G

L

Fpb

FAFAMIAEa

b

c

d

e

f

g

h

i

j

k

l

m

n

o

a

ARRAA

KCCYBFA

aT

0h

ARTICLE IN PRESS Model

R-5062; No. of Pages 6

Leukemia Research xxx (2013) xxx– xxx

Contents lists available at ScienceDirect

Leukemia Research

j o ur nal ho me page: www.elsev ier .com/ locate / leukres

ludarabine plus alemtuzumab (FA) front-line treatment in youngatients with chronic lymphocytic leukemia (CLL) and an adverseiologic profile

rancesca R. Mauroa,∗, Stefano Molicab, Luca Laurenti c, Agostino Cortelezzid,ngelo M. Carellae, Francesco Zaja f, Annalisa Chiarenzag, Francesco Angrilli h,rancesco Nobile i, Roberto Marasca j, Caterina Musolinok, Maura Brugiatelli l,lfonso Piciocchim, Marco Vignettim, Paola Fazim, Giuseppe Gentilea,aria S. De Proprisa, Irene Della Starzaa, Marilisa Marinelli a, Sabina Chiaretti a,

laria Del Giudicea, Mauro Nannia, Francesco Albanon, Antonio Cuneoo,nna Guarinia, Robin Foàa, on behalf of the GIMEMA (Gruppo Italiano MalattieMatologiche dell’Adulto) Working Party for chronic lymphoproliferative disorders

Hematology, Department of Cellular Biotechnologies and Hematology, “Sapienza” University, Rome, ItalyOncologia Medica, Azienda Ospedaliera Pugliese Ciaccio, Catanzaro, ItalyDepartment of Hematology, Universita’ Cattolica del Sacro Cuore, Rome, ItalyEmatology-BMT Unit, IRCCS Ca’Granda Ospedale Maggiore Policlinico Foundation, Milan, ItalyU.O.C. Ematologia 1, IRCCS San Martino-IST, Genova, ItalyHematology-BMT Unit, S. Maria Misericordia Hospital, University of Udine, ItalyDepartment of Hematology, Hospital Ferrarotto, University of Catania, ItalyDepartment of Hematology, Local Health Unit of Pescara, ItalyAzienda Ospedaliera “Bianchi-Melacrino-Morelli”, Reggio Calabria, ItalySection of Hematology, Department of Oncology, Hematology and Respiratory Diseases, University of Modena and Reggio Emilia, ItalyDivision of Hematology, Medicinal Chemistry Section, University of Messina, ItalyDepartment of Hematology, Azienda Ospedaliera Papardo, Messina, ItalyItalian Group for Adult Hematologic Diseases (GIMEMA), Rome, ItalyU.O. Ematologia con Trapianto, Università degli Studi Aldo Moro, Bari, ItalyDepartment of Hematology, Arcispedale Sant’Anna, Ferrara, Italy

r t i c l e i n f o

rticle history:eceived 25 August 2013eceived in revised form 9 November 2013ccepted 11 November 2013vailable online xxx

a b s t r a c t

In 45, ≤60 years old patients with CLL and an adverse biologic profile, a front-line treatment with Flu-darabine and Campath (Alemtuzumab®) was given. The overall response rate was 75.5%, the completeresponse rate (CR) 24.4% with the lowest CR rates, 16.7% and 8.3%, in 11q and 17p deleted cases. The3-year progression-free survival (PFS) and overall survival were 42.5% and 79.9%, respectively. PFS wassignificantly influenced by CLL duration, beta2-microglobulin, and improved by post-remissional stemcell transplantation. Front-line fludarabine and alemtuzumab showed a manageable safety profile and

eywords:hronic lymphocytic leukemiaLLoungiologyludarabinelemtuzumab

evidence of a benefit in a small series of CLL patients with adverse biologic features.© 2013 Published by Elsevier Ltd.

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

∗ Corresponding author at: Hematology, Department of Cellular Biotechnologiesnd Hematology, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy.el.: +39 06 499741; fax: +39 0644241984.

E-mail address: mauro@bce.uniroma1.it (F.R. Mauro).

