OCT embedded sections as a source of high quality RNA

5 6 7 8 9 10 11

p. q..

small numbers of CD44v positive cells, notapparent by antibody staining because of a lackof sensitivity or masking of the antigenic sites.10Combining the method described here withimmunohistochemistry on sequential sectionsmay permit a more detailed analysis of cellphenotype. In addition, PCR analysis followedby Southern blotting gives additional in-formation on the fingerprint pattern of variantexpression, which may have biological im-plications.We wish to thank the North East Cancer Research Campaignand the Tyneside Leukaemia Research Fund for their support.

1 2 3 4 5 6 7 8 9 10 11 12 13 1 EastJA, Hart IR. CD44 and its role in tumour progressionand metastasis. Eur J Cancer 1993;29A: 1921-2.

2 Ruiz P, Schwarzler C, Giunthert U. CD44 isoforms during-_ differentiation and development. Bioessays 1995;17:17-24.

3 Screaton GR, Bell MV, Jackson DG, Comelius FB, GerthU, Bell JI. Genomic structure of DNA encoding the__ - -___>--lymphocyte homing receptor CD44 reveals at least 12alternatively spliced exons. Proc Nat! Acad Sci USA 1992;89:12160-4.

4 Mackay CR, Terpe H-J, Stauder R, Marston WL, Stark H,Gunthert U. Expression and modulation of CD44 variantisoforms in humans. J Cell Biol 1994;124:71-82.

5 Matsumura Y, Tarin D. Significance ofCD44 gene productsfor cancer diagnosis and disease evaluation. Lancet 1992;340:1053-8.

6 Kaufmann M, Heider K-H, Sinn H-P, Von Minckwitz G,rmaldehyde agarose electrophoretic gel of RNA. Lanes 1 and 3, 3 plg RNA Ponta H, Herrlich P. CD44 variant epitopes in primary!d from OCT embedded sections of samples from two patients with non-Hodgkin's breast cancer and length of survival. Lancet 1995;345:ma; lane 2, A HindIII markers. (B) Agarose electrophoretic gel ofPCR products. 615-19.pas extracted from three different samples from patients with non-Hodgkin's 7 Chirgwin JM, Przybyla AE, McDonald R, Rutter WJ. Isol-ma as outlined in the methods section and reverse transcribed into cDNA. An ation of biologically active ribonucleic acid from sourcesof each was used as a substrate for PCR using three different amplimer sets. Lanes enriched in ribonuclease. Biochemistry 1979;18:5294-9.1; lanes 5-7, bcr; lanes 8-10, CD44; lanes 1 and 11, pBR322 AluI and OX1 74 8 Cross NCP, Feng L, Bungey J, Goldman JM. Minimalrespectively, as base pair markers. (C) Analysis of CD44 isoforms in non-

residual disease after bone marrow transplant for chronicrespectively, as base pair markers. (C;) Analysis of CDv44 isoforms in non- myeloid leukaemia detected by the polymerase chain re-

n's lymphoma samples after PCR and hybridisation with a radiolabelled oligoprobe action. Leuk Lymphoma 1993;11:39-43.n-variant part of the molecule (exon 4). Note: lane 3 in panel A, lanes 3, 6 and 9 Cross NCP, Melo JV, Feng L, Goldman JM. An optimizednel B and lane 9 in panel C represent RNA, PCR products and blot of the same multiplex polymerase chain reaction (PCR) for detectionion-Hodgkin's lymphoma sample, respectively. of bcr/abl fusion mRNAs in haematological disorders.

Leukemia 1994;8:186-9.10 Dall P, Heider K-H, Sinn H-P, Skroch-Angel P, Adolf G,

Kaufmann M, etal. Comparison ofimmunohistochemistryantibodies can detect variant expression in and RT-PCR for detection of CD44v expression, a new

prognostic factor in human breast cancer. Int J Cancercryostat sections, the PCR method may detect 1995;60:471-7.

