Andrea Camera

Ciro R Rinaldi

Salvatore Palmieri

Nicola Cantore

Giuseppina Mele

Vincenzo Mettivier

Eustachio Miraglia

Lucia Mastrullo

Francesco Grimaldi

Luigia Luciano

Anna Guerriero

Bruno Rotoli
email: rotoli@unina.it

Felicetto Ferrara

A O R N Cardarelli

Sequential continuous infusion of fludarabine and cytarabine associated with liposomal daunorubicin (DaunoXome®) (FLAD) in primary refractory or relapsed adult acute myeloid leukemia patients

Keywords: Refractory acute myeloid leukemia, Relapsed acute myeloid leukemia, Salvage treatment, DaunoXome, FLAD

Sequential continuous infusion of fludarabine and cytarabine associated with liposomal daunorubicin (DaunoXome®) (FLAD) in primary refractory or relapsed adult acute myeloid leukemia patients

Introduction

Although intensive chemotherapy, often including a stemcell transplant (SCT), is currently used in the treatment of acute myeloid leukemia (AML), a large proportion of patients relapse in their bone marrow or in extra-medullary sites, cure rate ranging from 30% to 40% [START_REF] Keating | The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis of the AML 8A study of the EORTC Leukaemia Cooperative Group and GIMEMA[END_REF][START_REF] Zittoun | Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) Leukemia Cooperative Groups[END_REF]. On the other hand, a minority of cases fail to achieve complete remission (CR) after a standard first line treatment. Age, cytogenetics, MDR-1 expression, previous or concomitant myelodysplasia, and first CR duration are the major prognostic factors influencing the outcome [START_REF] Burnett | The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial[END_REF][START_REF] Grimwade | The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties[END_REF][START_REF] Leith | Acute myeloid leukemia in the elderly: assessment of multidrug resistance (MDR1) and cytogenetics distinguishes biologic subgroups with remarkably distinct responses to standard chemotherapy. A Southwest Oncology Group study[END_REF][START_REF] Leith | Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/Pglycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study[END_REF][START_REF] Van Den Heuvel-Eibrink | MDR1 gene-related clonal selection and P-glycoprotein function and expression in relapsed or refractory acute myeloid leukemia[END_REF]. Primary refractory patients, and those who relapse early (i.e., first CR duration <6 months) cannot receive a transplant procedure, which is the best option with curative intent. The main objective in treating this group of poor prognosis patients is to adopt a salvage regimen having acceptable toxicity and capable of inducing high rate of complete remission, thus enabling patients to receive SCT.

Various salvage regimens, often including fludarabine associated with high-dose cytarabine (HDARAC) with or without an anthracycline, have been employed in this setting of AML patients. HDARAC increases intracellular availability of the active metabolite (Ara-CTP) and can rescue patients with refractory or relapsed leukemia; however, no survival advantage was found between poorrisk AML patients treated with HDARAC-containing regimens and those treated with investigational therapies [START_REF] Keating | The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis of the AML 8A study of the EORTC Leukaemia Cooperative Group and GIMEMA[END_REF]. Recent evidences suggest that fludarabine acts synergistically with cytarabine to increase intracellular Ara-CTP, if given soon before or simultaneously with cytarabine [START_REF] Gandhi | Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia during therapy[END_REF][START_REF] Vidarsson | Fludarabine and cytarabine as a sequential infusion regimen for treatment of adults with recurrent, refractory or poor prognosis acute leukemia[END_REF][START_REF] Ossenkoppele | The value of fludarabine in addition to ARA-C and G-CSF in the treatment of patients with high-risk myelodysplastic syndromes and AML in elderly patients[END_REF]. Such regimens (FLAG, Flag-Ida, FLANG) have been employed in several studies for treating AML in elderly patients and in those with a poor-risk disease, with CR rate ranging from 30% to 60% [START_REF] Clavio | High efficacy of fludarabine-containing therapy (FLAG-FLANG) in poor risk acute myeloid leukemia[END_REF][START_REF] Estey | Treatment of relapsed and refractory acute myelogenous leukemia[END_REF][START_REF] Montillo | Fludarabine, cytarabine, and G-CSF (FLAG) for the treatment of poor risk acute myeloid leukemia[END_REF][START_REF] Ferrara | Fludarabine, cytarabine, and granulocyte-colony stimulating factor for the treatment of high risk myelodysplastic syndromes[END_REF][START_REF] Byrne | Early allogeneic transplantation for refractory or relapsed acute leukaemia following remission induction with FLAG[END_REF][START_REF] Carella | Treatment of "poor risk" acute myeloid leukemia with fludarabine, cytarabine and G-CSF (FLAG regimen): a single center study[END_REF][START_REF] Jackson | A multicentre, open, non-comparative phase II study of a combination of fludarabine phosphate, cytarabine and granulocyte colony-stimulating factor in relapsed and refractory acute myeloid leukaemia and de novo refractory anaemia with excess of blasts in transformation[END_REF]. Unfortunately, toxicity is often relevant, and response duration is usually short, precluding most patients from benefiting of a transplant procedure.