145-2126/$ – see front matter © 2013 Published by Elsevier Ltd.ttp://dx.doi.org/10.1016/j.leukres.2013.11.009

1. Introduction

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

Chronic lymphocytic leukemia (CLL) is an incurable disease witha median age at the time of diagnosis between 65 and 70 years andwith about 22–30% of patients younger than 60 years [1,3]. The vari-able clinical course of the disease is strongly influenced by different

ING Model

L

2 ia Res

bitiwwteoitwc

aaadoawi

tit

dts

2

2

Carmpto

2

acaa1ue2t

st

2

dRm

ARTICLER-5062; No. of Pages 6

F.R. Mauro et al. / Leukem

iologic markers that do not appear to differ substantially accord-ng to age at diagnosis [4–8]. However, Dohner et al. observed thathe presence of deletion 11q was a profound stratifier of survivaln patients aged <55 years, but not in patients aged >55 years [9],

hile in all age patients 17p deletion and/or TP53 mutations areell known poor prognostic markers [10–12]. It has been estimated

hat 60% of patients ≤55 years require treatment and that the lifexpectancy of younger CLL patients is significantly shorter than thatf the age-matched general population [2,3]. A significant clinicalmprovement in the outcome of CLL patients has been obtained byhe combination of the anti-CD20 monoclonal antibody rituximabith fludarabine-based regimens, in particular, the fludarabine and

yclophosphamide regimen (FCR) [13,14].Campath (Alemtuzumab®) a chimeric anti-CD52 monoclonal

ntibody is effective in the management of CLL and has shownctivity regardless of the genetic risk groups [15–18]. Results from

single-group phase 2 study by Elter et al. suggested that the flu-arabine and alemtuzumab (FA) combination could improve theutcome of patients with relapsed or refractory CLL [19]. The sameuthors confirmed the efficacy of the FA combination in a studyhere this regimen was compared with fludarabine monotherapy

n patients with relapsed or refractory CLL [20].In order to prolong the response duration, a post-remissional

reatment with alemtuzumab [21,22] and, in younger and phys-cally fit patients with poor prognosis, the role of a stem cellransplant (SCT) [23–26] have also been explored.

The GIMEMA (Gruppo Italiano Malattie EMatologicheell’Adulto) promoted a prospective multicenter phase II trialo evaluate in CLL patients younger than 60 years the efficacy andafety of a treatment approach with the FA regimen.

. Design and methods

.1. Study design

A prospective, multicenter, phase II trial was designed for youngLL, with adverse biologic characteristics requiring front-line ther-py. The primary objectives of the study were to define the overallesponse (OR) and complete response (CR) rates after FA regi-en. The secondary objectives were to assess the toxicity, the

rogression-free survival (PFS), the overall survival (OS), the rela-ionship between the baseline clinical and biologic features and theutcome of the patients.

.2. Inclusion and exclusion criteria

Inclusion criteria were age ≤60 years, no prior treatment,dvanced Binet stage (C) or less advanced stage (B or A) [27] withlinical signs of active disease according to the NCI-WG criteria [28]nd a “high risk” (HR) biologic profile. For the purpose of this study,

HR biologic profile was defined by the presence of: (1), deletion7p (≥20%; HRa subset); or (2) deletion 11q with ≥1 additionalnfavorable factor (germline IGHV status, ZAP-70 and/or CD38xpression; HRb subset); or (3) germline IGHV or mutated VH3-1 and ≥2 unfavorable factors (ZAP-70 and/or CD38 expression,risomy 12; deletion 6q; HRc subset).

Institutional ethic committees approved the study. Patientsigned a written informed consent. The EudraCT number registra-ion of the study is 2005-002476-15.

.3. Study treatment

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

Patients received 4 monthly courses of the FA regimen (flu-arabine, 30 mg/m2 iv; alemtuzumab, 30 mg iv, days 1–3) [19,20].esponding patients with no evidence of residual disease at theolecular level (mol-CR) underwent a peripheral blood stem cell

PRESSearch xxx (2013) xxx– xxx

(PBSC) mobilization only, while patients with residual disease (par-tial response, PR; complete response, CR; cytometric completeresponse, cy-CR) underwent post-remissional therapy. In order ofpriority, 3 post-remissional treatment options were considered:a reduced intensity allogeneic PB SCT or, in the absence of a sib-ling donor, an autologous PB SCT or, in the absence of a sufficientharvest, treatment with alemtuzumab sc, 30 mg weekly for a max-imum of 12 weeks (Supplementary Table 1).

Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.leukres.2013.11.009.

2.4. Supportive treatment

All patients received bactrim and high dose valacyclovir pro-phylaxis (2 g/8 h) with weekly CMV antigenemia monitoring.Treatment was stopped in the presence of life-threatening adverseevents, persistent (≥4 weeks) grade ≥2 cytopenia, infection or othertoxicity.

