Acute leukemia presenting with extramedullary ... - Wiley Online Library

Letters Effective management of accelerated phase myelofibrosis with low-dose splenic radiotherapy A. Pardanani,1 P. Brown,2 M. Neben-Wittich,2 R. Tobin,3 and A. Tefferi1* The main cause of hepatosplenomegaly in primary (PMF), post polycythemia vera (post-PV MF), and post essential thrombocythemia (postET MF) myelofibrosis (MF) is extramedullary hematopoiesis (EMH). Drug-refractory symptomatic splenomegaly in MF is usually managed by splenectomy or involved-field radiotherapy. The latter is most effective in the treatment of MF-associated bone pain and pulmonary hypertension. Our previous experience with hepatosplenic radiotherapy in MF showed efficacy in the majority of treated patients but its utility was limited by the transient nature of its benefit and the occurrence of treatment-related pancytopenia. In an effort to address these issues, we have adopted an induction-maintenance treatment strategy that utilizes lower radiation doses—induction with 100 cGy total in four daily doses of 25 cGy and maintenance with either the same or lower intensity regimen. Herein, we report our most recent experience using this treatment plan in two cases, who in addition to their expected response from the standpoint of splenomegaly, also unexpectedly showed a marked response of their underlying accelerated phase disease, including clearance of circulating blasts and basophilia.

Case #1 AB is a 45-year-old male with post-PV MF (Table I). In October 2008, the patient was enrolled in a JAK1/2 inhibitor (INCB018424) clinical trial and at the time had massive splenomegaly (27 cm below left costal margin), 25 pound weight loss, and significant constitutional symptoms. He failed to exhibit a sustained response and the study medication was tapered with the intent to discontinue. During the taper, the patient experienced a systemic inflammatory response syndrome with multiorgan failure and pleural/pericar-

dial effusions, which was effectively managed by readjusting the INCB018424 dose and instituting, in addition, high-dose corticosteroid and hydroxyurea therapy. The patient continued to experience persistent/progressive hypercatabolic symptoms, leukocytosis/thrombocytosis, symptomatic splenomegaly, and circulating immature cells, and therefore, received three courses of intravenous cladribine therapy at 5 mg/m2/day 3 5 days on 4/15/2009, 5/7/2009, and 7/ 17/2009. At the same time, his recurrent pleural effusion required repeated thoracentesis and treatment with lung radiation (100 cGy in 1 fraction on 6/ 11/2009). Because of progressive disease despite the aforementioned therapeutic measures, gemtuzumab-ozogamycin was administered without benefit. Splenic radiotherapy at 100 cGy total dose in 4 daily fractions of 25 cGy was started on 9/15/2009, and improvement in dyspnea and early satiety commenced shortly afterward. The treatment was not associated with any significant decrease in blood counts, so the patient received a second course of splenic radiation starting 9/29/2009; he concurrently received 200 cGy in a single fraction to both legs for significant leg pain. Following this, he reported significant improvement in abdominal/leg pain (pain score from 9 to 4, on a scale of 0 to 10) such that he was able to begin ambulating, and also had near normalization of blood counts. He received a third course of splenic/leg radiotherapy at identical doses starting 10/27/2009, with full relief from abdominal/leg pain (spleen size 22 cm below left costal margin). In February 2010, he developed severe upper extremity pain and exhibited partial spleen regrowth; consequently he received a fourth course of splenic radiotherapy along with upper extremity radiation (200 cGy in single fraction) starting 2/22/2010.

TABLE I. Clinical and Treatment-Response Characteristics of Myelofibrosis Patients Treated with Low-Dose Fractionated Radiotherapy to

the Spleen

Sex/Age

Diagnosis

M/49

PPMF-a

F/43

PTMF-a

Prior therapies HU INCB018424 2-CdA GO

Gleevec HU

XRT Spleen

XRT LE

XRT Lungs

100 cGy in 4 fractions 3 4 courses (9/15/2009, 9/29/2009, 10/27/2009, 2/22/2010)

200 cGy single fraction (9/29/2009, 10/27/2009)

100 cGY single fraction (6/11/2009)

100 cGy in 4 fractions 3 1 (12/20/2009)

n.a.

n.a.

CBC/Diff pre-XRT Spleen Hgb 13.3 WBC 22.0 Plt 590 Neut 36% Lymph 5% Mon 5% Baso 14% Mmyelo 2% Myelo 19% Blasts 19% Mega 3% NuRBC 14% Hgb 10.3 WBC 134.6 Plt 115

Neut 57% Lymph 5% Mon 1% Eos 10% Baso 13% MMyelo 5% Myelo 6% Blasts 3% Mega 1% NuRBC 1%

25 cGy in 1 fraction 3 4 (every 4–5 weeks)

Spleen size pre-XRT Spleen 301 cm below LCM

F/u 9 months

CBC/Diff post-XRT Spleen Hgb 15.6 WBC 12.7 Plt 279

Spleen size post-XRT Spleen

Status

22 cm below LCM

Alive

15 cm below LCM, 1757cc

Alive

Neut 87% Lymph 7% Mono 3% Eos 1% Baso 2%

301 cm below LCM, 7025cc

6 months

Hgb 11.7 WBC 5.9 Plt 159 Neut 84% Lymph 11% Mon 1% Eos 1% Myelo 2% Blasts 1% Mega 1%

M indicates male; F, female; PPMF-a, accelerated phase of post-polycythemic myelofibrosis; PTMF-a, accelerated phase of post-thrombocythemic myelofibrosis; HU, hydroxyurea; 2-CdA, 2-Chlorodeoxyadenosine; GO, gemtuzumab-ozogamycin; XRT, radiation therapy; LE, lower extremities; CBC, complete blood count; Diff., CBC differential; F/u, follow-up; Hgb, hemoglobin (g/dL); WBC, white blood cell count (3 109/L); Plt, platelet count (3 109/L); Neut., neutrophil; Lymph, lymphocyte; Mon, monocyte; Baso, basophil; Mmyelo, metamyelocyte; Myelo, myelocyte; Mega, megakaryocyte; NuRBC, nucleated red blood cell; and LCM, left costal margin. C 2010 Wiley-Liss, Inc. V

American Journal of Hematology

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letters Complete blood count before course #1 was: Hemoglobin (Hgb) 13.3 g/ dL, white blood cell count (WBC) 22 3 109/L with 19% blasts, and platelet count (Plt) 590 3 109/L; before course #2: Hgb 13.6, WBC 20.8, Plt 572; before course #3: Hgb 14.2, WBC 6.1, Plt 160; and before course #4: Hgb 16.8, WBC 25.1, Plt 299. At last follow-up, the patient had marked improvement in his performance status such that he was ambulatory and able to return to work part-time. His blood counts had near normalized and he had complete resolution of peripheral blood leukoerythroblastosis included complete eradication of circulating blasts (Table I).

Case #2 TMB is a 42-year-old female, who was diagnosed with post-ET MF in June 2007, after 12 years of antecedent ET with an uncomplicated course (Table I). She had been followed expectantly without treatment with the exception of daily aspirin for thromboprophylaxis. In December 2009, she presented with subacute onset of an urticarial skin rash with progressive splenomegaly and leukocytosis, as well as with severe fatigue and marked abdominal discomfort. Her physical examination confirmed massive splenomegaly and the peripheral blood smear showed new onset eosinophilia and basophilia. A bone marrow biopsy confirmed post-ET MF without leukemic transformation, and molecular and cytogenetic studies were normal/negative. The patient received splenic radiation (100 cGy in 4 daily fractions of 25 cGy each) starting on 12/18/2009 with rapid control of myeloproliferation (Table I). Before treatment, Hgb was 10.3 g/dL, WBC 134.6 3 109/L, and Plt 115 3 109/L. At 3 weeks following completion of radiotherapy, Hgb was 7.4 g/dL, WBC 1.0 3 109/L, Plt 44 3 109/L; at 4 weeks: Hgb 6.7 g/dL, WBC 1.3 3 109/L, Plt 122 3 109/L; and at 5 weeks: Hgb 7.2 g/dL, WBC 2.9 3 109/L, Plt 150 3 109/L. The patient was transfused with 8 units of red cells and 4 units of platelets over a 6 week period for pancytopenia at the discretion of her physicians. It is important to note that the patient was being tapered off hydroxyurea therapy in the first 3 weeks of radiotherapy. At her 6 week assessment, spleen volume (based on CT) had decreased from 7025 cc to 2741 cc (60% decrease). Since then, the patient has received maintenance radiotherapy (single fraction of 25 cGy to the spleen) every 5 weeks. At last follow-up, the patient was markedly improved with complete relief from abdominal pain (spleen volume 1757 cc) and near normal blood counts with complete resolution of peripheral eosinophilia/basophilia and marked decrease in leukoerythroblastosis (Table I). She was able to go back to work full-time within 6 weeks of starting radiotherapy and start an exercise regimen.