A formulation of daunorubicin encapsulated in liposomal particles (liposomal daunorubicin, DaunoXome®) has an interesting pharmacokinetic profile: increased delivery to leukemic cells, decreased susceptibility to the membrane efflux pump, and reduced extra-hematological toxicity [START_REF] Gabizon | Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes[END_REF][START_REF] Gill | Phase I/II clinical and pharmacokinetic evaluation of liposomal daunorubicin[END_REF][START_REF] Cortes | Phase I study of liposomal daunorubicin in patients with acute leukemia[END_REF][START_REF] Fassas | Safety and early efficacy assessment of liposomal daunorubicin (DaunoXome) in adults with refractory or relapsed acute myeloblastic leukaemia: a phase I-II study[END_REF][START_REF] Sells | Reduction in toxicity of doxorubicin by liposomal entrapment[END_REF][START_REF] Guaglianone | Phase I and pharmacologic study of liposomal daunorubicin (DaunoXome)[END_REF]. We have combined HDARAC, fludarabine and DaunoXome® in a salvage regimen to be applied in a multicenter study for rescuing primary refractory and relapsed AML adult patients. For the latter drug, we scheduled three escalating dose levels, each one to be administered to a small cohort of patients. The aims of this trial were (1) to induce CR in primary refractory patients or a further remission in relapsed patients, (2) to evaluate the toxicity of a high-dose chemotherapy, including liposomal daunorubicin, (3) to evaluate the dose-limiting toxicity of DaunoXome® when associated with a sequential continuous infusion of fludarabine and HDARAC, and (4) to test the feasibility of post-remission consolidation by a SCT procedure.

Materials and methods

Eligibility criteria Adult patients older than 16 years, with primary refractory or relapsed AML, with exclusion of promyelocytic leukemia, were included in this study. Absence of heart, liver, or kidney damage, as well as no active infection, were required for inclusion in the trial. Adequate heart function was assessed by echocardiography (left ventricular ejection fraction ≥50%) and electrocardiogram. Written informed consent was obtained from all patients.

Disease evaluation Morphologic leukemia subtypes were evaluated on bone-marrow smears according to standard procedures, using the revised FAB criteria [START_REF] Bennett | Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group[END_REF] and according to the more recent WHO classification [START_REF] Vardiman | The World Health Organization (WHO) classification of the myeloid neoplasms[END_REF]. The disease status was assessed by bone-marrow cytology and flow cytometry analysis (FACSCalibur, Becton Dickinson). In addition, in several patients, gp170 expression was evaluated by incubating blasts with the MRK16 antibody (Kamiya Biochemical LTD) followed by flow cytometry analysis.

Cytogenetics Cytogenetic analysis was performed at diagnosis on 24-h cultured preparations of bone-marrow cells without stimulation. GTG-banded chromosomes were classified according to the International System for Human Cytogenetic Nomenclature [START_REF] Shibata | Recommended methods for cytological procedures in haematol-ogy. International Committee for Standardization in Haematology (ICSH)[END_REF]. At least ten metaphases were scored.

Treatment plan

The salvage regimen consisted of a loading dose of fludarabine (10 mg/m 2 ) administered intravenously over 15 min, followed after 3 h by cytarabine (390 mg/m 2 ) for 3 h on day 1, then by fludarabine (25 mg/m 2 ) administered intravenously on days 1 and 2, which was associated with cytarabine (1.9 g/m 2 ) on days 1 and 3 and both administered by continuous infusion over 24 h. DaunoXome was diluted in 250 ml of 5% dextrose and administered i.v. as 1-h infusion on days 2, 3, and 4, scheduled with a dose escalation as follows: the first group received 60 mg/m 2 (level 1), the second group received 80 mg/m 2 (level 2), and the third group was planned to receive 100 mg/m 2 (level 3). Each group was scheduled to consist of four patients from each participating institution.

Patients who achieved CR or partial remission (PR) were consolidated with a second FLAD course, in which liposomal daunorubicin was administered for two doses on days 2 and 3. Additional consolidation courses could be administered to patients in CR or PR while waiting for a SCT procedure. The salvage regimen is shown in Fig. 1.

All responder patients aged less than 55 years underwent an allogeneic SCT according to donor availability (patients with a HL-A identical sibling, who had achieved their first CR after the salvage protocol, underwent allogeneic SCT from identical sibling), while patients in second CR (with no identical sibling) were recruited for an unrelated marrow donor search. Auto-transplant was performed in patients aged 55 or older, and in some cases, by medical decision. Stem-cell transplants were performed at the various institutions according to the Center's own transplantation protocol, with conditioning regimens and GVHD prophylaxis given according to each institution's policy.

Concomitant medications Granulocyte colony stimulating factor (filgrastim or lenograstim at the dosage of 300 or 260 μg/day, respectively) was administered in patients with persisting neutropenia after the 20th day from the first day of therapy or in patients with neutropenia and proven infection until neutrophil recovery. Antiemetics, antibiotics, and antimycotics were also allowed according to clinical practice guidelines. Corticosteroids were used to control fever and chills during liposomal daunorubicin infusion. Packed red cells and platelets transfusions were used when hemoglobin was <8 g/dL and platelet count was below 10× 10 9 /L, respectively.

Toxicity All patients were carefully monitored during the salvage chemotherapy until hematological recovery. All events were graded according to WHO toxicity criteria and registered on the patient clinical record.

Response evaluation Hematological response to salvage chemotherapy was evaluated by bone-marrow cytology on day 28, or delayed for 1 or 2 weeks until blood count recovery. Patients who achieved CR (bone-marrow blasts <5%) were scheduled to receive one or more consolidation courses, followed by a SCT procedure, while patients in PR (bone-marrow blasts between 5% and 20%) were consid-ered for an additional salvage course. Patients showing leukemia persistence went off-study.