Clin Pathol 1996;49:259-262

Small cell variant of Ki-1 lymphoma associatedwith myelofibrosis and a novel constitutionalchromosomal translocation t(3;4)(ql3;ql2)

Department ofClinical Oncology,Sir Y K Pao CancerCentre, ChineseUniversity of HongKong, Prince of WalesHospital, Shatin,N. T., Hong KongW YeoN WongP J JohnsonN Wickham

Department ofAnatomical andCellular PathologyJ ChowW C Tsoi

Correspondence to:Dr Nicholas WickhamDepartment of ClinicalOncology, Prince ofWales Hospital, Shatin,N. T., Hong Kong.Accepted for publication18 October 1995

W Yeo, N Wong, J Chow, W C Tsoi, P J Johnson, N Wickham

AbstractAn unusual case of small cell variant ofKi-1 non-Hodgkin's lymphoma diagnosedone year after an original diagnosis ofidio-pathic myelofibrosis is reported. On thesecond occasion, the patient presentedwith fever, lymphadenopathy and hepa-tosplenomegaly. A lymph node biopsyspecimen confirmed a diagnosis of smallcell variant of Ki-l lymphoma. A repeatbone marrow biopsy specimen showedmyelofibrosis with no evidence of lymph-omatous infiltration, but cytogeneticstudies on blood, bone marrow and skin

fibroblasts revealed a novel chromosomaltranslocation t(3,4)(qI3;qI2).(J7 Clin Pathol 1996;49:259-262)

Keywords: Ki-1 lymphoma, myelofibrosis.

Small cell Ki-l lymphoma is a variant of Ki-1anaplastic large cell lymphoma (ALCL), whichhas been described recently.' The specific chro-mosomal translocation t(2;5) (p23;q35) hasbeen a consistent finding in association withboth variants of Ki- 1 lymphoma. 1-3 Lymphomais a recognised, albeit uncommon, cause ofmyelofibrosis. However, idiopathic myelo-

BA

1 2 3M

1 2 3 4

C

(A) Foextracte,lympho?RNA wlympho?aliquot,2-4, abHaeIII,Hodgkito a no?9 in paifrozen n

259

on April 3, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.49.3.259 on 1 March 1996. D

ownloaded from

Yeo, Wong, Chow, Tsoi, J7ohnson, Wickham

4s

Figure 1 (A) Lymph node histology showing polymorphic infiltrates with atypicallymphocytes and extramedullary haematopoiesis (haematoxylin and eosin, x 200); (Imarmw trphine biopsy specimen showing grade III myelofibrosis (inset reticdin stain)

Figure 2 Cytogenetics: metaphase chromosomes seen in peripheral blood showingt(3;4) (q13;q12).

fibrosis is an extremely rare finding iniation with lymphoma, with only threehaving been described previously.45 He]present a case of idiopathic myelofibros

%y patient who subsequently developed the smallcell variant of Ki-1 lymphoma and who has a

*. previously unreported constitutional chro-mosomal translocation t(3,4) (q1 3;q1 2).

* Case report*FF A 47 year old man presented in October 1993PF with a two day history of fever, sore throat and

swollen neck, and a four week history ofweightloss and night sweats. In January 1993 he had

+* been diagnosed as having idiopathic myelo-fibrosis, after a bone marrow biopsy was carriedout for an incidental finding of anaemia. Thepatient remained transfusion dependent, andrequired blood transfusions every two monthsto maintain his haemoglobin level above8 g/dl. On readmission to hospital, the patientwas pyrexial, with a temperature of 38-5°C,

NL and had enlarged tonsils with left-sided cervicalswelling, lymphadenopathy of both axillae andthe right groin, and hepatosplenomegaly. Ultra-sound examination of the neck showed ex-tensive cervical lymphadenopathy. Flexibleendoscopy showed inflammatory swelling ofthe lateral pharyngeal wall extending to thepiriform fossa. A computed tomography (CT)scan of the thorax was normal. Abdominalultrasound and CT scan confirmed hep-atomegaly with no focal lesion; the spleen was13 cm long, and two small nodes associatedwith the porta hepatis (0 9 cm) and coeliac axis(1 2 cm) were noted. The patient developedairway obstruction secondary to the pharyngealcellulitis and required tracheotomy. The cel-lulitis responded to combined broad spectrumantibacterial, antiviral and antifungal agents,and the tracheotomy wound healed un-eventfully. The bone marrow, left axillary node

3) bone and right groin node were biopsied.

