastric MALT lymphomas in B-CLL patients is higher than inthe general population or whether this association is purelycoincidental.

Department of Haematology, C A R L O S AG U I L A R

Hospital General del INSALUD,Paseo de Santa B´rbara,42.002-Soria,Spain.E-mail: caraguilar@excite.com.

REFERENCES

Cheson, B.D., Bennett, J.M., Rai, K.R., Grever, M.R., Kay, N.E.,Schiffer, C.A., Oken, M.M., Keating, M.J., Boldt, D.H., Kempin, S.J.

& Foon, K.A. (1988) Guidelines for clinical protocols for chronic

lymphocytic leukemia. Recommendations of the National Cancer

Institute Sponsored Working Group. American Journal of Hematol-ogy, 29, 152±163.

Mauro, F.R., Foa, R., Giannarelli, D., Cordone, I., Crescenzi, S.,

Pescarmona, E., Sala, R., Cerretti, R. & Mandelli, F. (1999).

Clinical characteristics and outcome of young chronic lymphocy-tic leukemia patients: a single institution study of 204 cases. Blood,

94, 448±454.

Montserrat, E., VinÄ olas, N., Reverter, J.C. & Rozman, C. (1988)Natural history of chronic lymphocytic leukemia: on the

progression and prognosis of early clinical stages. Nouvelle Revue

FrancËaise d'Hematologie, 30, 359±361.

Quaglino, D., Paterlini, P., De Pascuale, A., Cretara, G. & Venturoni,L. (1982) Association of chronic lymphocytic leukemia and

British Journal of Haematology, 2000, 108, 660±663

660 q 2000 Blackwell Science Ltd

multiple myeloma: report of a case and review of the literature.

Haematologica, 67, 576±588.

Robertson, L.E., Pugh, W., O'Brien, S., Kantarjian, H., Hirsch-

Ginsberg, C., Cork, A., McLaughlin, P., Cabanillas, F. & Keating,M.J. (1993) Richter's syndrome: a report on 39 patients. Journal of

Clinical Oncology, 11, 1985±1989.

Travis, L.B., Curtis, R.E., Hankey, B.F. & Fraumeni, Jr, J.F. (1992)

Second cancers in patients with chronic lymphocytic leukemia.

Journal of the National Cancer Institute, 84, 1422±1427.

Keywords: chronic lymphocytic leukaemia, gastric MALTlymphoma, second cancers, Helicobacter pylori.

CIRCADIAN VARIATION OF GRANULOCYTE COLONY-STIMULATING FACTOR LEVELS IN MAN

I read with great interest the paper by Jilma et al (1999). Acircadian rhythm of granulocyte colony-stimulating factor(G-CSF) levels in plasma is of great interest for the under-standing of white cell production.

Interestingly, the curve for G-CSF was almost identical tothe serum erythropoietin (S-Epo) curve that we established(Wide et al, 1989), with the peak in the evening at 20.00 hand the lowest levels in the early morning. In addition, themagnitude of the change was very similar; we found a 60%average difference between peak and nadir compared with54% found by Jilma et al (1999) for G-CSF.

The authors speculate that the diurnal variation incortisol levels may be responsible for the G-CSF rhythm.However, S-Epo levels are not in¯uenced by cortisol levels. Asboth cytokines show the same circadian rhythm, it isinteresting to speculate that some other factor in¯uencesboth S-Epo and plasma G-CSF. There must be someadvantage for the total bone marrow production in the

fact that both major cell-stimulating factors have the samediurnal rhythm.

Department of Haematology, G U N N A R B I RG E G AÊ R D

University Hospital, Uppsala,Sweden

REFERENCES

Jilma, B., Hergovich, N., Stohlawetz, P., Eichler, H.G., Bauer, P. &Wagner, O.F. (1999) Circadian variation of granulocyte colony

stimulation factor levels in man. British Journal of Haematology,

106, 368±370.

Wide, L., Bengtsson, C. & BirgegaÊrd, G. (1989) Circadian rhythm oferythropoietin in human serum. British Journal of Haematology,

72, 85±90.

Keywords: circadian, erythropoietin, G-CSF.