Statistical methods Disease-free survival (DFS) was calculated from the date of CR to the date of relapse, death, or last follow-up. Overall survival (OS) was defined as the time from inclusion in the study to death or last followup. Survival distributions were estimated by the method of Kaplan-Meier. Statistical differences in survival distributions were evaluated with the log-rank test. A twotailed p value of 0.05 or less was considered statistically significant. Data analysis was performed using the STATISTICA 7.0 software.

Results

Patient population From December 2000 to December 2006, 61 consecutive patients from seven institutions (see Appendix) entered the study. There were 35 men and 26 women, with a median age of 43 years (range, 21-72). FAB and WHO subtypes are shown in Table 1. Sixteen patients entered the trial because of leukemia persistence after first line induction therapy (eight of 16 patients had received more than one course of induction therapy), while 45 had had medullary leukemia relapse. For both refractory and relapsed patients, FACS analysis performed on bonemarrow leukemic cells confirmed the immunophenotype 2.

DaunoXome® dose escalation study With DaunoXome used at 80 mg/m 2 (i.e., second dose level), the toxicity was judged unacceptable. Indeed, four patients from two different centers treated with the salvage regimen, including the second dose level, experienced severe neutropenia (ANC<0.1×10 9 /L) lasting more than 30 days, associated with severe mucositis (grade IV WHO) and requiring total parenteral nutrition for several days. For these reasons, the protocol writing committee decided to stop the dose escalation program and fixed the DaunoXome dose at 60 mg/m 2 for all subsequent patients entering this study.

Response evaluation At hematological recovery after salvage therapy, CR was documented in seven of 16 patients (44%) with refractory leukemia and in 25 of 45 patients (56%) with leukemia relapse, with an overall response rate (ORR) of 52% (32 of 61). Twenty-two (36%) patients showed leukemia persistence, and seven patients (12%) died during chemotherapy: four because of infection, two because of disease progression, and one because of cerebral hemorrhage. These data are summarized in Table 3. Twentythree patients received one or more courses of consolidation therapy, with a median of two courses. Hematological relapse was observed in 20 of 32 cases (62.5%), with a median of 7.3 months (range, 0.6-54.1 months) from CR.

Eighteen patients had medullary relapse, whereas two patients experienced extra-hematological relapse: one in the left breast and the other in the left leg soft tissue. As for hematological response, no statistical difference was observed when patients were stratified for age (≤40 years vs >40 years). When patients were stratified according to their first CR duration (≤12 vs >12 months), a better CR rate (68%) was observed in the group of patients with a first CR >12 months (p=0.0002; Fig. 2). As for relapse after a transplant procedure, when this group of patients was stratified according to time from SCT (≤6 vs >6 months), a trend of better response was observed for patients who relapsed >6 months after the transplant. One patient showed both inv16 and trisomy 8. One patient showed both del 17 and trisomy 18. One patient showed both del 5 and iso 11.

One patient showed both del 7 and der 7. One patient showed both trisomy 8 and +M. + Additional chromosome,missing chromosome, del deleted chromosome, der derivated chromosome, inv inverted chromosome, iso isochromosome, +M additional unidentified genetic material Acute toxicity The main toxicity was myelosuppression. Median time to granulocyte (>0.5×10 9 /L) and platelet (>20×10 9 /L) recovery was 21 and 24 days, respectively. Fever (>38°C) for a median of 4 days (range, 1-20) occurred in 40 patients: six patients had positive blood cultures, while 34 had fever of unknown origin. Blood sepsis was fatal in four out of the six patients. Heart, liver, and enteric toxicity were negligible.

The main extra-hematological toxicity was stomatitis, occurring as grade II-III in 14 patients, while grade IV was observed in all patients (n=4) who had been treated with the salvage regimen containing the second dose level (i.e., 80 mg/m 2 ) of liposomal daunorubicin. Thus, in our hands, stomatitis represented the dose-limiting toxicity in this study. The data on toxicity are summarized in Table 4.

Stem-cell transplant Nineteen patients, 31% of the whole patient population (59% of those achieving CR), underwent a stem-cell transplant according to age and donor availability. Nine patients received a transplant from an HL-A identical sibling and five patients from a unrelated marrow donor; five patients were auto-transplanted, four from peripheral blood stem cells and one from bone marrow. Ten patients relapsed after the SCT procedure (four after auto-transplant, three after a HL-A identical sibling, and three after an unrelated donor transplant), with a median of 6.6 months. Among the transplanted patients, five are alive and are in continuous remission. Fig. 4 Disease-free survival, months in therapy responders, and only 2 months for resistants to salvage treatment. Median DFS was 7.3 months (range, 0.6-54.1; Fig. 4). Three patients died in CR: one from acute GVHD, one from pulmonary aspergillosis, and one from heart failure. Nine patients are alive, six of them in CCR after salvage, and three were rescued with additional cytotoxic treatment after a further relapse.