2.5. Efficacy assessments

Response was defined between the 8◦ and the 12◦ week from thelast fludarabine and alemtuzumab administration. Response wasassessed according to the 1996 NCI-WG guidelines [28] and CR wasconfirmed in all cases by bone marrow biopsy.

In addition, in all CR patients, computed tomography (CT) scanswere used. In patients with imaging confirmed CR, a centralizedevaluation of the minimal residual disease (MRD) was performedboth on PB and bone marrow (BM) by four color flow cytometry. Inpatients with no evidence of cytometric residual disease, molecularresponse was also assessed by polymerase chain reaction (PCR). Ina subset of patients a gene expression profile analysis (GEP) wasperformed as previously described [29]

Statistical analysis Differences in the distributions of prognosticfactors were analyzed by the �2 or Fisher’s exact test and by theKruskal–Wallis test. The probability of survival was estimated usingthe Kaplan–Meier method. The log-rank test was used to comparethe effect of treatment and risk factor categories, while confidenceintervals were estimated (95% CIs) using the Simon and Lee method.Logistic regression and Cox proportional hazard regression mod-els were performed to examine risk factors affecting treatmentresponse and survival outcomes. To ascertain the pure impact ofthe FA regimen and the prognostic effect of baseline clinical andbiologic variables patients who underwent SCT were censored atthe time of transplant.

3. Results

3.1. Study population

Between December 2005 and February 2009, the biologic profileof 86 consecutive young patients with CLL and a progressive diseasewas assessed. Forty-five patients (52%) showed a HR biologic pro-file and were treated with the FA regimen. The clinical and biologiccharacteristics of the patients are reported in Table 1. The distribu-tion of the three subsets of HR patients was: 26.7% for the group ofpatients with deletion 17p (HRa subset), 40% for the group includ-

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

ing patients with deletion 11q and 1 or more additional adversebiologic factors (HRb subset), and 33.3% for the group representedby cases with unmutated IGHV and 2 or more additional adversebiologic factors (HRc subset).

ARTICLE IN PRESSG Model

LR-5062; No. of Pages 6

F.R. Mauro et al. / Leukemia Research xxx (2013) xxx– xxx 3

Table 1Baseline clinical and biologic characteristics of patients.

Patients (45)

n %

Gender male/female 34/11 75.6/24.4Median age, years (range) 55.2 (29.8–60.8)ECOG performance status 0/1 42/3 95.3/6.7Median time from diagnosis, months 9.0 (0.6–639.8)Median lymphocyte count, ×109/L 62.0 (5.0–638)Binet’s stage A/B/C 6/30/9 13.3/66.7/20Lymph nodes of at least of 5 cm of

diameter16 35.6

Increased �2M 22 48.9Increased LDH 17 37.8CD19/CD38 ≥30% 22 48.8ZAP-70 ≥20% 27 60IGVH unmutated/mutated 42/3 93.3/6.7FISH abnormalities (hierarchcal model)

No abnormalities 3 6.713q− 5 11.112+ 6 13.36q− 1 2.211q− 18 40.017p− 12 26.7

Abbreviations: ECOG, Eastern Cooperative Oncology Group; �2M, beta2-mi

3

o

rt1wmld

dlpc≤v(tC

rta

iiia

da

sapCa

Table 2Response to FluCam by clinical and biologic characteristics.

ORn (%)

p-value CRn (%)

p-value

All patients 34 (75.5) – 11 (24.4) –Age>55 18/23 (78.3)

0.74/23 (17.4)

0.3≤55 16/22 (72.7) 7/22 (31.8)GenderMale 26/34 (76.5)

1.09/34 (75.6)

0.7Female 8/11 (72.7) 2/11 (18.2)PB lymphocytes, ×109/L<60 17/22 (77.3)

0.86/22 (27.3)

0.7≥60 17/23 (73.9) 5/23 (21.7)Binet’s stageA 4/6 (66.7)

0.52/6 (33.3)

0.7B 24/30 (80.0) 8/30 (26.7)C 6/9 (66.7) 1/9 (11.1)LDHNormal 22/28 (78.6)

0.77/28 (25.0)

1.0Increased 12/17 (37.8) 4/17 (23.5)ˇ2MNormal 18/23 (78.3)

0.79/23 (39.1)

0.02Increased 16/22 (72.7) 2/22 (9.1)Months from CLL diagnosis≤12 16/25 (64.0)

0.082/25 (8.0)

0.006>12 18/20 (90.0) 9/20 (45.0)Lymph nodes diameter<5 cm 24/29 (82.8)