INITIAL LABORATORY INVESTIGATIONSPeripheral blood count revealed a haemoglobinof 7-2 g/dl (normochromic normocytic pic-ture), a white cell count of 1 1 x 109/l and aplatelet count of 195 x 109/1. The peripheralblood smear was leukoerythroblastic withoccasional atypical immature mononuclearcells. The erythrocyte sedimentation rate was120 mm in the first hour. Renal function wasnormal but liver function was deranged:albumin, 19 g/l; total bilirubin, 32 gmol/l;alkaline phosphatase, 217 IU/1; alanine amino-transferase, 56 IU/1. The patient's totalprotein concentration was 57 g/l, his IgG levelwas 204g/l (normal range 5-3-16'3 g/l), IgAwas 2 3 g/l (normal range 0-8-4O0 g/l) and IgMwas 2 1 g/l (normal range 0 5-2-0 g/l). No para-

46XY, protein was detected. Viral serological studiesshowed that the antibody titre to Epstein-Barrvirus (EBV) nuclear antigen was >2, IgG anti-body titre to EBV viral capsid antigen (VCA)

assoc- was 160, and IgM antibody to EBV VCA wascases negative. Serological markers for cyto-

re, we megalovirus, hepatitis A and B, and HIV were,is in a negative.

%I,...3

260

.)

on April 3, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.49.3.259 on 1 March 1996. D

ownloaded from

Small cell variant of Ki-1 lymphoma

HISTOPATHOLOGICAL FINDINGSHistological examination of the lymph nodes(fig IA) showed fragments of lymphoid tissuewith noticeable paracortical and sinusoidalexpansion and vascular proliferation. The si-nusoids and perisinusoidal regions containedmany blastic lymphocytes with large irregularnuclei and prominent nucleoli. The para-cortical region contained mainly small lymph-oid cells with irregular nuclei and clear cy-toplasm. There was evidence of extramedullaryhaematopoiesis in the sinusoids (myeloid pre-cursors and megakaryocytes) and numerousplasma cells and plasmablasts were seen.

Immunocytochemistry showed the smallT lymphocytes to be CD3 +, CD43 +,CD45RO +, CD20- and the large lympho-blasts to be mostly CD30 +, CD3 -,

CD45RO +, CD45 -. These findings were

consistent with the small cell variant of Ki-1lymphoma.' Bone marrow biopsy specimenstaken in November and December showed50% cellularity, grade III reticulin myelofibrosis(fig 1 B), trilineage haematopoiesis, and normalnumbers of megakaryocytes. No myelo-dysplastic changes were noted and there was

no evidence of lymphoma.

CYTOGENETIC STUDIESCytogenetic studies were done on short termsynchronised cultures of unstimulated bonemarrow and phytohaemoagglutinin stimulatedperipheral blood samples. The metaphase chro-mosomes obtained were stained for G bandsusing Wright's stain and karyotyped accordingto ISCN 1991. Metaphase chromosomes seenin both bone marrow and peripheral bloodsamples showed 46XY, t(3;4)(ql3;q12) (fig 2).No other abnormalities, and specifically no

t(2;5) translocations could be identified andthere were no normal metaphases. As there wasno evidence ofeither bone marrow involvementor of circulating lymphoma cells, a con-stitutional chromosomal defect was suspected.Skin biopsy and analysis of metaphase chro-mosomes derived from the cultured skin fibro-blasts confirmed an identical translocation inall metaphases examined.