ANGIOGENIC GROWTH FACTORS AND ENDOSTATIN IN NON-HODGKIN'S LYMPHOMA

We read with interest the paper by Bertolini et al (1999)suggesting a prognostic value of pretreatment blood vascularendothelial growth factor (VEGF) levels in patients withlymphoma. Included in this study were 36 patients withmalignant lymphoma of seven histological subtypes withdifferent biological behaviour and various disease stages(Ann Arbor I, II, III and IV). Among them were eight patientswith extranodal involvement. This heterogeneous cohort wastreated with various chemotherapy regimens and, partly, withadditional radiation. Irrespective of the histological type of thelymphomas, progressive disease was observed predominantlyin patients with advanced stage disease, as one may expect.No information was given about the response to treatment,e.g. the quality and the duration of the remissions obtained.Nevertheless, Bertolini and colleagues reported that patientswith elevated pretreatment VEGF levels are at risk for treat-ment failure. In our opinion, no de®nitive conclusion aboutthe potential prognostic value of pretreatment VEGF valuesshould be deduced from this heterogeneous patient popula-tion. In lymphoma patients, we would rather propose tomeasure VEGF blood levels sequentially before and aftercytotoxic treatment to elucidate the kinetics of VEGF duringand after response to treatment. It would be interesting toknow whether the elevated VEGF levels in blood normalizewhen the patients achieve a remission.

The measurement of VEGF levels in the study carried out

by Bertolini et al (1999) was performed once before cytotoxictreatment in blood samples anticoagulated with EDTA. Inserum samples, the release of VEGF from activated plateletsin vitro is a well-known phenomenon (Verheul et al, 1997).The determination of VEGF in plasma samples might over-come this problem (Banks et al, 1998). Mostly, citrated plasmahas been used and data about the measurement of VEGF inEDTA plasma, as performed by Bertolini and co-workers, aresparse. There is evidence that EDTA activates platelets in vitro(Golanski et al, 1996), a phenomenon that may cause therelease of VEGF. Moreover, there are certain conditions inwhich VEGF can be released from platelets in vivo, such asplatelet consumption and platelet transfusions, which arenot uncommon in patients with advanced stage lymphomaand might contribute to the elevated VEGF plasma levelsreported by Bertolini et al (1999).

Division of Haematology and E B E R H A R D G U N S I L I U S

Oncology, A N D R E A S L. P E T Z E R

Innsbruck University Hospital, G U E N T H E R G A S T L

Anichstrasse 35,6020 Innsbruck, Austria

REFERENCES

Banks, R.E., Forbes, M.A., Kinsey, S.E., Stanley, A., Ingham, E.,

Walters, C. & Selby, P.J. (1998) Release of the angiogenic cytokine

661Correspondence

q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 660±663

vascular endothelial growth factor (VEGF) from platelets: signi®-

cance for VEGF measurements and cancer biology. British Journal

of Cancer, 77, 956±964.

Bertolini, F., Paolucci, M., Peccatori, F., Cinieri, S., Agazzi, A.,Ferrucci, P.F., Cocorocchio, E., Goldhirsch, A. & Martinelli, G.

(1999) Angiogenic growth factors and endostatin in non-

Hodgkin's lymphoma. British Journal of Haematology, 106, 504±

509.Golanski, J., Pietrucha, T., Baj, Z., Greger, J. & Watala, C. (1996)

Molecular insights into the anticoagulant-induced spontaneous

activation of platelets in whole blood ± various anticoagulants are

not equal. Thrombosis Research, 83, 199±216.

Verheul, H.M.W., Hoekman, K., Luykx-de Bakker, S., Eekman, C.A.,

Folman, C.C., Broxterman, H.J. & Pinedo, H.M. (1997) Platelet:transporter of vascular endothelial growth factor. Clinical Cancer

Research, 3, 2187±2190.

Keywords: vascular endothelial growth factor, non-Hodgkin'slymphoma.

REPLY TO GUNSILIUS ET AL

The letter from Dr Gunsilius and colleagues offers us theopportunity to comment further on the prognostic value ofangiogenic growth factors in non-Hodgkin's lymphoma(NHL). Our study (Bertolini et al, 1999) indicated that highpretreatment circulating levels of vascular endothelialgrowth factor (VEGF) and basic ®broblast growth factor(b-FGF) are predictive of a poor outcome in NHL. It must benoted that Salven et al (1997) reported on 82 NHL patients(including high- and low-grade NHL) in whom serum VEGFlevels above the median value at the time of diagnosis werehighly predictive of a poor survival after the 5-year follow-up. Moreover, the same group has recently con®rmed ourobservation that high pretreatment circulating b-FGF levelpredicts poor prognosis in NHL (Salven et al, 1999a). Tworecent papers gave further insight into this issue. First, Vaccaet al (1999) evaluated angiogenesis in 71 B-cell NHL patientsand suggested that the frequency of NHL tissue microvessels,most probably in¯uenced by angiogenic growth factors,increases simultaneously with pathological progression.Second, Bellamy et al (1999) have demonstrated that anumber of NHL cell lines produce very relevant amounts ofVEGF. Taken together, these data suggest that in NHLpatients high levels of circulating VEGF (and possibly b-FGF)may be generated by tumours with an angiogenic andaggressive phenotype. We agree with Dr Gunsilius andcolleagues that studies on VEGF kinetics after response totreatment are now warranted. The prospective data we havecollected so far (unpublished observations) show that VEGFlevels decrease in NHL patients responding to therapy.