0.210/29 (34.5)

0.07≥5 cm 10/16 (62.5) 1/16 (6.3)CD19/CD38<30% 18/23 (78.2)

1.06/23 (26.0)

1.0≥30% 16/22 (72.7) 5/22 (22.7)ZAP-70<20% 14/16 (87.5)

0.24/16 (25.0)

1.0≥20% 18/27 (66.7) 6/27 (22.2)IGHVMutated 2/3 (66.7)

1.01/3 (33.3)

1.0Unmutated 32/42 (76.2) 10/42 (23.8)High risks subgroupsHRa 7/12 (58.3)

0.31/12 (8.3)

0.07HRb 15/18 (83.3) 3/18 (16.7)HRc 12/15 (80.0) 7/15 (46.7)

Patients subgroups: HRa includes, patients with deletion 17p (≥20%); HRb, patients

3 years, deletion 17p vs deletion 11q vs trisomy 12 or deletion 13q,

icroglobulin; ZAP-70, zeta-chain-associated protein kinase 70; IGHV,mmunoglobulin heavy-chain; FISH, fluorescence in situ hybridization.

.2. Response to FA regimen

The majority of patients (89%) completed the planned 4 coursesf FA treatment.

Two patients withdrew from treatment because of severeeaction during the first alemtuzumab infusion. On an intention-o-treat basis, 34 HR patients (75.5%) responded to FA regimen with1 (24.4%) CRs and 23 (51.1%) PRs (Table 2). Out of the 11 patientsho achieved a CR, 8 (18%) showed no residual disease at the cyto-etric level (cy-CR) and 5 both at the cytometric and molecular

evel (mol-CR). A treatment failure was recorded in 9 cases (stableisease, 5; progressive disease, 4).

The effect of the clinical and biologic variables on response isetailed in Table 2. Patients with deletion 17p or 11q showed the

owest CR rates (HRa vs HRb vs HRc subset, 8.3% vs 16.7% vs 46.7%; = .07). The proportion of patients who obtained a CR was signifi-antly lower in the presence of a short interval from CLL diagnosis,12 months (p = .006), increased �2M levels (p = .02). At multi-ariate analysis, the interval between CLL diagnosis and treatmentp = .02), increased �2M (p < .027) and deletion 17p (p < .043) main-ained a significant and independent effect on the achievement ofR.

GEP analysis revealed a distinctive signature associated withesponse to treatment with 357 differentially expressed probesets,he majority being downregulated in patients who did not obtain

CR (Fig. 1A).Furthermore, DAVID functional annotation analysis evidenced

n patients who failed to reach a CR an overrepresentation, of genesnvolved in translation, proteasomal catabolic processes, RNA splic-ng, Ras signaling, cellular response to stress, DNA repair and B cellctivation (Fig. 1B).

In addition, in the cases who did not achieve a CR a significantownmodulation (p = .014) of the transcript encoding for the CD52ntigen was observed.

Out of the 29 patients who achieved a response with FA andhowed the evidence of residual disease, 14 underwent SCT and all

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

re alive (median time from SCT, 40 months). However, while the 6atients who had an allogeneic SCT are in persistent CR (2 in mol-R), 4 out of the 8 who had an autologous SCT have relapsed after

median time of 29 months.

with deletion 11q and 1 or more additional adverse biologic factors; HRc, IGHVunmutated patients with 2 or more additional adverse biologic factors.

3.3. Survival

The 3-year PFS and OS are 42.5% (95%CI: 27.4–65.9) and 79.9%(95%CI: 66.6–95.9), respectively. Baseline clinical and biologicparameters associated with a significantly higher PFS after FluCamwere a longer interval from CLL diagnosis (p = .0007), the absenceof bulky nodes (p = .02), or increased �2M levels (p = .05). Patientswith deletion 17p or 11q showed a shorter, though not significantlyinferior PFS (PFS at 3 years, 17p-, 51% vs 11q-, 28%, vs no deletion17p or 11q, 60%; p = .85; Fig. 2). At multivariate analysis, the sig-nificant and independent baseline factors influencing PFS were theinterval from CLL diagnosis (p = .012; HR: 3.2; 95% CI: 1.2–8.4) andthe presence of bulky nodes (p = .016; HR: 0.31; 95% CI: 0.12–0.77).A significantly longer PFS was observed in patients who achieved aCR (PFS at 3 years, CR vs PR, 80.0% vs 12.7%; p = .03) and in patientswho after response to FA underwent an allogeneic or autologousSCT as compared to patients who did not receive further treat-ment (p < .02; HR: 0.30; 95% CI: 0.11–0.84). Baseline variables witha significant effect on survival probability were the age of patients(p = .04), the interval from CLL diagnosis (p = .01), and the LDH value.Patients with deletion 17p showed the lowest OS probability (OS at

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

or no abnormalities, 69% vs 85% vs 83%). However, the differencedid not reach statistical significance (p = .22), presumably becauseof the limited sample size (Fig. 2).