Survival analysis

Discussion

In adults with AML, while remission rate has improved in the last decade, leukemic relapse remains the main cause of treatment failure. Indeed, in spite of the availability of several effective cytotoxic drugs and of various intensive regimens, including SCT, the outcome of refractory or relapsed AML patients is still disappointing [START_REF] Keating | The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis of the AML 8A study of the EORTC Leukaemia Cooperative Group and GIMEMA[END_REF]. Because of the synergistic activity of fludarabine with cytarabine, this combination has been recently employed to rescue patients with relapsed AML [START_REF] Vidarsson | Fludarabine and cytarabine as a sequential infusion regimen for treatment of adults with recurrent, refractory or poor prognosis acute leukemia[END_REF]. While it is well known that highdose cytarabine is able to overcome leukemic cell resistance, the efficacy of anthracyclines in salvage regimens, in combination with the mentioned drugs, is not yet established. Furthermore, patients with relapsed or primary refractory AML were frequently proven to be resistant to various cytotoxic drugs because of the emergence of multiple-drug resistant clones showing high expression of the gp-170 molecule and/or of alternative mechanisms of resistance, mainly affecting leukemic cell susceptibility to anthracyclines [START_REF] Leith | Acute myeloid leukemia in the elderly: assessment of multidrug resistance (MDR1) and cytogenetics distinguishes biologic subgroups with remarkably distinct responses to standard chemotherapy. A Southwest Oncology Group study[END_REF]. Several preclinical and clinical studies have shown that liposomally encapsulated daunorubicin has a peculiar pharmacokinetic and pharmacodynamic profile, with improved leukemic cell delivery, reduced susceptibility to gp-170 membrane efflux pump, and reduced toxicity [START_REF] Gabizon | Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes[END_REF][START_REF] Fassas | The use of liposomal daunorubicin (DaunoXome) in acute myeloid leukemia[END_REF][START_REF] Michieli | Liposome encapsulated daunorubicin doubles anthracycline toxicity in cell lines showing a non-PGP related multidrug resistance[END_REF].

Taking into account these data, we designed this multicenter trial for salvage treatment of adult patients with primary refractory or relapsed AML, which included the combination of sequential continuous infusion of fludarabine and HDARAC combined with liposomal daunorubicin.

The observed ORR (52%) can be considered excellent in this subset of patients, which included those with primary refractory disease after standard or high-dose Ara-C induction (44% CR), even if the best response rate was obtained in the group of relapsed patients. Similar results have been recently published by Clavio et al. [START_REF] Clavio | Combination of liposomal daunorubicin (DaunoXome), fludarabine, and cytarabine (FLAD) in patients with poor-risk acute leukemia[END_REF] using a salvage treatment with the same drugs as in this study but without continuous infusion in a variety of patients with poor-risk acute leukemias (ALL, AML resistant or relapsed, de novo AML in elderly people).

SCT procedures (autologous or allogeneic transplant) were performed in a high proportion of patients enrolled in this study, allowing them to attempt leukemia eradication; this opportunity was also given to a few patients with a HL-A identical sibling, who had not achieved CR after frontline induction treatment.

As foreseeable for relapsed patients, the response rate to salvage therapy correlated with the duration of the first CR. Indeed, when we stratified the patients according to first CR duration into two groups (≤12 and >12 months), we observed a better outcome for patients with first CR duration >12 months.

We also performed a dose-finding study to evaluate the maximum tolerated dose (MTD) of DaunoXome®, planning three escalating doses in the context of the intensive three-drug regimen. Although the number of patients was too small to draw any definitive conclusion, the cumulative dose of 180 mg/m 2 (60 mg/m 2 for 3 days) showed the best efficacy/toxicity ratio. Indeed, when we used the dose scheduled for the second level (80 mg/m 2 for 3 days), we observed severe stomatitis and unacceptable prolonged neutropenia in four patients. The MTD of DaunoXome® is reported to be much higher when used as a single cytotoxic agent [START_REF] Fassas | Safety and early efficacy assessment of liposomal daunorubicin (DaunoXome) in adults with refractory or relapsed acute myeloblastic leukaemia: a phase I-II study[END_REF].

On the whole, the association of Daunoxome cumulative dose 180 mg/m 2 with HDARAC and fludarabine caused acceptable hematological and extra-hematological toxicity, with early deaths occurring only in few cases. It is to be noticed that no patient showed severe heart or gut toxicity.

In conclusion, the association of fludarabine, high-dose cytarabine, and DaunoXome® proved to be effective in inducing high CR rate in poor-risk AML patients, without the occurrence of significant toxicity. This salvage regimen allowed to perform a SCT procedure in a high proportion of patients in CR. High relapse rate remains the major cause of unsatisfactory outcome in these patients, pointing out to the need of more eradicating chemotherapeutic and/or conditioning regimens.

Fig. 2

 2 Fig. 2 Response rate according to first CR duration

Fig. 3

 3 Fig. 3 Overall survival, months

Appendix:

  Participating centers and number (#) of patients enrolled Università Federico II, Napoli (Prof. Bruno Rotoli) #30 A.O.R.N. Cardarelli, XIX, Napoli (Dr. Felicetto Ferrara) #17 Ospedale Civile, Avellino (Dr. Nicola Cantore) #7 A.O.R.N. Cardarelli, TERE, Napoli (Dr. Vincenzo Mettivier) #2 Ospedale S. Giovanni Bosco, Napoli (Dr. Eustachio Miraglia) #2 Ospedale S. Gennaro, Napoli (Dr. Lucia Mastrullo) #2 Ospedale Civile, Arienzo (Caserta) (Dr. David Pagnini) #1

Table 1

 1 Patient characteristics

	Refractory Relapsed

Fludarabine 10 mg/m i.v. 15' short infusion 2 d 1 followed after 3 hr by Cytarabine 390 mg/m i.v. 3 hr infusion 2 d 1 followed by Fludarabine 25 mg/m i.v. 24 hr continuous infusion