Dr Gunsilius and colleagues also suggest that our VEGFmeasurements in EDTA±plasma samples may in part re¯ectVEGF release from activated platelets. Again, this issue hasbeen discussed in recent papers. Wynendaele et al (1999)have reported that, although EDTA is known to in¯uenceplatelet shape, no statistically signi®cant difference in VEGFlevels was observed in plasma samples collected with EDTAcompared with plasma samples collected with four differentanticoagulants (sodium citrate, theophylline, adenosine anddipyridamole) to obtain maximal platelet stabilization. Asindicated in the same paper (Wynendaele et al, 1999), thereis increasing evidence that in cancer patients serum VEGFlevels encompass both platelet-delivered VEGF and VEGFfrom other sources, most probably angiogenic tumourtissues. In this context, Salven et al (1999b) have demon-strated that leucocytes and platelets from cancer patients

contain higher levels of VEGF than those from healthycontrols, possibly acting as scavengers of circulating VEGFproduced by the tumour tissue. Accordingly, in cancerpatients, the VEGF reservoir circulating in leucocytes andplatelets may play a role in tumour angiogenesis andmetastasis formation, and it may be clinically useful toevaluate it. Along this line of investigation, we are followingthe suggestion of Salven et al (1999b) and prospectivelyevaluating VEGF levels in both whole blood and plasma fromNHL patients.

*Haematology Oncology, F R A N C E S C O B E RT O L I N I *

and ²Medical Oncology, M A R A PA O L U C C I*

IRCCS European Institute F E D RO P E C C AT O R I *

of Oncology, S AV E R I O C I N I E R I *

Milan, Italy A L B E RT O AG A Z Z I *

P I E R F R A N C E S C O F E R RU C C I *

E M I L I A C O C O RO C C H I O *

A RO N G O L D H I R S C H ²

G I OVA N N I M A RT I N E L L I *

REFERENCES

Bellamy, W.T., Richter, L., Frutiger, Y. & Grogan, T.M. (1999)

Expression of vascular endothelial growth factor and its

receptors in hematopoietic malignancies. Cancer Research, 59,728±733.

Bertolini, F., Paolucci, M., Peccatori, F., Cinieri, S., Agazzi, A.,

Ferrrucci, P.F., Cocorocchio, E., Goldhirsch, A. & Martinelli, G.

(1999) Angiogenic growth factors and endostatin in non-Hodgkin's lymphoma. British Journal of Haematology, 106, 504±

509.

Salven, P., Teerenhovi, L. & Joensuu, H. (1997) A high pretreatmentserum vascular endothelial growth factor concentration is

associated with poor outcome in non-Hodgkin's lymphoma.

Blood, 90, 3167±3172.

Salven, P., Teerenhovi, L. & Joensuu, H. (1999a) A high pretreat-ment serum basic-®broblast growth factor concentration is an

independent predictor of poor prognosis in non-Hodgkin's

lymphoma. Blood, 94, 3334±3339.

Salven, P., Orpana, A. & Joensuu, H. (1999b) Leukocytes andplatelets of patients with cancer contain high levels of

vascular endothelial growth factor. Clinical Cancer Research,

5, 487±491.Vacca, A., Ribatti, D., Ruco, L., Giacchetta, F., Nico, B., Quondamatteo,

F., Ria, R., Iurlaro, M. & Dammacco, F. (1999) Angiogenesis extent

662 Correspondence

q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 660±663

and macrophage density increase simultaneously with patholo-

gical progression in B-cells in non-Hodgkin's lymphoma. British

Journal of Cancer, 79, 965±970.

Wynendaele, W., Derua, R., Hoylaerts, M.F., Pawinski, A., Waelkens, E.,de Bruijn, E.A., Paridaens, R., Merlevede, W. & van Oosterom, A.T.

(1999) Vascular endothelial growth factor measured in platelet

poor plasma allows optimal separation between cancer patients

and volunteers: a key to study an angiogenic marker in vivo?

Annals of Oncology, 10, 965±971.

Keywords: angiogenic growth factors, non-Hodgkin's lym-phoma, VEGF levels.