PROGRESSFever persisted until chemotherapy with a

standard CHOP (cyclophosphamide, adria-mycin, vincristine, prednisolone) regimen com-menced, when it resolved within 24 hours.Following the first cycle, further chemotherapywas delayed, initially because ofprolonged pan-cytopenia and then because the patient declinedfurther treatment and defaulted from followup for four months. Subsequently, repeatedadmissions have been required for intravenousantibiotic therapy for septicaemia, usually dueto severe skin sepsis with cellulitis. Despitehaving received only one course ofCHOP over18 months ago, the lymphoma continues torun an indolent course. The lymphadenopathyhas regressed, although both clinical ex-

amination and abdominal ultrasound confirmthe persistence of hepatosplenomegaly. Theintra-abdominal nodes have not increased in

size. The patient has remained anaemic re-quiring intermittent red cell support. Repeatbone marrow examination six months afterthe only course of chemotherapy confirmed ahypocellular bone marrow with fibrosis and noevidence of lymphoma. The white cell countcurrently remains low, between 3 0 and4 0 x 109/1, but the platelet count is normal.However, there is notably reduced bone mar-row reserve, with severe pancytopenia de-veloping rapidly following any infection.

DiscussionSmall cell Ki-I lymphoma accounts for ap-proximately 10% of cases of Ki-1 ALCL. Smalllymphoid cells predominate in the lymph nodebiopsy specimen with a minority componentof large blastic lymphocytes.' Ki-I ALCL isclassified as a high grade lymphoma but has asurprisingly variable clinical course, with epi-sodes of apparent clinical remission, and pro-longed survival despite multiple recurrences.2No specific association is known between

primary myelofibrosis and lymphoma. In thetwo case reports describing three patients withconcomitant or subsequent lymphoma as-sociated with myelofibrosis, there was no bonemarrow involvement with lymphoma.45 Bonemarrow involvement in Ki-I ALCL is in anycase unusual, affecting approximately 10-15%of cases overall, and may be associated withhistiocytosis rather than fibrosis.6 In a clinico-pathological study of 41 patients with Ki-1ALCL by Chott et al7 bone marrow in-volvement was described in seven patients, allofwhom fell within a subgroup of patients witha monomorphic infiltrate of predominantlysmaller cells. There was no distinctive patternto the involvement in these cases, being eitherdiffuse or focal, and ranging from a low degreeof infiltration to complete effacement.The specific chromosomal translocation

t(2;5)(p23;q35) has been a consistent findingin association with Ki-1 ALCL.2 3 Of ninepatients who were documented originally ashaving small cell Ki-1 lymphoma variant, onehad cytogenetic studies performed on involvedskin, and four had cytogenetic studies per-formed on bone marrow. The same trans-location, together with multiple variableabnormalities affecting chromosome 7 and thesex chromosomes, was found in all cases.' Thetranslocation in our patient, 46XY t(3;4)(ql3;q12) has not been described previously. Thefact that it was isolated consistently in all meta-phases examined from both blood and bonemarrow, despite lack of evidence of mor-phological involvement by lymphoma, sug-gested that it was constitutional and this hasbeen confirmed by chromosomal analysis ofthe patient's skin fibroblasts.The significance of this cytogenetic finding

is unknown. However, the c-kit proto-oncogenethat encodes the membrane tyrosine kinasereceptor for stem cell factor, and the gene forplatelet derived growth factor (PDGF) receptorA are localised to bands qi 1-13 ofchromosome4 in humans.8 Also, the small inducible genefamily that encodes platelet factor 4, ,B-throm-boglobulin and other related proteins which

261

on April 3, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.49.3.259 on 1 March 1996. D

ownloaded from

Yeo, Wong, Chow, Tsoi, J7ohnson, Wickham

are involved in the processes of coagulation,inflammation, immune response and woundrepair, have been localised to chromosomebands 4ql2-ql3.9 It is intriguing to speculatethat this constitutional genetic abnormality mayhave not only contributed to this patient's my-elofibrosis by affecting PDGF production,10but that it may have played a role in the de-velopment of his lymphoma and the patternof his repeated septic episodes. The patient'satypical course and response to therapy maytherefore reflect an unusual pattern of a raredisease modified by an unique underlying con-stitutional chromosomal aberration.

AddendumSince the original submission of this article, areport has appeared of a non-constitutional(3;4) translocation occurring in associationwith a myeloproliferative disorder. (Myint H,Chacko J, Mould S, Ross F, Oscier DG. Karyo-typic evolution in a granulocytic sarcoma de-veloping in a myeloproliferative disorder witha novel (3;4) translocation. BrJYHaematol 1995;90:462-4.)