  Cytogenetics Evaluable metaphases were obtained at diagnosis in 49 of 61 patients. A normal karyotype was observed in 31 cases. Various chromosome translocations were present in eight patients, two of them showing the Ph chromosome [t(9;22)]. Hyper-and hypodiploidy were detected in six and three patients, respectively. Other anomalies were observed in six cases, as shown in Table

	2		d 1, 2
	Cytarabine 1.9 g/ m i.v. 24 hr continuous infusion 2	d 1, 2, 3
	DaunoXome 60 mg/ m level 1 2	i.v. 1 hr infusion	d 2, 3, 4
	80 mg/ m level 2 2
	100 mg/ m level 3 2
	Fig. 1 The FLAD regimen
	assessed at diagnosis. Multiple drug-resistance expression
	was evaluated in 30 cases, showing a low expression of
	gp-170, ranging from 3% to 7%. Fifty-seven patients had
	received as front-line treatment a standard three-drug
	regimen, including daunorubicin, or idarubicin, associated
	with standard or high dose of cytarabine and etoposide
	according to the EORTC/GIMEMA AML 10 or AML 12
	multicenter trials. One patient had received liposomal
	daunorubicin in association with cytarabine, and three
	patients had already been treated with the association of
	fludarabine and HDARAC. Thirty-one patients (51% of the
	whole study population) had relapsed after a SCT proce-
	dure, 26 after auto-transplant, and five after allogeneic
	marrow transplant (one of them had received also one
	course of DLI). For relapsed patients, the median duration
	of the first CR was 7 months (range, 2-69 months).

Table 2

 2 Cytogenetic data (n=49)

	Number

Table 3

 3 Response to salvage treatment

		Refractory	Relapsed	Total
	n	16	45	61
	CR	7 (44%)	25 (56%)	32 (52%)
	Leukemia persistence	8 (50%)	14 (31%)	22 (36%)
	Early deaths	1 (6%)	6 (13%)	7 (12%)

Table 4

 4 Toxicity after the salvage regimen

	Hematological toxicity ANC>0.5×10 9 /L PLT>20×10 9 /L Extra-hematological toxicity Heart function	Days to recovery, median 21 24	Grade (WHO) 1	Number 3	Cumulative Proportion Surviving
			2	2
	Hepatic		1 2	8 4	OVERALL SURVIVAL (n = 61)
			3	3
	Oral		1	4
			2	1 1
			3	4
			4	6
	Enteric		1	7
			2	7
			3	1
	FUO			34
	Sepsis			6


Ana Isabel Mendes

Ana Ferro

Rute Martins

Isabel Picanço

Susana Gomes

Rute Cerqueira

António Manuel Correia

Robalo Nunes

Jorge Esteves

Rita Fleming

Robalo António

Jorge Nunes

Rita Esteves

Paula Fleming

Faustino

P Faustino
email: paula.faustino@insa.min-saude.pt

: M Correia

A R Nunes

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes

Keywords: Hereditary hemochromatosis, HFE, non-C282Y, Population screening

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of adult-onset, common among Caucasians of Northern European ancestry, which leads to iron overload in several organs, if left untreated. Molecular studies have shown that HH is prevalently due to a founder missense mutation, C282Y, in the HFE gene on chromosome 6p21.3 [START_REF] Feder | A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis[END_REF]. Although C282Y in the homozygous condition remains the most frequent patients' genotype, other HFE mutations such as H63D and S65C have been described in compound heterozygosity with C282Y [START_REF] Feder | A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis[END_REF][START_REF] Mura | HFE mutations analysis in 711 hemochromatosis probands: Evidence for S65C implication in mild form of hemochromatosis[END_REF]. In Southern Europe, a large percentage of individuals (35-45%) with HH phenotypes are neither C282Y homozygous nor C282Y/H63D compound heterozygous. Other very rare or private HFE variants have been reported in affected individuals, contributing to the HH genetic heterogeneity [START_REF] Piperno | Heterogeneity of hemochromatosis in Italy[END_REF][START_REF] De Villiers | Spectrum of mutations in the HFE gene implicated in haemochomatosis and porphyria[END_REF][START_REF] Piperno | Two novel nonsense mutations of HFE gene in five unrelated Italian patients with hemochromatosis[END_REF][START_REF] Pointon | Uncommon mutations and polymorphisms in the hemochromatosis gene[END_REF][START_REF] Gac | Complete scanning of the hereditary hemochromatosis gene (HFE) by use of Denaturing HPLC[END_REF][START_REF] Beutler | A previously undescribed nonsense mutation of the HFE gene[END_REF][START_REF] Swinkels | A novel (Leu183Pro) mutation in the HFEgene co-inherited with the Cys282Tyr mutation in two unrelated Dutch hemochromatosis patients[END_REF][START_REF] Dupradeau | An unusual case of hemochromatosis due to a new compound heterozygosity in HFE (p.[Gly43Asp; His63Asp] +[Cys282Tyr]): structural implications with respect to binding with transferrin receptor 1[END_REF]. Moreover, an association of HH with other genes, such as the transferrin receptor 2 (TFR2) [START_REF] Camaschella | The gene encoding transferrin receptor 2 is mutated in a new type of hemochromatosis mapping to 7q22[END_REF][START_REF] Roetto | New mutations inactivating transferrin receptor 2 in hemochromatosis type 3[END_REF][START_REF] Lee | Mutation analysis of the transferrin receptor-2 gene in patients with iron overload[END_REF][START_REF] Biasiotto | Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload[END_REF][START_REF] Mattman | Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation[END_REF][START_REF] Gérolami | Early-haemochromatosis caused by a novel combination of TFR2 mutations (p.R396X/c.1538-2 A>G) in a woman of Italian descent onset[END_REF], the hemojuvelin (HJV) [START_REF] Papanikolaou | Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis[END_REF][START_REF] Gac | The recently identified type 2 A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype[END_REF][START_REF] Lanzara | Spectrum of hemojuvelin gene mutations in 1q-linked juvenile hemochromatosis[END_REF][START_REF] Lee | Genetic abnormalities and juvenile hemochromatosis: mutations of the HJV gene encoding hemojuvelin[END_REF][START_REF] Huang | Identification of a novel mutation (C321X) in HJV[END_REF][START_REF] Van Dijk | Effect of the new HJV-L165X mutation on penetrance of HFE[END_REF][START_REF] Ka | Phenotypic and functional data confirm causality of the recently identified hemoju-velin p.r176c missense mutation[END_REF][START_REF] De Diego | New HJV mutation in a patient with hyperferritinemia and H63D homozygosity for the HFE gene[END_REF][START_REF] Aguilar-Martinez | Juvenile hemochromatosis caused by a novel combination of hemojuvelin G320V/R176C mutation in a 5-year old girl[END_REF] and the hepcidin (HAMP) [START_REF] Biasiotto | Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload[END_REF][START_REF] Roetto | Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis[END_REF][START_REF] Merryweather-Clarke | Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis[END_REF][START_REF] Delatycki | A homozygous HAMP mutation in a multiply consanguineous family with pseudo-dominant hemochromatosis[END_REF][START_REF] Jacolot | HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype[END_REF][START_REF] Roetto | Screening hepcidin for mutations in juvenile hemochromatosis: identification of a new mutation (C70R)[END_REF][START_REF] Matthes | Severe hemochromatosis in a Portuguese family associated with a new mutation in the 5′-UTR of the HAMP gene[END_REF] genes, has also been documented. These latter two genes are mainly associated with the juvenile form of the disease (Juvenile Hemochromatosis, JH). The purpose of the current study was to search for uncommon HFE, TFR2, HJV, or HAMP mutations liable to explain the iron overload phenotype in Portuguese individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The result of this study will allow clarifying the importance of these genes in the routine HH genetic diagnosis in the Portuguese population.