ALA-10 MUTATIONS IN THE FACTOR IX PROPEPTIDE AND HAEMORRHAGE IN A PATIENT TREATED

WITH WARFARIN

Oral anticoagulant therapy is dangerous, with 1±2% ofpatients suffering a major haemorrhage each year (Palaretiet al, 1996). Two mutations that are associated withhaemorrhage in patients treated with warfarin have beendescribed at Ala-10 in the factor IX propeptide, i.e. Ala to Thr(Chu et al, 1996; Oldenburg et al, 1997) and Ala to Val(Oldenburg et al, 1997). These mutations interfere with arecognition site for g-glutamylcarboxylase, the enzyme thatcarboxylates the glutamic acids in the gla-domain. Thismeans that when warfarin therapy is given to maintain atherapeutic International Normalized Ratio (INR), withfactors VII, X and prothrombin 20±40% of normal, thefactor IX level falls to <3%. These reports raised the questionof whether male patients on warfarin should have anactivated partial thromboplastin time (aPTT) screening test.In our anticoagulant clinic, we decided against this afterPeters et al (1997) screened 750 patients (412 males) andfound no cases, and we simply resolved to measure the aPTTin patients who haemorrhaged without an excessive INR.This policy was recently supported by another group (vander Meer et al, 1999), who screened 734 patients and againfound no cases.

We report a further case. A 56-year-old man presentedwith an axillary vein thrombosis secondary to carcinoma ofthe stomach. He was initiated on warfarin therapy, butsuffered extensive bruising and a right leg haematomawithin 4 weeks of commencement. His INR was consistentlymaintained between 2 and 3 but his aPTT was dispropor-tionately prolonged, ranging from 104 to 170 s [normalrange (NR) 22±34 s]. When his INR was 2´2, his factor VIIlevel was 20 u/dl (NR 50±150 u/dl) and his factor IXlevel <1 u/dl (NR 50±150 u/dl). The warfarin was discon-tinued, his bruising stopped and the factor IX level rose to65 u/dl. Sequencing of the patient's factor IX gene revealedthe G6346A mutation that results in the Ala-10!Thrsubstitution.

Problems due to mutations at Ala-10 are likely to bemanifest soon after warfarin therapy is initiated, thereforethe screening of long-term stable patients may be selectingagainst the likelihood of ®nding these mutations which maynot be so rare as supposed. This case shows that the aPTTshould be measured in patients on warfarin who bleeddespite a therapeutic INR, and one should be particularly

vigilant soon after treatment is initiated. If this problem isidenti®ed, warfarin must be discontinued. Vitamin K willnormalize coagulation, but if more urgent reversal isrequired for serious haemorrhage then prothrombin com-plex concentrates are likely to be more effective than FFP in apatient with very low levels of factor IX. In patients who needcontinued anticoagulation, we would recommend using lowmolecular weight heparin. Family studies should be under-taken to identify other male carriers of the mutation so thatthey can be warned of the risk of anticoagulant therapy.

Department of Haematology, P E T E R BA K E R

Oxford Radcliffe Hospitals, K E V I N C L A R K E

Oxford, PAU L G I A N G R A N D E

UK DAV I D K E E L I N G

REFERENCES

Chu, K., Wu, S.M., Stanley, T., Stafford, D.W. & High, K.A. (1996) A

mutation in the propeptide of factor IX leads to warfarin sensitivityby a novel mechanism. Journal of Clinical Investigation, 98, 1619±

1625.

van der Meer, F.J., Vos, H.L. & Rosendaal, F.R. (1999) No indicationfor APTT screening in patients on oral anticoagulant therapy.

Thrombosis and Haemostasis, 81, 364±366.

Oldenburg, J., Quenzel, E.M., Harbrecht, U., Fregin, A., Kress, W.,

Muller, C.R., Hertfelder, H.J., Schwaab, R., Brackmann, H.H. &Han¯and, P. (1997) Missense mutations at ALA-10 in the factor

IX propeptide: an insigni®cant variant in normal life but a decisive

cause of bleeding during oral anticoagulant therapy. British

Journal of Haematology, 98, 240±244.Palareti, G., Leali, N., Coccheri, S., Poggi, M., Manotti, C.A.D.A.,

Pengo, V., Erba, N., Moia, M., Ciavarella, N., Devoto, G., Berrettini,

M. & Musolesi, S. (1996) Bleeding complications of oral antic-oagulant treatment: an inception-cohort, prospective collabora-

tive study (ISCOAT). Italian Study on Complications of Oral

Anticoagulant Therapy. Lancet, 348, 423±428.

Peters, J., Luddington, R., Brown, K., Baglin, C. & Baglin, T. (1997)Should patients starting anticoagulant therapy be screened for

missense mutations at Ala-10 in the factor IX propeptide? [letter;

comment]. British Journal of Haematology, 99, 467±468.

Keywords: oral anticoagulant, Ala-10 mutations, factor IXlevels, INR.

663Correspondence

q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 660±663