Discussion Radiotherapy is an attractive treatment option in both hepatosplenic and nonhepatosplenic EMH [1–3]. In a Mayo Clinic study of 23 MF patient undergoing splenic irradiation, a median dose of 2.8 Gy (range 0.3–14) was given in a median of 7.5 fractions (range 2–17) [4]. Approximately 94% of the patients achieved an objective response in spleen size and the median duration of response was 6 months. Approximately 44% of treated patients experienced treatment-related and sometimes life threatening cytopenia [4]. In another Mayo Clinic study [5], MF patients with (five patients) or without (nine patients) associated ascites received hepatic irradiation at a median dose of 1.5 Gy (range 0.5–10) given in a median of 6 fractions. Reduction in liver size was documented in 35% of the patients (median response duration was 3 months). Treatment-associated cytopenia occurred in the majority of the patients [6]. Leukemic transformation of myelofibrosis frequently occurs through an intermediate ‘‘accelerated’’ phase in its evolution from chronic phase of the disease. Although there is no uniform consensus definition for accelerated phase myelofibrosis at the present time, it is characterized by rapid clinical

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progression with survival less than a year; other characteristics that describe this phase include increased peripheral blood/bone marrow myeloblasts, thrombocytopenia, and possibly, specific cytogenetic abnormalities [7,8]. Akin to accelerated phase of chronic myeloid leukemia, this phase of myelofibrosis is characterized by increasing refractoriness to conventional therapies, such as HU, and consequently represents a significant therapeutic challenge; the choice and intensity of therapy in this setting is generally determined on a case-by-case basis after consideration of patient and disease characteristics. Although there are published reports of the efficacy of splenic radiotherapy in myelofibrosis patients, the current case report is unique in that it is restricted to patients with accelerated phase of the disease. We demonstrate the feasibility and efficacy of low-dose splenic radiotherapy utilizing a strategy of induction with 100 cGy in 4 daily 25 cGy fractions, followed by maintenance phase of intermittent radiotherapy at the same or lower dose (i.e., 25 cGy). The response to splenic radiotherapy was rapid in both cases, with decreased spleen size and WBC count becoming clearly evident within the first few weeks after radiotherapy; the treatment was extremely well tolerated with no evident extramedullary toxicity. Myelosuppression can ensue from splenic radiotherapy; for case #2, Grade 3 cytopenias were observed with induction radiotherapy, but not seen later during the maintenance phase; the cytopenias were likely related to concurrent HU therapy as well as the large initial radiation treatment field that covered the spine and pelvis. With subsequent discontinuation of HU and smaller treatment dose delivered via oblique fields that were tapered off at the bone marrow, myelosuppression did not recur. We believe the currently described treatment strategy, which represents a significant therapeutic advance that is worthy of being tested in a prospective fashion in larger group of patients with accelerated phase myelofibrosis.

1

Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota; 2Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota; 3Rocky Mountain Oncology Center, Casper, Wyoming *Correspondence to: Ayalew Tefferi, MD, Mayo Clinic, Rochester, MN 55905 E-mail: [email protected] Conflict of interest: Nothing to report. Published online 22 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21799

References 1. Jyothirmayi R, Coltart S. An audit of the indications for and techniques of palliative splenic radiotherapy in the UK. Clin Oncol (R Coll Radiol) 2005;17: 192–194. 2. Koch CA, Li CY, Mesa RA, Tefferi A. Nonhepatosplenic extramedullary hematopoiesis: associated diseases, pathology, clinical course, and treatment. Mayo Clin Proc 2003;78:1223–1233. 3. Weinmann M, Becker G, Einsele H, Bamberg M. Clinical indications and biological mechanisms of splenic irradiation in chronic leukaemias and myeloproliferative disorders. Radiother Oncol 2001;58:235–246. 4. Elliott MA, Chen MG, Silverstein MN, Tefferi A. Splenic irradiation for symptomatic splenomegaly associated with myelofibrosis with myeloid metaplasia. Br J Haematol 1998;103:505–511. 5. Tefferi A, Jimenez T, Gray LA, et al. Radiation therapy for symptomatic hepatomegaly in myelofibrosis with myeloid metaplasia. Eur J Haematol 2001;66: 37–42. 6. Riesterer O, Gmur J, Lutolf U. Repeated and preemptive palliative radiotherapy of symptomatic hepatomegaly in a patient with advanced myelofibrosis. Onkologie 2008;31:325–327. 7. Tam CS, Kantarjian H, Cortes J, et al. Dynamic model for predicting death within 12 months in patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis. J Clin Oncol 2009;27:5587–5593. 8. Huang J, Li CY, Mesa RA, et al. Risk factors for leukemic transformation in patients with primary myelofibrosis. Cancer 2008;112:2726–2732.


letters

Alteration of cohesin genes in myeloid diseases Julien Rocquain,1 Ve´ronique Gelsi-Boyer,1–3 Jose´ Ade´laı¨de,1 Anne Murati,1,2 Nadine Carbuccia,1 Norbert Vey,3,4 Daniel Birnbaum,1 Marie-Joelle Mozziconacci,1,2* and Max Chaffanet1 New genes involved in leukemogenesis, such as ASXL1 and TET2, have been identified recently using genomic analyses of DNA from patient samples. We have studied by array-comparative genomic hybridization (aCGH) a series of 167 samples including myelodysplastic syndromes, chronic myelomonocytic leukemias, and acute myeloid leukemias. We found a deletion of the RAD21 and STAG2 genes, which encode two components of the cohesin complex. We propose that these alterations may compromise the cohesin complex and its regulation of the transcription of genes. Several new genes involved in myeloid leukemogenesis, including ASXL1, CBL, and TET2, have been identified following genomics studies using aCGH or single nucleotide polymorphism-arrays combined with sequence analyses [1–3]. Mutation analyses have identified other genes, such as IDH1 [4]. No alteration has been identified yet in many cases of malignant myeloid disease and many other genes remain to be discovered. Using aCGH, we searched for genome alterations in a series of 167 malignant myeloid diseases. We describe here two cases with loss of genes encoding components of the same protein complex. About two-thirds of the samples did not show any aCGH copy number aberrations (CNA): 41 of 63 MDSs (65%) (Supporting Information Table I), 37 of 53 CMMLs (70%) (Supporting Information Table II), 35 of the 51 AMLs (69%) (Supporting Information Table III). This high proportion of noCNA profile in AMLs is probably due to our selection of cases with normal karyotype. In all cases, losses of whole or part of chromosome (e.g., 20q losses) were found in agreement with the results of the karyotyping (except for HD-0627 where the del20q could only be suspected on the aCGH profile). Small deletions were more interesting. They were rare but indicative. We found a deletion of TET2 in one myelodysplastic syndrome (MDS) and in one chronic myelomonocytic leukemia (CMML), and a deletion of ASXL1 in one MDS and in one acute myeloid leukemia (AML; Table I). We also found deletions of NF1, RB1, RUNX1, and UTX. Some of these CNAs have been described in our previous reports [1,5,6]. We here focused on two particular CNAs. CMML HD-0259 was diagnosed in 2007 in a 70 year-old patient with no particular antecedent. aCGH showed a small heterozygous deletion at 8q24 (chr8: 117768619-117969545 Mb), which comprised three genes including RAD21 (Fig. 1A). The CMML evolved in an M5 FAB AML (HD-0402) in 2008. aCGH again revealed the RAD21 loss but no additional alteration. The patient died in 2008, 6 months after acute transformation. AML HD0120 (M6 FAB) was diagnosed in 2005 in a 55 year-old patient without any antecedent. aCGH detected a small deletion centered on the STAG2 gene, at Xq25 (chrX: 122787860-122970717 Mb) (Fig. 1B). The patient died in 2007, 5 months after relapse. In both cases, the karyotype did not show any abnormality and no other aCGH alteration was noticed. HD-0120 had an IDH1 mutation (R132C). HD-0259 was the only CMML case with an NPM1 exon 12 mutation; a FLT3-ITD was additionally detected in the corresponding acute phase (HD-0402). There were no mutations in the other studied genes associated with leukemogenesis. We searched for mutations of RAD21 in 95 samples but did not find any.

At first look, deletions of RAD21 and STAG2 could appear as isolated cases with limited interest. However, close examination revealed the involvement of a new pathway of leukemogenesis. Indeed, the two genes happen to encode subunits of the same nuclear complex, cohesin [7]. Cohesin is made of four evolutionary-conserved core subunits, SMC1, SMC3, RAD21, and STAG1/STAG2 (Fig. 2), and plays a major role in chromosome biology [7]. Abnormal functioning of cohesin compromises faithful segregation of sister chromatids during cell division. Mutations in cohesin genes are responsible for the Cornelia de Lange syndrome (CdLs), a multisystem anomaly disorder with abnormal phenotypical and behavioral features, and may be associated with chromosome instability in colorectal cancer [7,8]. However, we did not detect any aneusomy in the two cases. But cohesin has other roles. Cohesin participates in double-strand DNA break repair and long-distance regulation of gene expression. In zebrafish, monoallelic Rad21 inactivation downregulates Runx1 expression [9]. Cohesin is necessary for the function of the enhancerblocking transcriptional insulator CTCF, which is an interactor of nucleophosmin and a positive regulator of tumor suppressor loci [10–12]. Cohesin can also acts on transcription independently of CTCF [13]. Thus, loss of RAD21 or STAG2 could participate in leukemogenesis through enhancement of inactivation of NPM1, RUNX1, and other tumor suppressor genes, such as RB1 or CDKN2A/B. Loss of RAD21 co-occurred with an NPM1 mutation in case HD0259 showing that the two alterations can indeed cooperate. Abnormal repression of transcription could be a general mechanism of leukemogenesis. Alternatively, cohesin may silence oncogenes. The activity of cohesin/CTCF is regulated by polo kinase 1, which interacts with CEP170 [5] and is coordinated with epigenetic marks [10–12], which is in agreement with current views of oncogenesis [14,15]. Thus, abnormal cohesin function may be involved in the same pathway as TET2 or ASXL1. To extend and validate our results, we surveyed published genomic studies of myeloid malignancies for alterations of cohesin genes. We found that one similar small deletion at 8q24 including RAD21 [16] and one at Xq25 including STAG2 [17] have been found in independent series of 157 and 86 AMLs, respectively. The method used to detect these CNAs allowed the conclusion that the deletions were acquired. In these studies, the involvement of cohesin was not suggested. In array studies of myeloid diseases [1,5,6,16,17], recurrence is overall low but quite essential for identifying candidate target genes. Even if rare (1%), alterations of cohesin genes are truly recurrent and found in three independent series [16,17 this work]. These results point for the first time to cohesin as a new player in leukemogenesis and contribute to establish the comprehensive catalog of pathogenic genes. They provide an incentive to look thoroughly for alterations (mutations?) in the genes encoding members of the protein complex and its associated network (e.g., CTCF), as well as to define the function of cohesin in hematopoiesis.