ARTICLE IN PRESSG Model

LR-5062; No. of Pages 6

4 F.R. Mauro et al. / Leukemia Research xxx (2013) xxx– xxx

F the hia .

3

cr(cAri(Rd(

4

ctww

ig. 1. (A) Gene expression profile. Relative levels of gene expression: red representsnnotation analysis of differentially expressed genes from the 2 subsets of patients

.4. Safety

A grade III–IV granulocytopenia was recorded after 18% ofourses and in 38% of patients. However, a severe infection wasecorded in a lower proportion of patients, 13%. Three patients6.7%) experienced a CMV reactivation that responded to ganci-lovir iv, while no case of symptomatic CMV infection was observed.lemtuzumab-related grade 3–4 adverse infusion reactions wereecorded in 2 patients (4.4%) who discontinued treatment. Dur-ng the follow-up, a second malignancy was recorded in 2 casesprostate carcinoma, Kaposi sarcoma) and 1 patient developed aichter’s syndrome. Seven patients have died (15.5%). The cause ofeath was an infection in 1 patient and disease progression in 6CLL, 5 cases, Richter’s syndrome, 1).

. Discussion

We know today a large number of genetic abnormalities asso-

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

iated with the pathogenesis and prognosis of CLL [30–33]. Athe time this study was designed, in the pre-FCR era, thereas evidence that both deletions 17p and 11q were associatedith a poor outcome after treatment, and a treatment strategy

ghest level of expression and blue the lowest level of gene expression. (B) Functional

including fludarabine combined with the monoclonal antibodyalemtuzumab was judged as an appropriate first line treatmentapproach for young CLL patients with an overall poor prognosticlikelihood.

About half of the younger patients we analyzed at the timeof first-line treatment showed an adverse biologic profile. On anintention-to-treat basis, a response was obtained after FA by 75.5%of these patients with a CR rate of 24.4%, including 18% of cytomet-ric CRs and a sizable number of molecular CRs. A shorter intervalfrom diagnosis and beta2-microglobulin, were significantly asso-ciated with an adverse outcome. The genetic profile identified adifferent pattern of response to FA regimen. Patients with unmu-tated IGHV and no deletion 11q or 17p showed the best responsein terms of OR, 80%, and CR, 47%, rates while patients with deletion11q showed a very low CR rate, 17%, and patients harboring a dele-tion 17p the worse response in terms of both, OR, 58% and CR, 8%,rates.

Interestingly, GEP analysis revealed a significant downmodu-

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

lation of transcripts involved in the DNA repair and/or apoptosisregulation and a significant downmodulation of the transcriptencoding for the CD52 antigen in patients who obtained only a PRor no response.

ARTICLE ING Model

LR-5062; No. of Pages 6

F.R. Mauro et al. / Leukemia Res

Fig. 2. HRa subset, patients with deletion 17p (≥20%). HRb subset, deletion 11qand ≥1 additional adverse biologic factor; HRc subset, IGHV unmutated and ≥2a6

rptpwtTib[

bwotrp

care to patients, collected data, and gave the final approval of the

dditional adverse biologic factors. (A) PFS at 3 years: HRa, 51%; HRb, 28%; HRc,0%; p = .85. (B) OS at 3 years: HRa, 69%; HRb, 85%; HRc, 83%; p = .22.

If we consider the results of the CLL8 study [14], with the FCRegimen, being today the gold standard front-line therapy for fitatients with CLL, patients with unmutated IGHV and no dele-ion 11q or 17p showed a similar CR rate after FA. Unfortunately,atients with deletion 17p did not show a better response rateith FA, and patients with deletion 11q revealed a very unsatisfac-

ory CR rate and they appear to respond better to the FCR regimen.his finding further suggests that treatment combinations includ-ng rituximab and alkylating agents such as cyclophosphamide orendamustine are more effective in patients with the 11q deletion34,35].