Materials and methods

Selection of participants

The studied population consisted of 215 patients referred to the Human Genetics Centre of the National Institute of Health Dr. Ricardo Jorge in Lisbon, Portugal, for HH genetic testing. The criteria for patient's inclusion were to be adult, not related and presenting serum ferritin level >400 ng/ml or transferrin saturation >50% (unless currently treated by phlebotomy). All these patients were previously diagnosed as non-C282Y or non-H63D homozygous and non-compound heterozygous.

Fifty control samples from unrelated healthy individuals were also studied. Investigations were undertaken with the written informed consent of all participants.

Molecular studies

Polymerase chain reaction (PCR) amplification of HFE exons 2, 3 and 4, and of TFR2 exons 2, 4, 5 and 6 (including intron/exon boundaries) was performed for each patient's DNA, as described by Le Gac et al. 2001 [START_REF] Gac | Complete scanning of the hereditary hemochromatosis gene (HFE) by use of Denaturing HPLC[END_REF] and Roetto et al. 2001 [START_REF] Roetto | New mutations inactivating transferrin receptor 2 in hemochromatosis type 3[END_REF]. Screening for mutations in PCR-amplified DNA was performed by using both single-strand conformation polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC). Purified PCR products presenting altered profiles were directly sequenced using the ABI Prism BigDye Terminator v1.1 Cycle Sequencing Kit in the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Codifying regions of HJV and HAMP genes were screened by dHPLC as described or were directly sequenced [START_REF] Gac | The recently identified type 2 A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype[END_REF][START_REF] Lanzara | Spectrum of hemojuvelin gene mutations in 1q-linked juvenile hemochromatosis[END_REF][START_REF] Merryweather-Clarke | Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis[END_REF][START_REF] Jacolot | HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype[END_REF].

In silico studies Potential splice signals of mutated DNA sequences were analyzed using the SpliceView software (http://l25.itba.mi. cnr.it/~webgene/wwwspliceview.html) and multiple sequence alignment of HFE protein from mouse, rat, human and dog was accomplished by using the ClustalW v1.82 software (http://www.ebi.ac.uk/clustalw).

Results and discussion

In this study, the mutational screening of HFE and TFR2 was performed by using two different methodologies, SSCP and dHPLC. The analysis of HFE exon 2 revealed five abnormal profiles. The respective sequencing allowed the identification of the known S65C in compound heterozygosity with H63D in two individuals, in compound heterozygosity with C282Y in other individual, and in another one in the heterozygous condition. The other abnormal profile was due to a T→G transversion in heterozygosity at codon 46 (Fig. 1a). Consequently, leucine is replaced by tryptophan originating a novel mutation, L46W. It was found in a 45-year-old male presenting a transferrin saturation=68% and ferritin=444 ng/ml.