Materials and Methods Panels of samples. We collected samples from 63 MDS, 53 CMML, and 51 AML. According to the WHO classification, the MDS panel comprised five refractory anemia (RA), 13 RA with ring sideroblasts (RARS), six refractory cytopenia with multilineage dysplasia (RCMD), 16 RA with excess of blasts type 1 (RAEB1), 19 RA with excess of blasts type 2

TABLE I. Summary of Results MDS

CMML

AML

Total

Total number of studied samples 63 53 51 167 No CNA 41 (65%) 37 (69.8%) 35 (68.6%) 113 (67.7%) ASXL1 loss 1 0 1 2 NF1 loss 0 2 0 2 RB1 loss 0 2 0 2 TET2 loss 1 1 0 2 Cohesin component loss 0 1 1 2


(RAEB2), and 4 MDS-unclassified (MDS-U) cases. The 53 CMML panel comprised 24 myelodysplastic (MD) forms (leucocytosis count 13 3 109/L) (6). The AML panel comprised 39 cases of primary AMLs and 12 of secondary AMLs. It included 40 cases with normal karyotype and 10 cases with trisomy 8 and 1 with 20q deletion as a sole karyotypic abnormality. All patients signed an informed consent and the study was approved by our institutional review board.

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Figure 1. aCGH profiles showing loss of genes encoding cohesin subunits. A. CMML HD-0259 shows a loss of one copy of the RAD21 gene (arrows). B. AML HD-0120 shows the complete loss (male patient) of the STAG2 gene (arrows). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Sequencing. We searched for mutations in 12 genes that have a demonstrated role in leukemogenesis: ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2, and WT1. We also sequenced RAD21 in 47 MDS, 38 CMML, and 10 AML samples. Sequencing was done as described [1]. The sequence of all primers is available on simple request.

Author Contributions JA did the genomic profiling and data analysis. JR and NC did the sequencing experiments and data analysis. VGB, AM, and MJM are biologists and were responsible for biological features collections. NV is physician in charge of the patients and clinical data. DB, MJM, and MC did project planning, integrated data analysis and manuscript writing. 1

Figure 2. The mitotic cohesin complex is constituted of four core subunits (SMC1, SMC3, RAD21, and either STAG1 or STAG2), which embrace DNA and chromatin (Reproduced from Ref. 7, with permission from Cold Spring Harbor Laboratory Press). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Array-comparative genomic hybridization. Genomic imbalances were analyzed by using 244K CGH Microarrays (Hu-244A, Agilent Technologies, Massy, France) as previously described [1,5]. Some of the data have already been reported [1,5,6].

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Centre de Recherche en Cance´rologie de Marseille, Laboratoire d’Oncologie Molećulaire, UMR891 Inserm, Institut Paoli-Calmettes, Marseille, France; 2 De´partement de BioPathologie, Institut Paoli-Calmettes, Marseille, France; 3 Faculte´ de Me´decine, Universite´ de la Me´diterraneé, Marseille, France; 4 De´partement d’He´matologie, Institut Paoli-Calmettes, Marseille, France Additional supporting information may be found in the online version of this article. Grant sponsor: Inserm, Institut Paoli-Calmettes. Grant sponsor: Association pour la Recherche contre le Cancer Grant number: 4992, AM. Grant sponsor: Fondation de France (comite´ leuce´mie, DB) *Correspondence to: Marie-Joelle Mozziconacci, Institut Paoli-Calmettes, 232 Bd de Sainte-Marguerite, 13009 Marseille, France E-mail: [email protected] Conflict of interest: Nothing to report. Published online 18 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21798


letters References 1. Gelsi-Boyer V, Trouplin V, Ade´laı¨de J, et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 2009;145:788–800. 2. Makishima H, Cazzolli H, Szpurka H, et al. Mutations of E3 ligase Cbl family members constitute a novel common pathogenic lesion in myeloid malignancies. J Clin Oncol 2009;27:6109–6116. 3. Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid cancers. N Engl J Med 2009;360:2289–2301. 4. Mardis ER, Ding L, Dooling DJ, et al. Recurrent mutations found by sequencing an acute myeloid leukemia genome. N Eng J Med 2009;361:1058–1066. 5. Gelsi-Boyer V, Trouplin V, Ade´laı¨de J, et al. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes. BMC Cancer 2008;8:299–314. 6. Carbuccia N, Trouplin V, Gelsi-Boyer V, et al. Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias. Leukemia 2010;24: 469–473. 7. Peters JM, Tedeschi A, Schmitz J. The cohesin complex and its roles in chromosome biology. Genes Dev 2008;22:3089–3114. 8. Barber TD, McManus K, Yuen KWY, et al. Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers. Proc Natl Acad Sci USA 2008;105:3443–3448.

9. Horsfield JA, Anagnostou SH, Kuang-Hsien Hu J, et al. Cohesin-dependent regulation of Runx genes. Development 2007;134:2639–2649. 10. Recillas-Targa F, De La Rosa-Velazquez IA, Soto-Teyes E, Benitez-Bribiesca L. Epigenetic boundaries of tumour suppressor genes promoters: The CTCF connection and its role in carcinogenesis. J Cell Mod Med 2006;3:552–566. 11. Zlatanova J, Caiafa P. CTCF and its protein partners; divide and rule? J Cell Sci 2009;122:1275–1294. 12. Hakimi MA, Bochar DA, Schmiesing JA, et al. A chromatin remodelling complex that loads cohesin onto human chromosomes. Nature 2002;418:994–998. 13. Schmidt D, Schwalie PC, Ross-Innes CS, et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res 2010;20:578–588. 14. Acquaviva C, Gelsi-Boyer V, Birnbaum D. Myelodysplastic syndromes: Lost between two states? Leukemia 2010;24:1–5. 15. Figueroa ME, Lugthart S, Li Y, et al. DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemias. Cancer Cell 2010;17: 13–27. 16. Bullinger L, Kro¨nke J, Scho¨n C, et al. Identification of acquired copy number alterations and uniparental disomies in cytogenetically normal acute myeloid leukemia using high-resolution single-nucleotide polymorphism analysis. Leukemia 2010;24:438–449. 17. Walter MJ, Payton JE, Ries RE, et al. Acquired copy number alterations in adult acute myeloid leukemia genomes. Proc Natl Acad Sci USA 2009;106: 12950–12956.

Clinical significance of clonality and Epstein-Barr virus infection in adult patients with hemophagocytic lymphohistiocytosis Jae-Sook Ahn,1y Sung-Yoon Rew,1y Myung-Geun Shin,2,3 Hye-Ran Kim,3 Deok-Hwan Yang,1 Duck Cho,2 Soo-Hyun Kim,2 Soo Young Bae,1 Se Ryeon Lee,4 Yeo-Kyeoung Kim,1 Hyeoung-Joon Kim,1 and Je-Jung Lee1,3* We assessed the clinical significance of T or B cell clonality and Epstein-Barr virus (EBV) infection in adult patients with hemophagocytic lymphohistiocytosis (HLH) to identify factors related to prognosis. A total of 30 adult patients with diagnosed HLH were included in the study. In all patients, EBV-DNA in peripheral blood was examined by quantitative real-time polymerase chain reaction and bone marrow cells were examined for clonal rearrangement of T cell receptor gamma (TCRG) and immunoglobulin heavy chain (IGH) genes. TCRG clones were detected in 10 patients (33.3%) and IGH clones were detected in 8 patients (26.7%). We found no correlation between clonality and patient outcome. The patients less than 1,000 copies (mL)21 of EBVDNA showed a significantly higher clinical response (P 5 0.008) and longer overall survival (P 5 0.01) than those with high viral load of EBV-DNA. Our results suggest that TCRG and IGH rearrangement do not have any clinical significance in adult patients with HLH, but that high viral load of EBV-DNA may be a risk factor for poor outcomes. In HLH, high viral load of EBV-DNA should thus suggest a prompt approach with aggressive therapeutic interventions. In adults, HLH is often associated with a variety of infections, malignant neoplasms, drugs, autoimmune diseases, and various immunodeficiencies [1–4]. Lymphomas have been reported in secondary HLH associated with malignant disease, mostly in adults. Another common cause of secondary HLH is EBV associated HLH. The EBV genome can be detected in more than 80% of patients with T/NK cell lymphoma and may play a major role in the development of lymphoma-associated HLH. In fact, a substantial percentage of HLH may relapse or progress to T cell lymphoma in months to years [5]. Unfortunately, it is difficult to make a diagnosis of lymphoma-associated HLH, because fatal conditions delayed to perform the tissue biopsy and it took a long time to confirm diagnosis. A lack of histological proof of lymphoma can delay the choice of appropriate treatment for lymphomaassociated HLH at the initial stage. So, we hypothesized that early diagnosis may be supported by molecular study of the EBV genome and the clonal rearrangement of immunoglobulin (IG) and T cell receptor (TCR) genes. In this study, we assessed the clinical significance of T or B cell clonality and EBV infection in adult patients with HLH to identify factors related to prognosis. Thirty patients were enrolled in this study. There were 13 men and 17 women. The median age of the patients was 43 years (range, 17–75 years). Eighteen patients had viral infections (16 with EBV, 1 with cytomegalovirus, and 1 with Hantaan virus). Four patients had a malignant lymphoma (2 with