Even though the analysis of a small cohort of 45 patients shoulde consider as exploratory and no conclusions can be drawn, it isorth noting that the introduction of a post-remissional allogeneic

r autologous SCT showed a benefit in prolonging response after

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

reatment. This observation is in line with that of two studies thateported a benefit in the outcome of CLL patients when the SCT waserformed in an early phase of the disease [25,26].

PRESSearch xxx (2013) xxx– xxx 5

The safety profile of FA was acceptable. Infections were lessfrequent than expected considering the immunodepressive effectof both agents. The low cumulative dose of alemtuzumab, theextended prophylaxis and the absence of prior treatments played afavorable effect in restraining infections. Interestingly, on high dosevalacyclovir prophylaxis, no CMV symptomatic infections wereobserved and a relatively low proportion of patients developed aCMV reactivation.

In conclusion, in this study about half of young CLL patientsrequiring therapy showed adverse biologic features. In this sub-set of patients FA regimen given as front-line treatment revealeda manageable safety profile and provided evidence of a patientbenefit. Further confirmatory phase III studies in larger series ofpatients comparing FA with the gold standard regimen for CLL andan extended follow-up to evaluate survival end points and long-term toxicity are required.

Acknowledgements

We thank the patients who agreed to participate in thisstudy. We also thank Antonella Graziosi and Laura Collada Alifor the support in the study organization and data acquisi-tion, and all the study investigators who participated in thisstudy: Giovanna Meloni and Anna Paola Iori, Hematology, Depart-ment of Cellular Biotechnologies and Hematology, “Sapienza”University, Rome, Italy; Francesco Lauria and Francesco Forconi,Hematology, Department of Clinical Medicine and Immunolog-ical Sciences, University of Siena and Department of Oncology,Azienda Ospedaliera Universitaria Senese, Siena, Italy; DavideSoligo, Hematology-BMT Unit, IRCCS Ca’Granda Ospedale Mag-giore Policlinico Foundation, Milan, Italy; Alberto Bosi and StefaniaCiolli, Divisione di Ematologia, Università di Firenze, Firenze,Paolo Corradini, Division of Hematology, Istituto Nazionale deiTumori, Milano, Italy; Rosanna Mirabelli, Oncologia Medica,Azienda Ospedaliera Pugliese Ciaccio, Catanzaro, Italy; VincenzoLiso, Giorgina Specchia, Rita Rizzi, U.O. Ematologia con TrapiantoUniversità degli Studi Aldo Moro, Bari, Italy; Alessandro Levis,Division of Hematology, Sant’Antonio e Biagio Hospital, Alessan-dria, Italy; Massimo Massaia and Marta Coscia, Dipartimentodi Medicina e Oncologia Sperimentale, Sezione di Ematologia,University of Torino, Italy; Fortunato Morabito and Massimo Gen-tile, Hematology Section, Cosenza Hospital, Cosenza, Italy; SergioAmadori and Francesco Buccisano, Hematology, University TorVergata, Rome, Italy; Giovanni Del Poeta, Hematology, OspedaleS. Eugenio, Rome, Italy; Sergio Storti, Istituto di Ematologia-Campobasso, Italy; Nicola Di Renzo, Divisione di Ematologia, Lecce,Italy.

Funding sources: This work was supported by Genzyme who pro-vided alemtuzumab and a research grant, by Associazione Italianaper la Ricerca sul Cancro (AIRC), Special Program Molecular Clin-ical Oncology, 5 × 1000, No. 10007, Milan, Italy; and by RomAIL(Associazione Italiana contro le Leucemie, Rome section).

Author’s contributions: R.F. designed the research, wrote themanuscript and gave final approval of the manuscript. F.R.M.designed the research, provided clinical care to patients, col-lected data, analyzed and interpreted data, wrote the manuscriptand gave final approval of the manuscript. A.G. designed theresearch, performed research, collected data, analyzed and inter-preted data, and gave final approval of the manuscript. S.M., A.C.,L.L., A.M.C., F.Z., A.C., F.A., F.N., R.M., C.M., M.V., P.F., G.G., M.B.,M.S.D., I.D.S., M.M., I.D.G., S.C., M.N., G.S., A.C. provided clinical

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

manuscript.Conflict of interest: The authors declare no competing financial

interests.

ING Model

L

6 ia Res

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ARTICLER-5062; No. of Pages 6

F.R. Mauro et al. / Leukem

eferences

[1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics. CA Cancer J Clin2010;60(5):277–300.