Although three abnormal dHPLC profiles of HFE exon 3 were observed, only two of them were detected by SSCP. One was due to a G→A transition at codon 129 (Fig. 1b), resulting in an aspartic acid to asparagine alteration and, consequently to the novel D129N mutation. It was detected in a 35-year-old man in compound heterozygosity with H63D. The patient presented transferrin saturation=79.6% and ferritin=135 ng/ml. Both L46W and D129N patients' families were not available to be studied, so it was not possible to observe a familiar segregation associated to the iron overload phenotype. The L46W mutation is localized near the flexible HFE α1 domain loop Q40-S45, which interacts with helix 1 of the transferrin receptor 1 (TfR1) helical domain [START_REF] Lebron | Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor[END_REF][START_REF] Bennett | Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor[END_REF]. On the other hand, the D129N mutation modifies a residue located in HFE α2 domain which interacts with helix 2 of TfR1. Therefore, these two novel mutations could affect HFE affinity for TfR1 and consequently might interfere with its proper functioning. Moreover, it was observed that both L46 and D129 are highly conserved residues (Fig. 2a andb), so they should be important for protein function. Splicing analysis suggested that both mutations do not affect splicing.

As L46W was found only in heterozygosity, it is unlikely that this mutation, by itself, justifies the patient iron overload. In addition, this individual has alcoholic habits that probably also contribute to his iron burden. Concerning the D129N/ H63D patient, attending to the novel mutation putative functional effects and to the absence of known external contributor factors, it might be a pathologic genotype.

Sequencing of the other two HFE exon 3 abnormal profiles revealed two novel alterations at tyrosine 138: C→T and C→G, giving rise to Y138Y (only detected by dHPLC) and Y138X, respectively. The former alteration that does not change the amino acid, was detected in heterozygosity in a 69-year-old man who presents transferrin saturation=45% and ferritin=1466 ng/ml. This mutation causes a minor alteration of the consensus value of a potential donor site (from 82 to 79). However, because this site is not commonly used, it is unlikely that this mutation affects splicing. So it may be an alteration without any pathologic significance and does not justify the patient's iron overload.

On the opposite, the nonsense mutation (Y138X) (Fig. 1c) was detected in compound heterozygosity with C282Y in a 63-year-old man presenting transferrin saturation = 90% and ferritin=1,000 ng/ml. This novel mutation originates a premature stop codon and consequently the correspondent mRNA will be committed to the nonsense-mediated mRNA decay mechanism. Therefore, this patient genotype (Y138X/ C282Y) entirely justifies his HH phenotype.

Two altered HFE exon 4 profiles were found. Sequencing revealed in one case a novel heterozygous A→T transversion at codon 230 (Fig. 1d), substituting a tyrosine for a phenylalanine (Y230F). The patient's family study revealed that Fig. 2 Multiple sequence alignment of HFE and TFR2 protein from mouse, rat, human and dog using the ClustalW v1.82 software. Considered residues are indicated by a rectangle; identical residues by an asterisk (*); conserved substitutions by a colon (:) and semiconserved substitutions by a dot (.) Fig. 1 Sequencing results showing the mutations found in the HFE, TFR2 and HJV genes although his father is a compound heterozygous for Y230F and H63D, he presents normal iron parameters (Table 1). Y230F alters a residue positioned on a β-strand of the α3 domain which binds to β2-microglobulin [START_REF] Lebron | Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor[END_REF][START_REF] Bennett | Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor[END_REF]. However, the Y230 position is originally occupied by a phenylalanine in some species (Fig. 2c), suggesting that tyrosine at position 230 may not be essential. Also, this mutation induces a small alteration of a potential acceptor site consensus value (from 81 to 85) but probably without consequences. Taking into consideration all these facts, it is unlikely that this mutation is contributing for the iron overload in this patient.

The other abnormal profile found in HFE exon 4 was due to the known E277K [START_REF] Bradbury | Spectrum and haplotypes of the HFE hemochromatosis gene in Iran: H63D in beta-thalassemia major and the first E277K homozygous[END_REF]35] mutation, found in compound heterozygosity with H63D in a patient. His family study showed that his brother presents the same genotype and also a high serum ferritin level (Table 2). The mutation does not alter splicing signals and E277 is highly conserved between species (Fig. 2d). Although the E277K has been previously described as a neutral polymorphism [START_REF] Bradbury | Spectrum and haplotypes of the HFE hemochromatosis gene in Iran: H63D in beta-thalassemia major and the first E277K homozygous[END_REF], in this study, it was detected in compound heterozygosity with H63D in two brothers both presenting altered iron parameters. Due to its HFE α3 domain localization, this amino acid alteration might perturb the binding to β2-microglobulin, affecting HFE correct processing. Altogether these facts suggest some deleterious effect of this mutation, but only functional studies could determine its real significance.

The screening of TFR2 revealed that three out of the 215 patients had altered dHPLC profiles. The direct sequencing identified one heterozygous transversion C→G at codon 238, which causes an amino acid substitution from isoleucine to methionine, I238M. This alteration was already reported as a polymorphism by Lee et al, 2001 [START_REF] Lee | Mutation analysis of the transferrin receptor-2 gene in patients with iron overload[END_REF].

Another TFR2 variant corresponds to a novel C→G mutation at codon 280 (Fig. 1e) which originates a phenylalanine to leucine substitution (F280L). It was found in double heterozygosity with H63D in a 29-year-old man presenting a transferrin saturation=80% and ferritin=111 ng/ml. This mutation does not alter splicing signals and F280 is a highly conserved residue (Fig. 2e). It is located in the apical region of TFR2, so the amino acid change might perturb protein function and, consequently, it's binding to transferrin. Considering the patient's alcoholic habits, the cause of his iron overload is probably the combined action of genetic and environmental factors. The F280L mutation requires functional studies to complete its characterization.