a diffuse large B cell lymphoma, 1 with an extranodal NK/T cell lymphoma, and 1 with a peripheral T cell lymphoma). One patient also had Adult-onset Still’s disease (AOSD). The remaining 7 patients (idiopathic HLH) had neither an underlying infection nor neoplasm. Thirteen (43.3%) of 30 patients were showed high viral copies for EBV-DNA [1000 (mL)21], including one patient with lymphoma-associated HLH and one AOSD-associated HLH. There were no significant differences in laboratory and clinical findings between high-and low-viral load groups. The results for clonal detection by use of the specific primer set are summarized in Table I. TCRG clones were detected in 10 (33.3%) of 30 cases. In four cases, clonality was demonstrated by more than one primer set. IGH clones were detected in 8 (26.7%) of 30 cases. In one case, clonality was demonstrated by two primer sets. Among the cases of TCRG clonality, three cases were simultaneously detected by IGH rearrangement. One of eight cases with IGH clones and three of 10 cases with TCRG clones showed exceeded 1000 (mL)21 of EBV-DNA. Of the 28 patients treated, the overall clinical response rate was 50% (14 patients) and the rate of inactive disease was 39.3% (11 patients). We analyzed, the response rate according to whether exceeded 1000 (mL)21 of EBV-DNA. As shown in Table II, the patients with < 1000 copies (mL)21 of EBV-DNA had a significantly higher clinical response rate than 1000 copies (mL)21 (P 5 0.008). With a median follow-up of 4.6 months (range, 0.1– 93.1 months), the median overall survival of the 28 patients was 6.9 months and the estimated overall survival rate at 1 year was 47.6 ± 9.7%. When we analyzed the factors affecting survival, none of the clinical features or laboratory findings was found to affect survival except for excess viral load of EBV-DNA. Patients with 1000 (mL)21 copies of EBV-DNA showed poor overall survival compared with those with low EBV-DNA load (P 5 0.010) (Fig. 1). However, the clonality of TCRG and IGH did not have any prognostic role in patients with HLH (Fig. 2). EBV-infected LMP-1 expressed T cells may escape from TNF-a-induced cytokine injuries and, therefore, EBV-infected T cells tend to survive and proliferate, explaining the relapsing or disease progression from HLH to T cell lymphoma [5]. The prognosis of lymphoma-associated HLH is fatal and the overall survival rate to be less than 10% [6]. It is difficult to make a diagnosis of lymphoma-associated HLH from other causes of HLH. Detection of lymphocyte clonality by analysis of rearranged TCR and IG plays a critical role in the diagnosis and differential diagnosis of lymphoma [7–9]. The molecular study of clonal rearrangement for TCR and IGH may support the early diagnosis of lymphoma and may even contribute to detecting hidden malignancies of lymphoid lineage. In EBV-associated HLH, clonality study by

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letters TABLE I. Summary Data of EBV-DNA and Clonality in 30 Patients with HLH

Age/Sex

Cause

EBV-DNA RQ PCR titer (copies mL21)

1 2 3

58/F 42/M 21/F

EBV Idiopathic EBV

5,384,232 0 16,997,475

4

20/F

EBV

175,487

Case No.

5

59/F

Idiopathic

0

6 7 8

75/M 17/M 42/M

EBV EBV Hantan virus

1,490 187,147 0

9 10 11 12 13 14 15 16 17 18

62/F 22/F 27/F 71/F 66/M 58/M 57/F 25/M 48/F 70/M

EBV Idiopathic EBV EBV EBV EBV EBV Lymphoma AOSD EBV

1,468 0 794 163 452 412,515 7,333,612 92 17,755 120

19 20 21 22 23 24 25 26 27 28 29 30

30/F 27/F 43/F 50/F 61/M 17/F 26/F 36/F 50/M 34/M 56/M 46/M

CMV EBV EBV EBV Idiopathic Idiopathic Idiopathic EBV Idiopathic NK/T cell lymphoma DLBCL DLBCL

0 1,499,055 9,885 63 0 0 0 46,840 0 143,315 452 87

TCRG rearrangement 2 Vg9 1 Jg1.1/2.1 2

IGH rearrangement

OS (ms)

Death Survival Death

0.5 53 3

Survival

45

2 2 DH1–6 2 DH1–6 DH7 2 2 FR2 2 2 2 2

Etoposide 1steroid1IVIG NA Steroid1IVIG Steroid Etoposide1steroid1IVIG Etoposide1steroid1IVIG1CsA Etoposide1steroid1IVIG NA Steroid Etoposide1steroid1IVIG

Survival NA Survival Death Death Death Death NA Survival Death

0.23 NA 26 0.5 1 2 0.1 NA 17 0.2

Etoposide1steroid Etoposide1steroid1IVIG Etoposide1steroid1IVIG Steroid Steroid1IVIG1CsA Steroid1IVIG Etoposide1steroid1IVIG Etoposide1steroid1IVIG Alem-CHOP CHOP ? Allo-BMT R-CHOP ? Auto SCT R-CHOP ? Auto SCT

Survival Death Death Survival Survivala Survival Death Death Survivala Death Survival Survival

93 2 0.3 10 8 40 0.4 1 2 7 32 9

2 2

2 Vg10 1 Jg 1.1/2.1 Vg10 1 Jg 1.1/2.1, Vg9 1 Jg1.1/2.1 Vg1–8 1 Jg1.1/2.1 2 2 2 Vg10 1 Jg 1.1/2.1 2 2 Vg9 1 Jg1.3/2.3 2 Vg10 1 Jg1.3/2.3, Vg9 1 Jg1.3/2.3 2 2 2 2 Vg9 1 Jg1.1/2.1 2 2 2 2 2 2 Vg10 1 Jg1.3/2.3, Vg1–8 1 Jg1.3/2.3, Vg1–8 1 Jg1.1/2.1

Outcome

Conservative Conservative Etoposide1steroid1 IVIG ? CHOP Etoposide1steroid1 IVIG1CsA ? Allo-BMT Etoposide1steroid1 IVIG1CsA ? Alem-CHOP Conservative Etoposide 1steroid1IVIG Conservative

2 DH1–6 2

Vg1–8 1 Jg1.1/2.1, Vg9 1 Jg1.3/2.3 2

Therapeutic method

2 2 2 2 2 2 DH7 2 2 2 FR2 FR3, DH1–6

a

Survival

15

Death Death Survival

0.1 7 57

a

Patient survived with active disease. F, female; M, male; EBV, Epstein Barr virus; AOSD, adult-onset Still’s disease; CMV, cytomegalovirus; DLBCL, diffuse large B cell lymphoma; PCR, polymerase chain reaction; TCRG, T cell receptor gamma; IGH, immunoglobulin heavy chain; OS, overall survival; IVIG, intravenous immunoglobulin; CHOP, cyclophosphamide 1 Adriamycin 1 vincristine 1 prednisone; CsA, cyclosporin A; Allo-BMT, allogeneic bone marrow transplantation; Alem, alemtuzumab; NA, not available; Auto-SCT, autologous stem cell transplantation.

TABLE II. Response and Clinical Outcome of 28 Patients with HLH EBV-DNA 1000 copies mL21 (n 5 13)

EBV-DNA 150 8 16 >150 27 >150

Figure 1. Time course of creatinine first and second indicate periods of eltrombopag administration, respectively. C, cephalexin; A, azithromycin; L, levothyroxine.

for drug-induced nephrotoxicity, eltrombopag was tapered with discontinuation of the medication on September 2, at which point his serum creatinine was 2.7 mg/dL. The median platelet count [interquartile range (IQR)] during the 3 weeks immediately prior to therapy was 49 (42–50) cells/mm3 as compared with 63 (58–65) cells/mm3 while on eltrombopag (P 5 0.004). Immature platelet fractions did not differ between pretherapy and on-therapy measurements (7.2% vs. 5.0%, respectively), and the median INR prior to and during therapy was similar (2.1 [2.1–2.6] vs. 2.45 [2.1–2.9], respectively; P 5 0.7). His creatinine remained fairly stable over the ensuing months, and on December 1 with a creatinine of 2.7 mg/dL, another attempt at eltrombopag therapy was initiated at a dose of 25 mg daily and later increased to 50 mg daily. By January 15, 2010, the creatinine had increased to 3.8 mg/dL and eltrombopag was again tapered. The creatinine peaked at 4.4 mg/dL 4 days after eltrombopag was discontinued, improved to 3.6 within a week, and then reached a nadir of 3.2 mg/dL by April. A statistically significant difference in platelet count was not seen with this course of therapy (45 [30–66] cells/mm3 pretherapy vs. 38 [36–46] cells/mm3; P 5 0.7), and immature platelet fractions were similar to pre- and on-therapy (5.8% vs. 5.2%, respectively). Similarly, the pretherapy INR did not differ from the on-therapy INR (3.3 [2.6–3.7] vs. 3.25 [2.7–3.9], respectively; P 5 0.8). As the relationship of 1/creatinine versus time is roughly linear, the rate of change (slope) in 1/creatinine during time on and off eltrombopag can be compared. While on eltrombopag from July 10, 2009 to August 31, 2009, kidney function declined at a rate of 20.0032 dL/mg/day when compared with an improvement of 0.00036 dL/mg/day after eltrombopag was discontinued. The median creatinine on therapy was 2.3 mg/dL (2.1–2.7) vs. 2.7 (2.5–2.8) after discontinuation of eltrombopag (P 5 0.002). While on therapy again from December 1, 2009 to January 22, 2010, kidney function declined at a rate of 20.0022 dL/mg/day when compared with an improvement of 0.00044 dL/mg/day after cessation of eltrombopag. The median creatinine on eltrombopag was 3.1 mg/dL (2.9–3.5) vs. 3.5 (3.3–3.6) after discontinuation of the medication (P 5 0.02). Thus, while a permanent decrease in kidney function occurred during administration of eltrombopag, the function stabilized without evidence of ongoing deterioration in the weeks following discontinuation of the medication. As seen in Fig. 1, the second course of eltrombopag was notable for intercurrent use of cephalexin, azithromycin, and levofloxacin for the treatment of cellulitis and bronchitis.