[2] Shanafelt TD, Rabe KG, Kay NE, Zent CS, Jelinek DF, Reinalda MS, et al. Age atdiagnosis and the utility of prognostic testing in patients with chronic lympho-cytic leukemia. Cancer 2010;116(20):4777–87.

[3] Mauro FR, Foa R, Giannarelli D, Cordone I, Crescenzi S, Pescarmona E, et al.Clinical characteristics and outcome of young chronic lymphocytic leukemiapatients: a single institution study of 204 cases. Blood 1999;94(2):448–54.

[4] Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. IgV gene mutationstatus and CD38 expression as novel prognostic indicators in chronic lympho-cytic leukemia. Blood 1999;94(6):1840–7.

[5] Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H)genes are associated with a more aggressive form of chronic lymphocyticleukemia. Blood 1999;94(6):1848–54.

[6] Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70expression as a surrogate for immunoglobulin variable-region mutations inchronic lymphocytic leukemia. N Engl J Med 2003;348(18):1764–75.

[7] Dohner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L, et al.Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl JMed 2000;343(26):1910–6.

[8] Del Giudice I, Mauro FR, De Propris MS, Santangelo S, Marinelli M, PeragineN, et al. White blood cell count at diagnosis and immunoglobulin variableregion gene mutations are independent predictors of treatment-free survivalin young patients with stage A chronic lymphocytic leukemia. Haematologica2011;96(4):626–30.

[9] Dohner H, Stilgenbauer S, James MR, Benner A, Weilguni T, Bentz M, et al.11q deletions identify a new subset of B-cell chronic lymphocytic leukemiacharacterized by extensive nodal involvement and inferior prognosis. Blood1997;89(7):2516–22.

10] Dohner H, Fischer K, Bentz M, Hansen K, Benner A, Cabot G, et al. p53 genedeletion predicts for poor survival and non-response to therapy with purineanalogs in chronic B-cell leukemias. Blood 1995;85(6):1580–619.

11] Cordone I, Masi S, Mauro FR, Soddu S, Morsilli O, Valentini T, et al. p53 expres-sion in B-cell chronic lymphocytic leukemia: a marker of disease progressionand poor prognosis. Blood 1998;91(11):4342–9.

12] Zenz T, Krober A, Scherer K, Häbe S, Bühler A, Benner A, et al. MonoallelicTP53 inactivation is associated with poor prognosis in chronic lymphocyticleukemia: results from a detailed genetic characterization with long-termfollow-up. Blood 2008;112(8):3322–9.

13] Keating MJ, O’Brien S, Albitar M, Lerner S, Plunkett W, Giles F, et al. Early resultsof a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, andrituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol2005;23(18):4079–88.

14] Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, et al.Addition of rituximab to fludarabine and cyclophosphamide in patients withchronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet2010;376(9747):1164–74.

15] Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J, et al. Therapeutic role ofalemtuzumab (Campath-1H) in patients who have failed fludarabine: resultsof a large international study. Blood 2002;99(10):3554–61.

16] Karlsson C, Lundin J, Kimby E, Kennedy B, Moreton P, Hillmen P, et al. PhaseII study of subcutaneous alemtuzumab without dose escalation in patientswith advanced-stage, relapsed chronic lymphocytic leukaemia. Br J Haematol2009;144(1):78–85.

17] Stilgenbauer S, Zenz T, Winkler D, Bühler A, Schlenk RF, Groner S, et al.Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocyticleukemia: clinical results and prognostic marker analyses from the CLL2H

Please cite this article in press as: Mauro FR, et al. Fludarabine plus alemlymphocytic leukemia (CLL) and an adverse biologic profile. Leuk Res (201

study of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol2009;27(24):3994–4001.

18] Hillmen P, Skotnicki AB, Robak T, Jaksic B, Dmoszynska A, Wu J, et al.Alemtuzumab compared with chlorambucil as first-line therapy for chroniclymphocytic leukemia. J Clin Oncol 2007;25(35):5616–23.

[

PRESSearch xxx (2013) xxx– xxx

19] Elter T, Borchmann P, Schulz H, Reiser M, Trelle S, Schnell R, et al. Fludarabinein combination with alemtuzumab is effective and feasible in patients withrelapsed or refractory B-cell chronic lymphocytic leukemia: results of a phaseII trial. J Clin Oncol 2005;23(28):7024–31.

20] Elter T, Gercheva-Kyuchukova L, Pylylpenko H, Robak T, Jaksic B, Rekhtman G,et al. Fludarabine plus alemtuzumab versus fludarabine alone in patients withpreviously treated chronic lymphocytic leukaemia: a randomised phase 3 trial.Lancet Oncol 2011;12(13):1204–13.