1 Kinney MC, Collins RD, Greer JP, Whitlock JA, SioutosN, Kadin ME. A small-cell-predominant variant of prim-ary Ki-1 (CD30 +) T-cell lymphoma. Am J Surg Pathol1993;17:859-68.

2 Bitter MA, Franklin WA, Larson RA, McKeithan TW,Rubin CM, Le Beau MM, et al. Morphology in Ki-1(CD30)-positive non-Hodgkin's lymphoma is correlatedwith clinical features and the presence of a unique chro-mosomal abnormality, t(2;5)(p23;q35). Am Y Surg Pathol1990;14:305-16.

3 Le Beau MM, Bitter MA, Larson RA, Doane LA, EllisED, Franklin WA, et al. The t(2;5)(p23:q35): a recurringchromosomal abnormality in Ki-l positive anaplastic largecell lymphoma. Leukemia 1989;3:866-70.

4 Epstein RJ, Joshua DE, Kronenberg H. Idiopathic my-elofibrosis complicated by lymphoma. Report oftwo cases.Acta Haematol 1985;73:40-4.

5 Jennings WH, Li CY, Kiely JM. Concomitant myelofibrosiswith agnogenic myeloid metaplasia and malignant lymph-oma. Mayo Clin Proc 1983;58:617-19.

6 Wong KF, Chan JK, Ng CS, Chu YC, Lam PW, YuenHL. Anaplastic large cell Ki-I lymphoma involving bonemarrow: Marrow findings and association with reactivehaemophagocytosis. Am J Hematol 1991;37: 112-19.

7 Chott A, Kaserer K, Augustin I, Vesely M, Heinz R, Oeh-linger W, et al. Ki-l-positive large cell lymphoma. Aclinicopathologic study of 41 cases. Am J Surg Pathol1990;14:439-48.

8 Andre C, Martin E, Comu F, Hu WX, Wang XP, GalibertF. Genomic organization of the human c-kit oncogene:evolution of the receptor tyrosine kinase subclass III.Oncogene 1992;7:685-91.

9 Tunnacliffe A, Majumdar S, Yan B, Poncz M. Genes forbeta-thromboglobin and platelet factor 4 are closely linkedand form part of a cluster of related genes on chromosome4. Blood 1992;79:2896-900.

10 Reilly JT. Pathogenesis of idiopathic myelofibrosis: Role ofgrowth factors. J Clin Pathol 1992;45:461-4.

Department ofPathology, QueenMary Hospital,Hong KongK Y LamA C L Chan

Department ofMedicineM S Wat

Correspondence to:Dr K Y Lam, Departmentof Pathology, Queen MaryHospital, Pokfulam Road,Hong Kong.Accepted for publication18 October 1995

J Clin Pathol 1996;49:262-264

Langerhans cell histiocytosis forming an

asymptomatic solitary nodule in the spleen

K Y Lam, A C L Chan, M S Wat

AbstractA case of solitary Langerhans cell histio-cytosis (LCH) in the spleen of a 29 yearold Chinese man, discovered incidentallyat necropsy, is reported. This is the first

documented case of LCH confined to thespleen and suggests that LCH should beincluded in the differential diagnosis ofspace occupying lesions in the spleen.(_J Clin Pathol 1996;49:262-264)

Figure 1 A slice of the sectioned spleen showing a white nodule (arrow) underneath thesplenic capsule.

Keywords: Langerhans cell histiocytosis, spleen.

The common denominator of all forms of Lan-gerhans cell histiocytosis (LCH), formerlyknown as histiocytosis X, is the presence ofhistiocytes phenotypically similar to the Lan-gerhans cell of the epidermis. The disease hasa wide range of clinical and pathological pre-sentations. Almost every organ in the body canbe involved in LCH, the favoured sites beingthe skin, bone, lungs, and lymph nodes.' Whilesplenic infiltration is part of the multisystemdisease in LCH, histiocytosis confined to thespleen, to our knowledge, has never been docu-mented in the literature.

Case reportAn otherwise healthy 29 year old Chinese mandeveloped sudden onset retrosternal chest pain,shortness of breath and sweating. He then de-

262

on April 3, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.49.3.259 on 1 March 1996. D

ownloaded from