We report the first case to our knowledge of an APS patient with apparently immune thrombocytopenia who developed acute renal injury during eltrombopag therapy. Eltrombopag is an oral, low molecular weight, synthetic agonist of the thrombopoietin receptor that increases platelet counts by stimulating megakaryocyte proliferation through activation of the Janus kinase 2—Signal transducer and activator of transcription 5 (Jak2-Stat5) pathway. It is approved for use in ITP, and randomized controlled studies have demonstrated efficacy in patients with ITP and in patients with thrombocytopenia in the setting of hepatitis C–associated cirrhosis [8,9]. The thrombopoietin receptor is not believed to be expressed in the kidney, although low levels of mRNA can be detected by reverse transcriptase-polymerase chain reaction in human kidney tumor cell lines [10]. Approximately, 30% of the drug is excreted by the kidneys after hepatic metabolism, and studies in both healthy individuals and patients with ITP have shown no effects of eltrombopag on platelet function [11,12]. Eltrombopag appears to be well tolerated with the most commonly reported side effects being headache, nausea, and vomiting. It does carry a black box warning of risk for hepatotoxicity but no episodes of renal dysfunction have been noted in the published trials [13]. While the renal injury observed in this patient cannot be ascribed to eltrombopag with certainty, the time course and recurrence on rechallenge with the drug are concerning for eltrombopag-associated nephrotoxicity. While antibiotics and systemic infection are common etiologies of acute kidney injury, the change in creatinine during the second course of eltrombopag closely mirrors the dates of eltrombopag administration. No hypotension or hypoxemia was documented in the course of his outpatient care, and of note, no concomitant antibiotics were administered during the first period of eltrombopag usage. As such, the clinically limited, intercurrent infections during the second course were not felt to be the likely etiology of renal function decline. A small increase in creatinine was also observed in late October 2009 between the two courses of eltrombopag. This was in the setting of intravenous diuresis for volume overload, and the creatinine returned to baseline following transition to an oral diuretic regimen. Potential mechanisms of eltrombopag nephrotoxicity remain speculative. As his baseline kidney dysfunction was clinically consistent with thrombosis of small intrarenal vessels, reconstitution of circulating platelets may predispose to worsening disease in the setting of inadequate anticoagulation. This hypothesis is supported by studies suggesting platelet activation by antiphospholipid antibodies may also contribute to the thrombocytopenia associated with APS. Although the platelet count was statistically significantly increased only during the first course of eltrombopag therapy, increased production in association with simultaneous consumption within the kidney during the second exposure cannot be excluded. The INR was statistically unchanged both prior to and during treatment with eltrombopag. Particularly during the first course, the INR of 2.1 was less than his target INR range of 3.0–4.0 and may have been insufficient to prevent intrarenal thrombosis. In addition, nephrotoxicity may have been a manifestation of drug-induced interstitial nephritis and not dependent on the pharmacological mechanism of eltrombopag’s action. Only 50% of patients with drug-induced interstitial nephritis have pyuria, and the absence of rash and fever does not significantly lessen the likelihood of interstitial nephritis [14]. A formal diagnosis often requires kidney biopsy which was not possible given the thrombocytopenia and need for ongoing anticoagulation. Eltrombopag appears to be highly specific for the thrombopoietin receptor and as this receptor is not believed to be expressed in the kidney, it is unlikely that potential eltrombopag nephrotoxicity is a manifestation of intrarenal signaling [15]. As such, this case demonstrates a clinical association between eltrombopag administration and nephrotoxicity in a patient with APS and ITP. While a definitive association with eltrombopag cannot be made, healthcare providers should be alert to the possibility of worsening kidney function in patients with APS and thrombocytopenia treated with eltrombopag. Clinical circumstances often require the use of medications in patient populations in whom the safety of the medication has not been studied and such circumstances always warrant additional caution and close observation.

Methods Statistical analysis was performed with Stata 11.0 (StataCorp; College Station, TX). Serum creatinine, platelet count, and INR were analyzed by Wilcoxon rank-sum testing and are reported as median (interquartile range). P 0.05 was considered statistically significant.

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letters 1 Department of Medicine, Division of Nephrology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Department of Medicine, Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, Maryland *Correspondence to: C. John Sperati, 1830 E Monument St, Rm 416, Baltimore, MD 21205. E-mail: [email protected] Conflict of interest: Nothing to report. Published online 10 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21789

References 1. Giannakopoulos B, Passam F, Ioannou Y, Krilis SA. How we diagnose the antiphospholipid syndrome. Blood 2009;113:985–994. 2. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. 3. Cervera R, Piette JC, Font J, et al. Antiphospholipid syndrome: Clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients. Arthritis Rheum 2002;46:1019–1027. 4. Amigo MC, Garcia-Torres R, Robles M, et al. Renal involvement in primary antiphospholipid syndrome. J Rheumatol 1992;19:1181–1185. 5. Uthman I, Godeau B, Taher A, Khamashta M. The hematologic manifestations of the antiphospholipid syndrome. Blood Rev 2008;22:187–194. 6. Kavanaugh A. Danazol therapy in thrombocytopenia associated with the antiphospholipid antibody syndrome. Ann Intern Med 1994;121:767–768.

7. Trappe R, Loew A, Thuss-Patience P, et al. Successful treatment of thrombocytopenia in primary antiphospholipid antibody syndrome with the anti-CD20 antibody rituximab—Monitoring of antiphospholipid and anti-GP antibodies: A case report. Ann Hematol 2006;85:134–135. 8. McHutchison JG, Dusheiko G, Shiffman ML, et al. Eltrombopag for thrombocytopenia in patients with cirrhosis associated with hepatitis C. N Engl J Med 2007;357:2227–2236. 9. Bussel JB, Cheng G, Saleh MN, et al. Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura. N Engl J Med 2007;357:2237–2247. 10. Graf G, Dehmel U, Drexler HG. Expression of thrombopoietin and thrombopoietin receptor MPL in human leukemia-lymphoma and solid tumor cell lines. Leuk Res 1996;20:831–838. 11. Jenkins JM, Williams D, Deng Y, et al. Phase 1 clinical study of eltrombopag, an oral, nonpeptide thrombopoietin receptor agonist. Blood 2007;109:4739– 4741. 12. Erhardt JA, Erickson-Miller CL, Aivado M, et al. Comparative analyses of the small molecule thrombopoietin receptor agonist eltrombopag and thrombopoietin on in vitro platelet function. Exp Hematol 2009;37:1030– 1037. 13. GlaxoSmithKline. Promacta (Eltrombopag Tablets): US Prescribing Information. Available at:http://us.gsk.com/products/assets/us_promacta.pdf. Accessed on May 19, 2010. 14. Rossert J. Drug-induced acute interstitial nephritis. Kidney Int 2001;60:804– 817. 15. Erickson-Miller CL, Delorme E, Tian SS, et al. Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist. Stem Cells 2009;27:424–430.

Screening coagulation testing using the APTT: Which reagent to choose? Francine R. Dembitzer,1 Yvelisse Suarez,1 Louis M. Aledort,2 and Ellinor I.B. Peerschke1* APTT testing is integral to hemostasis testing. A prolonged result, however, can be difficult to interpret, depending on the APTT reagent’s sensitivity to the lupus anticoagulant. This often generates additional laboratory testing for both factor deficiencies and the presence of a lupus anticoagulant, and in so doing, delays patient management. We have found it useful to provide APTT testing with both a lupus anticoagulant sensitive and insensitive reagent, to facilitate the rapid exclusion of significant factor deficiencies. The following case report illustrates the utility of this approach and provides a backdrop for necessary discussions between laboratories and clinicians regarding which APTT reagent best meets their clinical need for screening hemostasis testing. The APTT is traditionally used to screen for significant coagulation factor deficiencies. APTT reagents are available from a variety of manufacturers and generally detect deficiencies in intrinsic pathway coagulation factors below 35%. Reagents, however, vary in their sensitivity to circulating anticoagulants, particularly the lupus anticoagulant [1]. As lupus anticoagulants are not associated with bleeding but rather with an increased risk for thrombosis [2,3], laboratories need to decide whether to routinely perform the screening APTT with lupus anticoagulant sensitive or insensitive reagents. The decision is further impacted by practical considerations involving time required to evaluate a prolonged screening APTT before patients are cleared for surgery or other invasive procedures. In our center, we have found that offering APTT testing with both a lupus anticoagulant sensitive and insensitive reagent allows for the rapid rule out of a factor deficiency and provides presumptive evidence for the presence of a lupus anticoagulant. To illustrate the utility of this approach, we present the following case report. The patient is a 49-year-old woman (height 50 5@, weight 170 lbs) with a dilated cardiomyopathy and stage D systolic heart failure who required a biventricular pacemaker. She had no bleeding history and is a candidate for a heart transplant. At the time of presentation, her blood pressure was 100/ 70 and pulse 68. She had an S3 gallop and a soft systolic murmur with signs of cardiac decompensation. Her preoperative evaluation was remarkable for a prolonged APTT (132.3 sec., reference range 23.6–35.7 sec) using a lupus anticoagulant sensitive reagent. A Hematology consult was called, and the

726

APTT was repeated with a lupus insensitive reagent. The result was within the reference interval (31.1 sec., reference range 23.2–32.0 sec). The patient was cleared for surgery by the hematology consultant, and the pacemaker was placed without bleeding or other complications. The laboratory evaluation of a prolonged APTT screening test is time consuming. It usually begins with a mixing study. Patient plasma and pooled normal plasma are combined in a 1:1 ratio, and an APTT is performed on the mix [4,5]. Results of this APTT mix are used to differentiate between an APTT prolongation due to the presence of a circulating anticoagulant, such as a lupus anticoagulant, or factor deficiency(cies). Because of the inherent instability of plasma, mixing studies require manual technical intervention and are not usually available on automated analyzers. The mix is made manually when indicated with freshly thawed pooled plasma, and depending on laboratory staffing and practices, may only be performed in a special coagulation laboratory that does not operate 24 h per day, thus limiting availability. Moreover, mixing study results are often difficult to interpret [4], especially when a partial correction occurs. In these situations, comparison of screening APTT results obtained with a lupus anticoagulant sensitive and insensitive reagent can be extremely informative to rapidly rule out significant intrinsic pathway factor deficiencies. APTT reagents vary in sensitivity to lupus anticoagulant depending on their phospholipid concentration and source [1]. The choice of a particular APTT reagent, therefore, prioritizes the need to detect either a lupus anticoagulant or factor deficiency, particularly in a situation where the rapid identification of factor deficiencies is essential, i.e., preoperatively. In our laboratory, APTT screening is offered using both lupus anticoagulant sensitive (STA1 PTT, Stago/Roche) and insensitive (Dade1 Actin1 FS Activated PTT, Siemens/Dade-Behring) reagents. First line APTT screening is performed with a lupus anticoagulant sensitive reagent. The mixing study is performed with the same reagent but includes a second APTT screening test using the lupus anticoagulant insensitive reagent. The concomitant use of the lupus anticoagulant insensitive reagent permits for both the rapid screening of a possible lupus anticoagulant and will readily identify a factor deficiency or deficiencies. Each laboratory must decide the best approach to APTT testing based on local patient care considerations. Is it more important to detect factor deficiencies rapidly than to identify the possibility of a lupus anticoagulant? Is


letters lupus anticoagulant testing, including APTT screening with a LA sensitive