21] Schweighofer CD, Ritgen M, Eichhorst BF, Busch R, Abenhardt W, Kneba M,et al. Consolidation with alemtuzumab improves progression-free survival inpatients with chronic lymphocytic leukaemia (CLL) in first remission–long-term follow-up of a randomized phase III trial of the German CLL Study Group(GCLLSG). Br J Haematol 2009;144(1):95–8.

22] Lin TS, Donohue KA, Byrd JC, Lucas MS, Hoke EE, Bengtson EM, et al. Consolida-tion therapy with subcutaneous alemtuzumab after fludarabine and rituximabinduction therapy for previously untreated chronic lymphocytic leukemia: finalanalysis of CALGB 10101. J Clin Oncol 2010;28(29):4500–6.

23] Schetelig J, van Biezen A, Brand R, Lucas MS, Hoke EE, Bengtson EM,et al. Allogeneic hematopoietic stem-cell transplantation for chronic lym-phocytic leukemia with 17 p deletion. A retrospective European group forblood and marrow transplantation analysis. J Clin Oncol 2008;26(31):5094–100.

24] Dreger P, Döhner H, Ritgen M, Böttcher S, Busch R, Dietrich S, et al. Allogeneicstem cell transplantation provides durable disease control in poor-risk chroniclymphocytic leukemia: long-term clinical and MRD results of the GCLLSG CLL3Xtrial. Blood 2010;116(14):2438–47.

25] Sutton L, Chevret S, Tournilhac O, Diviné M, Leblond V, Corront B, et al. Autol-ogous stem cell transplantation as a first-line treatment strategy for chroniclymphocytic leukemia: a multicenter, randomized, controlled trial from theSFGM-TC and GFLLC. Blood 2011;117(23):6109–19.

26] Dreger P, Döhner H, McClanahan F, Busch R, Ritgen M, Greinix H,et al. Early autologous stem cell transplantation for chronic lymphocyticleukemia: long-term follow-up of the GCLLSG CLL3 trial. Blood 2012,http://dx.doi.org/10.1182/blood-2011-09-378505.

27] Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, et al. Anew prognostic classification of chronic lymphocytic leukemia derived froma multivariate survival analysis. Cancer 1981;48(1):198–206.

28] Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, et al. NationalCancer Institute-sponsored Working Group lymphocytic leukemia: revisedguidelines for diagnosis and treatment. Blood 1996;87(12):4990–7.

29] Chiaretti S, Tavolaro S, Marinelli M, Messina M, Del Giudice I, Mauro FR, et al.Evaluation of TP53 mutations with the AmpliChip p53 research test in chroniclymphocytic leukemia: correlation with clinical outcome and gene expressionprofiling. Genes Chromosomes Cancer 2011;50(4):263–74.

30] Guarini A, Marinelli M, Tavolaro S, et al. ATM gene alterations in chroniclymphocytic leukemia patients induce a distinct gene expression profile andpredict disease progression. Haematologica 2012;97(1):47–55.

31] Stilgenbauer S, Eichhorst BF, Busch R, et al. Biologic and clinical markers foroutcome after fludarabine (F) or F plus cyclophosphamide (FC): comprehensiveanalysis of the CLL4 trial of the GCLLSG. Blood 2008;112(11):2089. Abstract2089.

32] Schnaiter A, Paschka P, Rossi M, et al. NOTCH1, SF3B1 and TP53 mutations infludarabine-refractory CLL patients treated with alemtuzumab: results fromthe CLL2H trial of the GCLLSG. Blood 2013 [Epub ahead of print].

33] Gaidano G, Foà R, Dalla-Favera R. Molecular pathogenesis of chronic lympho-cytic leukemia. J Clin Invest 2012;122(10):3432–8.

34] Ding W, Ferrajoli A. Evidence-based mini-review: the role of alkylating agentsin the initial treatment of chronic lymphocytic leukemia patients with the 11qdeletion. Hematol Am Soc Hematol Educ Program 2010;2010:90–2.

tuzumab (FA) front-line treatment in young patients with chronic3), http://dx.doi.org/10.1016/j.leukres.2013.11.009

35] Fischer K, Cramer P, Busch R, Böttcher S, Bahlo J, Schubert J, et al. Ben-damustine in combination with rituximab for previously untreated patientswith chronic lymphocytic leukemia: a multicenter phase II trial of the Ger-man Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 2012;30(26):3209–16.