Published online 4 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21780

reagent better performed as part of a specialized thrombophilia work up? Even lupus anticoagulant sensitive APTT reagents are not universally sensitive to lupus anticoagulants [6]. Opinions and needs will vary. Regardless of the testing algorithms selected, a laboratory choosing to detect a lupus anticoagulant with their screening APTT, might consider reflex testing with a lupus anticoagulant insensitive reagent when the screening APTT is prolonged, to achieve a more rapid evaluation of factor deficiencies, particularly in patients with suspected bleeding or in a pre-surgical setting. Alternatively, a laboratory might offer a lupus anticoagulant-insensitive APTT for hemostasis screening and use the lupus sensitive APTT reagent only when LA testing is ordered. 1 Department of Pathology, The Mount Sinai School of Medicine New York, New York; 2Division of Hematology, Department of Medicine The Mount Sinai School of Medicine, New York *Correspondence to: Ellinor I.B. Peerschke, Department of Pathology, The Mount Sinai School of Medicine, New York, New York *E-mail: [email protected] Conflict of interest: Nothing to report.

References 1. Brandt JT, Triplett DA, Rock WA, et al. Effect of lupus anticoagulants on the activated partial thromboplastin time. Arch Pathol Lab Med 1991;115:109– 114. 2. Giannakipoulos B, Passam F, Ioannou U, et al. How we diagnose the antiphospholipid syndrome. Blood 2009;113:985–994. 3. Devreese K, Hoylaerts MF. Laboratory diagnosis of the antiphospholipid syndrome: A plethora of obstacles to overcome. Eur J Haematol 2009;83: 1–16. 4. Ledford-Kraemer MR. Laboratory testing for lupus anticoagulants: Preexamination variables, mixing studies, and diagnostic criteria. Semin Thromb Hemost 2008;34:380–388. 5. Tripodi A. Laboratory testing for lupus anticoagulants: Diagnostic criteria and use of screening, mixing, and confirmatory studies. Semin Thromb Hemost 2008;34:373–378. 6. Favaloro EJ, Wong RCW. Laboratory testing and identification of antiphospholipid antibodies and the antiphospholipid syndrome: A potpourri of problems, a compilation of possible solutions. Semin Thromb Hemost 2008;34:389–410.

Hepatosplenic T cell lymphoma responsive to 20 -deoxycoformycin therapy Michael Bennett,1* Estella Matutes,2 and Philippe Gaulard3 Hepatosplenic T cell lymphoma (HSTL) is a rare condition usually with an aggresssive course and a poor prognosis even after extensive treatment. We describe here a patient who presented with hemophagocytosis. The lymphoma had unusual phenotypic features, an indolent course and responded to 20 -deoxycoformycin therapy as a single agent. We suggest that this therapy be used in further cases as part of the treatment strategy. HSTL is a rare neoplasm of cytotoxic T cells that was recognized initially in 1990 [1]. Although first described as having a gd phenotype, a minority of cases were instead found to have ab receptors [2–4]. The malignant cells in this condition usually express cytotoxic proteins such as TIA1 and granzyme M but are negative for granzyme B and perforin [5–7]. Isochromosome 7q is present in most cases [8,9]. Somatic TRG@ and TRB@ gene rearrangements are found in both gd and ab variants. Biallelic TRG@ rearrangements are specific for gd cases. The cells appear as medium-sized lymphocytes with abundant pale cytoplasm; a few granules may be present but these are not prominent. There is a characteristic sinusoidal infiltration in the spleen, liver, and bone marrow. Patients present with marked splenomegaly, hepatomegaly but with no lymphadenopathy [5,10]. The disease with the gd phenotype occurs predominantly in young adult males. In the ab form, the female incidence increases. The course is aggressive [5,10,11]. Treatment for this condition is disappointing with a median overall survival of about 1 year even after aggressive regimes of treatment [10,12]. Our case was a 45-year-old caucasian woman who presented with B symptoms, pancytopenia, and a massive splenomegaly palpable in the abdomen 16 cm below the costal margin without lymphadenopathy. Initially, Hb was 9.6 g/dL, WBC 1.95 3 109/L, absolute neutrophil count was 0.69 3 109/L, and platelets were 66 3 109/L. The LDH (lactate dehydrogenase) was normal and there was no evidence of disturbed hepatic or renal function apart from a slightly raised serum alkaline phosphatase 160 U/L (normal 40–150). A bone marrow aspirate and trephine showed no diagnostic features. Splenectomy was performed 2 months later. The excised spleen weighed 1.65 Kg. Initial histology findings were of an increase in macrophages with erythrophagocytosis in the red pulp and a diagnosis of hemophagocytic syndrome was entertained.


Post splenectomy, there was an improvement in peripheral blood parameters and the B symptoms resolved. The patient was asymptomatic for 1 year and then started to complain of lassitude and developed an increasing lymphocytosis. These cells were medium to large in size with condensed chromatin, no nucleoli, occasional nuclear projections, abundant pale cytoplasm, and a faint granulation. On flow cytometry, they were positive for CD2, CD3, CD7, CD16, CD56, CD94, CD161, CD158b, and TIA1 and negative for CD5, CD4, CD8, CD57, CD25, HLA DR, CD1a, TdT, CD34, CD52, TCR a/b, TCR g/d, and B cell markers. Analysis by PCR (polymerase chain reaction) showed TRB@ and TRG@ rearrangements consistent with a monoclonal T cell population. A further bone marrow examination and a review of the splenic histology revealed a marked intrasinusoidal infiltration by T cells characteristic of HSTL (Fig. 1). Malignant cells were granzyme B positive. The phenotype otherwise on the histological sections was similar to that of the peripheral blood except for CD56, which was not detected in histological sections and positive in the blood. In situ hybridization with EBER (EBV-encoded RNA) probes did not show EBV (Epstein-Barr virus) association. FISH (fluorescent in situ hybridization) analysis on histological sections and on peripheral blood cells did not show evidence of an isochromosome 7q. Later investigation showed a TCR (T cell receptor) delta rearrangement confirming the gd origin of the lymphoma. At 2 years from the initial presentation, mild elevations of serum SGOT (serum glutamic oxaloacetic transaminase) (55 U/L normal 0–37), SGPT (serum glutamic pyruvate transaminase) (47 U/L normal 0–43), LDH (644 U/ L normal 230–480), and alkaline phosphatase (165 U/L normal 40–150) were noted. The white cell count had by then risen to 94.26 3 109/L with an absolute lymphocyte count of 80.37 3 109/L, neutrophils 2.52 3 109/L, Hb 14.0 g/dl, and platelets 185 3 109/L. Lymphocyte morphology appeared unchanged. At this stage, treatment with 20 -deoxycoformycin as a single agent was started (see Fig. 2). This was given at a dose of 4 mg/ m2 and caused a rapid cell lysis within a few days accompanied by a nonseptic febrile reaction. Within 1 month liver function tests became normal. Treatment was continued on a 2–5 weekly basis for 10 months, and the patient remained asymptomatic with an absolute lymphocyte count below 2.0 3 109/L. During this time, repeat PCR for TRG@ in peripheral blood lymphocytes showed an oligoclonal population. An attempt to increase the

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Figure 2.

Absolute lymphocyte count and response to therapy.

The use of 20 -deoxycoformycin has been reported in five patients with HSTL and shown to have cytotoxic activity. Its efficacy has been supported by in vitro studies [17]. The response here as a single agent was dramatic with a complete although temporary clearance of peripheral lymphocytosis and normalization of the liver abnormalities. She even responded to a further two courses of treatment before eventually succumbing to complications following allogeneic transplantation. The effect of 20 -deoxycoformycin was superior to combination therapy with ESHAP. This case supports the activity of 20 -deoxycoformycin in HSTL, which was superior to combination chemotherapy that is used in high-grade lymphomas. It should be considered as initial therapy for this condition. 1

Figure 1. Histopathology. A Spleen: Sheets of medium-sized lymphoid cells in sinuses mixed with histiocytes. Erythrophagocytosis is marked with arrows. H&E stain. Magnification 3100. B Spleen: Lymphoid cells expressing CD3. Magnification 3100. C Spleen: Lymphoid cells not expressing CD5. Magnification 3100. D Bone marrow: Sinusoidal infiltration by small to medium-sized lymphocytes expressing the TIA1 antigen. Magnification 3200. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

interval between the treatments resulted in an increase in the counts, which again responded to a more aggressive schedule of 20 -deoxycoformycin administration. It was decided to proceed to allogeneic stem cell transplantation. Prior to this combination, chemotherapy with ESHAP (etoposide, methylprednisone, cytarabine and cisplatin) was administered to try to reduce the bulk of malignant cells. This treatment caused profound pancytopenia but lymphocytosis returned within 3 weeks. Further therapy with 20 -deoxycoformycin was again successful in controlling the lymphocytosis. High dose therapy with BEAM (BCNU, etoposide, cytarabine, melphalan) was then administered followed by allogeneic stem cell transplantation from an HLA (human leukocyte antigen) compatible sibling. She died of acute graft versus host disease 2 months after transplantation and 43 months from the date of initial presentation. An association with hemophagocytosis and HSTL has been described before [13] and the two conditions may be difficult to differentiate [14]. The indolent course of this patient is unusual for HSTL which is usually aggressive, only a few more chronic courses have been described [15]. The histopathological features of preferential sinusoidal infiltration in the spleen and bone marrow were in favor of HSTL, but there were unusual phenotypic features, in particular, granzyme B expression and a lack of isochromosome 7q. The TCR silent phenotype was also unusual although TRG@ and TRB@ rearrangements were demonstrated. Single cases of this have been previously reported sometimes related with cytological progression over time [1,10]. In agreement with previous findings [16], there was coexpression of several NK markers including CD16, CD56, CD94, CD161, KIR (killer immunoglobulin-like receptor), CD158b, and TIA-1.

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Department of Haematology, Ha’Emek Medical Centre, Afula, Israel; 2 Department of Haematology, The Royal Marsden Hospital, London, United Kingdom; 3Department of Pathology, Henri Mondor Hospital, Paris, France *Correspondence to: Michael Bennett, Ha’Emek Medical Centre, Afula 18101, Israel E-mail: [email protected] Received for publication 16 May 2010; Accepted 19 May 2010 Conflict of interest: Nothing to report Published online 4 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21774

References 1. Farcet JP, Gaulard P, Marolleau JP, et al. Hepatosplenic T-cell lymphoma: Sinusal/sinusoidal localization of malignant cells expressing the T-cell receptor gamma delta. Blood 1990;75:2213–2219. 2. Macon WR, Levy NB, Kurtin PJ, et al. Hepatosplenic alphabeta T-cell lymphomas: A report of 14 cases and comparison with hepatosplenic gammadelta Tcell lymphomas. Am J Surg Pathol 2001;25:285–296. 3. Suarez F, Wlodarska I, Rigal-Huguet F, et al. Hepatosplenic alphabeta T-cell lymphoma: An unusual case with clinical, histologic, and cytogenetic features of gammadelta hepatosplenic T-cell lymphoma. Am J Surg Pathol 2000;24: 1027–1032. 4. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues; IARC:Lyon, France: 2008. 5. Cooke CB, Krenacs L, Stetler-Stevenson M, et al. Hepatosplenic T-cell lymphoma: A distinct clinicopathologic entity of cytotoxic gamma delta T-cell origin. Blood 1996;88:4265–4274. 6. Felgar RE, Macon WR, Kinney MC, et al. TIA-1 expression in lymphoid neoplasms. Identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation. Am J Pathol 1997;150:1893–1900. 7. Krenacs L, Smyth MJ, Bagdi E, et al. The serine protease granzyme M is preferentially expressed in NK-cell, gamma delta T-cell, and intestinal T-cell lymphomas: Evidence of origin from lymphocytes involved in innate immunity. Blood 2003;101:3590–3593. 8. Alonsozana EL, Stamberg J, Kumar D, et al. Isochromosome 7q: The primary cytogenetic abnormality in hepatosplenic gammadelta T cell lymphoma. Leukemia 1997;11:1367–1372. 9. Wlodarska I, Martin-Garcia N, Achten R, et al. Fluorescence in situ hybridization study of chromosome 7 aberrations in hepatosplenic T-cell lymphoma: Isochromosome 7q as a common abnormality accumulating in forms with features of cytologic progression. Genes Chromosomes Cancer 2002;33:243– 251. 10. Belhadj K, Reyes F, Farcet JP, et al. Hepatosplenic gammadelta T-cell lymphoma is a rare clinicopathologic entity with poor outcome: Report on a series of 21 patients. Blood 2003;102:4261–4269.


letters 11. Weidmann E. Hepatosplenic T cell lymphoma. A review on 45 cases since the first report describing the disease as a distinct lymphoma entity in 1990. Leukemia 2000;14:991–997. 12. Falchook GS, Vega F, Dang NH, et al. Hepatosplenic gamma-delta T-cell lymphoma: Clinicopathological features and treatment. Ann Oncol 2009;20:1080–1085. 13. Chin M, Mugishima H, Takamura M, et al. Hemophagocytic syndrome and hepatosplenic gammadelta T-cell lymphoma with isochromosome 7q and 8 trisomy. J Pediatr Hematol Oncol 2004;26:375–378. 14. Nosari A, Oreste PL, Biondi A, et al. Hepato-splenic gammadelta T-cell lymphoma: A rare entity mimicking the hemophagocytic syndrome. Am J Hematol 1999;60:61–65.

15. Weirich G, Sandherr M, Fellbaum C, et al. Molecular evidence of bone marrow involvement in advanced case ot Tgammadelta lymphoma with secondary myelofibrosis. Hum Pathol 1998;29:761–765. 16. Morice WG, Macon WR, Dogan A, et al. NK-cell-associated receptor expression in hepatosplenic T-cell lymphoma, insights into pathogenesis. Leukemia 2006;20:883–886. 17. Aldinucci D, Poletto D, Zagonel V, et al. In vitro and in vivo effects of 20 -deoxycoformycin (Pentostatin) on tumour cells from human gammadelta1 T-cell malignancies. Br J Haematol 2000;110:188–196.

Acute leukemia presenting with extramedullary diseases and completely normal hemogram: An extremely unusual manifestation unique to pre-B ALL Chih-Cheng Chen,1,2,3 Hsu-Huei Weng,4 Cih-En Hwang,1 Chang-Hsien Lu,1,2 Ping-Tsung Chen,1,2 and Jyh-Pyng Gau5,6* Acute lymphoblastic leukemia (ALL) is a clonal hematological disease characterized by inadequate normal hematopoiesis secondary to excessive proliferation of leukemic blasts and their impaired differentiation. As a result, patients usually manifested symptoms related to bone marrow failure. It’s very uncommon for ALL patients to present with normal hemogram. Herein, we describe two patients who presented with excruciating bone pain at orthopedic clinics. Osteopathy involving multiple bones was noted initially, but acute leukemia was never considered as one of the differential diagnoses because of the completely normal hemogram in both cases. Consequently, the diagnosis of leukemia was slightly delayed. Upon literature review, we found that ALL patients with solely extramedullary diseases and nearly normal hemogram had exclusively pre-B disease. We also propose a putative hypothesis for this interesting finding. Osteopathy is not an uncommon initial manifestation of ALL. In a pediatric population, radiological abnormalities in the musculoskeletal system were demonstrated in about 40% of ALL patients [1]. Bone pain, resulting from either bone erosion, periosteal lesions, or massive proliferation of blasts in the medullary canal and under the periosteum, is one of the most common presenting symptoms of ALL. In patients with ALL, who presented with prominent skeletal symptoms, they tended to have no lymphoadenopathy, organomegaly, or leukocytosis. As a result, the diagnosis of leukemia is often delayed [2–4]. Here, we described our two unique cases of ALL, who presented musculoskeletal manifestations with completely normal hemograms. There were no other leukemia-related symptoms that could have led to the suspicion of the disease. Early accurate diagnosis in these patients can be extremely difficult. We would like to use our cases as a reminder that acute leukemia may be taken into account in the differential diagnosis of bone lesions, even in patients with normal hemograms. The first case was a 35-year-old male, who experienced 2-week-long migratory pain in abdomen, neck, and back. Other than exquisitely sensitive tenderness in the back and sacral area, physical examination upon admission was negative. There were no palpable peripheral lymph nodes or hepatosplenomegaly. Lab studies showed completely normal hemogram with a white cell differential count of 71% neutrophils and 26% lymphocytes, although there were markedly elevated levels of alkaline phosphatase (263 U/L; normal < 100 U/L) and lactate dehydrogenase (LDH, 978 U/L; normal < 213 U/L). Although imaging studies, including X-ray examination of the spine, pelvis and sacrum, and abdominal sonography failed to detect any abnormality, a bone scan revealed numerous foci of increased radioactivity scattered in the skull, sternum, rib cage, spine, and bilateral sacroiliac regions, suggesting metastatic bone lesions (Fig. 1A). Systemic work-up failed to identify any primary tumor from other organs. Ten days after admission, follow-up blood work revealed pancytopenia with WBC: 2410/ mL, Hb: 10.9 g/dL, and platelets: 16,000/mL. A bone marrow study showed a markedly hypercellular marrow with over 95% blasts. Surface marker analysis revealed that these malignant cells were positive for CD 19, CD10, and HLA-DR, suggesting a pre-B phenotype. Immunohistochemistry showed positive stains for


CD34 and TdT. Moreover, chromosomal abnormalities of t(9;22) and 47 XY, 1X were found and reverse transcription-polymerase chain reaction (RT-PCR) detected bcr-abl (e1a2) fusion gene. The final diagnosis was Ph-chromosome positive pre-B ALL. The patient was treated with chemotherapy combined with imatinib and achieved complete remission. He subsequently underwent successful allogeneic peripheral blood stem cell transplantation (PBSCT) and has been in stable condition since. A follow-up bone scan performed a year after initial diagnosis showed complete regression of bone lesions (Fig. 1B). The second case was also a 35-year-old male, who suffered from progressive lower back pain. Neither palpable peripheral lymphadenopathy nor hepatosplenomegaly was noted on physical examination. His hemogram, including white cell differential count, and biochemical data (including LDH and alkaline phosphatase) were completely normal. Magnetic resonance imaging (MRI) of the lumbosacral spine revealed ill-defined masses with various degrees of enhancement involving the L1, L4, L5, and sacral vertebral bodies with cortical destruction (Fig. 1C). Prominent cortical breakthrough of these masses could be seen at the prevertebral body region of S2 through S4 (Fig. 1C, black arrows). Other systemic work-up, including whole-body computed tomography, failed to identify any additional abnormalities. Computed tomography-guided biopsy obtained tumor tissue mainly consisted of lymphoblasts. Random bone marrow biopsy over the left iliac crest showed markedly cellular marrow consistent with acute leukemia. On immunophenotyping, these blasts stained positive for CD34, CD10, CD19, CD38, and HLA-DR. Cytogenetic study disclosed clonal hyperploidy changes: 52–61, X, 2Y, 1X, 11, 1iso(1)(q10), 14, 15, 17, 18, 18, 110, 114, 118, 119, 220, 121. The final diagnosis was pre-B ALL. The patient received induction chemotherapy, which resulted in complete remission, with rapid resolution of clinical symptoms. Follow-up MRI at the time of remission disclosed apparent regression of spinal lesions with marked reduction of cortical breakthrough by the leukemic infiltrates (Fig. 1D, white arrow). The patient has been doing well after successful allogeneic PBSCT. Our cases are unique as both patients presented musculoskeletal manifestations with completely normal hemograms. Early diagnosis of acute leukemia in such patients can be extremely difficult as physicians would not consider it during differential diagnosis. Laboratory data would be of limited diagnostic help, as happened with our second case, who had a normal biochemical profile. The association between bone pain and hematological findings at ALL diagnosis has been noted before [3]. Jonsson et al. identified 52 cases (18%) from 296 ALL patients, who experienced prominent bone pain that overshadowed other manifestations of acute leukemia [3]. The aforementioned cases have hemogram values closer to normal than others; this often led to a delayed accurate diagnosis. It is very rare for ALL patients to present extramedullary lesions and a completely normal hemogram. We searched the English literature and found scattered reports of ALL with near-normal hemograms at presentation. Excluding those diagnosed before immunophenotyping became com-

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Figure 1. (A) The bone scan study of the first case at diagnosis. It shows numerous foci of increased radioactivity in various skeletal regions, suggesting metastatic bone lesions. (B) Bone scan images of the same patient obtained at one year after diagnosis. It shows complete regression of bone lesions. (C) Magnetic resonance imaging (MRI) of lumbosacral spine of the second case. It shows ill-defined masses with various degrees of enhancement involving the L1, L4, L5 and sacral vertebral bodies with cortical destruction. Prominent cortical breakthrough of these masses can be seen at the pre-vertebral body region of S2 through S4 (black arrows). (D) Follow-up MRI of the second patient at the time of remission. It shows apparent regression of spinal lesions and marked reduction of cortical breakthrough caused by leukemic infiltrates (white arrow).

mon, the list of such cases is very short (Table I). Interestingly, almost all of them had immunophenotypes consistent with pre-B ALL, as with our cases. It seems that in these reported pre-B ALL cases, the actively proliferating lymphoblasts ‘‘skip’’ peripheral blood and directly disseminate into the extramedullary space or organs. The actual cause of this phenomenon

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is unknown, but several relevant facts have been noted in the past few years. Chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), have been prominently linked to extramedullary organ infiltration of ALL [5–7]. Overexpression of CXCR4 could lead to transendothelial migration of ALL blasts [6]. SDF-1/CXCR4 signaling is especially impor-


letters TABLE I. Characteristics of Cases of ALL with Near-Normal Hemograms at Presentation Case

Age

1 2 3 4 5 6 7 8 9 10 11

47 44 20 22 36 21 70 2.5 4 35 35

Sex F F M M M M F M M M M

Immunophenotype Pre-B Pre-B Pre-B Pre-B Pre-B (presumed)e Not specifiedf Bg Pre-B Pre-B Pre-B Pre-B

Initial CBCa

Specific initial presentation Renal masses Renal masses Malignant pleural effusion Osteoblastic bone lesions Leukemia cutis Leukemia cutis Leukemia cutis Limping with bone pain Bone pain Bone mass and paraspinal lesions Extensive bony involvement

Hb 11.7g/dL Hb 10.4g/dL Normal Normal Platelet 142 3 103/mL Hb 9.5 g/dL Hb 8.8 d/DL Normal Normal Normal

Other findings

Ref. b

Chr: complex karyotype Normal karyotype Normal karyotypec Normald CD10:76%; HLA-DR:93%

Chr: complex karyotypeh Chr: 47, XY, 1X, t(9;22)

12 12 13 14 15 16 17 18 18 Present cases Present cases

a

Only abnormal data are listed. Chr (Chromosome): 45, XX, del(9)(p22), t(12;22)(q24;q11), der(12)t(12;17)(p13;q11), 217, del(20)(q13). Blasts in pleural effusion: normal karyotype; Cytogenetic study of bone marrow cells failed. d CBC findings not described in detail, but authors stated that initial laboratory evaluation was unremarkable. e Assumption based on limited immunophenotyping data (only CD10 and HLA-DR data available). f Only limited immunophenotyping data available: CD45(1), CD20(2). g Immunophenotyping not done; immunohistochemical study showed CD45(1), CD20(1), CD3(2), CD45RO(2), CD34(2), and CD68(2). h Karyotype: 52–61, X, 2Y, 1X, 11, 1iso(1)(q10), 14, 15, 17, 18, 18, 110, 114, 118, 119, 220, 121. b c

tant in the regulation of leukemic pre-B cells as the chemokine enhances their adhesion, whereas its inhibitors attenuate the chemotaxis and migration of these cells [8–10]. The assumption here is that in the early stages of pre-B ALL disease, some leukemic blasts ‘‘leak’’ into the peripheral blood. Through cellular SDF-1/CXCR4 signaling, these ‘‘leaked’’ pre-B lymphoblasts migrate through the endothelium and enter the extramedullary space or organs, taking them as their new ‘‘habitat.’’ Other than CXCR4 signaling, another molecule potentially involved is vascular endothelial growth receptor 1. Fragoso et al. demonstrated that it is an important regulator in the onset of ALL extramedullary diseases [11]. Whether or not its activation specifically modulates the behavior of pre-B ALL cells is not clear, yet it is quite interesting that the four cell lines used in their experiments were either B-cell precursors or Pre-B cells [11]. Immunohistochemical staining on the patients’ cells shall help to prove our hypothesis. Unfortunately, there was insufficient tissue in retrospect to assay our patients’ leukemia cells for the expression of CXCR4 and other chemokine receptors. In conclusion, our cases serve as a reminder that ALL diagnosis should be considered for all patients with unexplained bone pain, bone lesions, or other musculoskeletal manifestations, even if the hemogram is completely normal. Almost all ALL patients with solely extramedullary diseases and nearly normal hemogram had exclusively pre-B disease. Further study of extramedullary ALL could lead to important information about the role of chemokines in leukemia cell trafficking. 1

Division of Hematology/Oncology, Department of Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan; 2Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan; 3School of Nursing, Chang Gung Institute of Technology, Chiayi, Taiwan; 4Department of Diagnostic Radiology, Chang Gung Memorial Hospital, Chiayi, Taiwan; 5Division of Hematology/Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; 6School of Medicine, National Yang-Ming University, Taipei, Taiwan *Correspondence to: Jyh-Pyng Gau, Division of Hematology/Oncology, Department of Medicine, Taipei Veterans General Hospital, Taiwan E-mail: [email protected] Conflict of interest: Nothing to report. Published online 23 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.21801

References 1. Sinigaglia R, Gigante C, Bisinella G, et al. Musculoskeletal manifestations in pediatric acute leukemia. J Pediatr Orthop 2008;28:20–28.


2. Blatt J, Martini SL, Penchansky L. Characteristics of acute lymphoblastic leukemia in children with osteopenia and vertebral compression fractures. J Pediatr 1984;105:280–282. 3. Jonsson OG, Sartain P, Ducore JM, Buchanan GR. Bone pain as an initial symptom of childhood acute lymphoblastic leukemia: Association with nearly normal hematologic indexes. J Pediatr 1990;117:233–237. 4. Ribeiro RC, Pui CH, Schell MJ. Vertebral compression fracture as a presenting feature of acute lymphoblastic leukemia in children. Cancer 1988;61:589–592. 5. Crazzolara R, Bernhard D. CXCR4 chemokine receptors, histone deacetylase inhibitors and acute lymphoblastic leukemia. Leuk Lymphoma 2005;46:1545–1551. 6. Crazzolara R, Kreczy A, Mann G, et al. High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia. Br J Haematol 2001;115:545–553. 7. Juarez J, Dela Pena A, Baraz R, et al. CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment. Leukemia 2007;21:1249–1257. 8. Juarez J, Bradstock KF, Gottlieb DJ, Bendall LJ. Effects of inhibitors of the chemokine receptor CXCR4 on acute lymphoblastic leukemia cells in vitro. Leukemia 2003;17:1294–1300. 9. Shen W, Bendall LJ, Gottlieb DJ, Bradstock KF. The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhesion and facilitates engraftment of leukemic precursor-B cells in the bone marrow. Exp Hematol 2001; 29:1439–1447. 10. Spiegel A, Kollet O, Peled A, et al. Unique SDF-1-induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling. Blood 2004;103:2900–2907. 11. Fragoso R, Pereira T, Wu Y, et al. VEGFR-1 (FLT-1) activation modulates acute lymphoblastic leukemia localization and survival within the bone marrow, determining the onset of extramedullary disease. Blood 2006;107:1608– 1616. 12. Au WY, Kwong YL, Ma SK. Aleukemic leukemia masquerading as renal masses: a rare presentation of adult lymphoblastic leukemia. Am J Hematol 2000;65:328–329. 13. Pei SN, Kuo CY, Ma MC, Wang MC. Mediastinal mass and malignant pleural effusion in an aleukemic case with pre-B acute lymphoblastic leukemia. J Pediatr Hematol Oncol 2009;31:139–141. 14. Recine M, Castellano-Sanchez AA, Sheldon J, et al. Precursor B-cell lymphoblastic lymphoma/leukemia presenting as osteoblastic bone lesions. Ann Diagn Pathol 2002;6:236–243. 15. Zengin N, Kars A, Ozisik Y, et al. Aleukemic leukemia cutis in a patient with acute lymphoblastic leukemia. J Am Acad Dermatol 1998;38:620–621. 16. Taniguchi S, Hamada T, Kutsuna H, Ishii M. Lymphocytic aleukemic leukemia cutis. J Am Acad Dermatol 1996;35:849–850. 17. Kishimoto H, Furui Y, Nishioka K. Guess what. B-cell acute lymphoblastic leukemia with aleukemic leukemia cutis. Eur J Dermatol 2001;11:151– 152. 18. Lefevre Y, Ceroni D, Laedermann A, et al. Pediatric leukemia revealed by a limping episode: A report of four cases. Orthop Traumatol Surg Res 2009;95: 77–81.

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