The other TFR2 abnormal profile found corresponded to the rare IVS5 -9T→A alteration [START_REF] Mattman | Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation[END_REF]. It was detected in double heterozygosity with H63D in a 38-year-old man presenting transferrin saturation=51% and ferritin=186 ng/ml. This mutation produces a small reduction of the native acceptor splicing site consensus value (88 to 84) and its effect on splicing can be hypothesized.

The 5′UTR -25G→A (24) mutation was found in homozygosity in the HAMP gene in a 50-year-old man who presented, at the age of 28, a severe juvenile hemochromatosis phenotype (transferrin saturation=70%; cardiomyopathy, hepatomegaly, intense hepatic hemosiderosis) [START_REF] De Lacerda | Hemocromatose cardíaca: uma "miocardia" curável[END_REF]. This mutation creates a new initiation codon at position +14 related to the cap site, which induces a shift of the open reading frame. Homozygosity for this mutation was previously reported as the cause of JH in two other individuals of Portuguese origin [START_REF] Matthes | Severe hemochromatosis in a Portuguese family associated with a new mutation in the 5′-UTR of the HAMP gene[END_REF][START_REF] Porto | A Portuguese patient homozygous for the -25G>A mutation of the HAMP promoter shows evidence of steady-state transcription but fails to up-regulate hepcidin levels by iron[END_REF]. As this third affected individual had a birth place located near the first one described (Matthes T, personal communication), probably they are related to the same HAMP mutational event.

Two of the HJV alterations found are apparently asymptomatic. The A310G is described as a polymorphism in ensEMBL (www.ensembl.org) and GeneCards® (www. genecards.org) databases. The other one, a G→A transition at codon 275, does not alter the amino acid residue (E275E) and does not seem to affect splicing. On the other hand, the third alteration observed, the IVS2 +395 C→G (Fig. 1f), was detected in heterozygosity in a 29-year-old man presenting transferrin saturation=59% and ferritin=661 ng/ml. It is located at six deoxyribonucleotides from the beginning of exon 3 and induces an alteration of the consensus value of the native acceptor site (from 89 to 85), which becomes lower than another potential splice site (86). Consequently, some competition between these two splice sites might occur, altering the splicing variants produced. Even if the mutation deleterious effect is confirmed in future functional studies, it is improbable that its heterozygosity is the only cause of the individual iron overload. All novel mutations found were searched for and not found in 50 healthy individuals, so they cannot be considered polymorphisms.

In conclusion, in this study, several alterations were found in HFE, TFR2, HJV, and HAMP genes, screened in 215 Portuguese individuals presenting iron overload, previously characterized as non-C282Y or non-H63D homozygous or compound heterozygous. However, only a few number of these mutations can be directly associated with HH or JH, revealing a modest contribution of the novel variants in this phenotype development, and suggesting that their screening in routine diagnosis is not cost-effective. Nevertheless, the identification and study of these novel mutations is important to enlarge the knowledge of the HH genetic heterogeneity. Additionally, their functional study can be significant for a better understanding of the correspondent proteins role in the iron metabolism and in HH development.

Table 1

 1 Clinical data of the HFE Y230F family

		Propositus Father	Mother Brother
	Age (years)	30	55	54	23
	Sex	M	M	F	M
	Cardiomyopathy -	+	-	-
	Diabetes mellitus -	-	+	-
	Iron (μg/dl)	162	76	69	63
	Transferrin	75.8	35.8	26.3	32.1
	saturation (%)
	Ferritin (ng/ml)	531	165	74	258
	HFE genotype	Y230F/wt Y230F/H63D wt/wt	H63D/wt

wt Wild type, + presence,absence

Table 2

 2 Clinical data of the HFE E277K family

		Propositus	Brother	Sister I	Sister II
	Age (years)	63	67	59	46
	Sex	M	M	F	F
	Cardiomyopathy -	+	-	-
	Iron (μg/dl)	185	115	77	30
	Transferrin	60.7	39.5	25	9
	saturation (%)
	Ferritin (ng/ml) 620	812	72	20.8
	HFE genotype	E277K/H63D E277K/H63D H63D/wt wt/wt
	wt Wild type, + presence, -absence

Acknowledgements We thank G. Le Gac (Establissement Français du Sang-Bretagne, Brest, France), G. Biasiotto (Università di Brescia, Italy), F. Daraio (Università di Torino, Italy) e A. Roetto (Università di Torino, Italy) for having kindly supplied mutant controls. We also thank the Unidade Laboratorial de Utilização Comum of the National Institute of Health Dr. Ricardo Jorge for technical support.

This work was partially supported by Fundação para a Ciência e a Tecnologia, Programa de Financiamento Plurianual do CIGMH. This work was awarded by Comissão de Fomento da Investigação em Cuidados de Saúde, Ministério da Saúde P.I. no. 130/2007.


Lars Fischer
email: lars.fischer@charite.de

Agnieszka Korfel

Philipp Kiewe

Martin Neumann

Kristoph Jahnke

Eckhard Thiel

: M Neumann

: K Jahnke

Systemic high-dose methotrexate plus ifosfamide is highly effective for central nervous system (CNS) involvement of lymphoma

Keywords: Neoplastic meningitis, CNS lymphoma, Ifosfamide, Methotrexate

Patients with malignant central nervous system (CNS) involvement of lymphoma have a poor prognosis with intrathecal chemotherapy and radiation. In this paper, we report the results we obtained in such patients by intravenous chemotherapy with high-dose methotrexate and ifosfamide (HDMTX/IFO). The study involved a review of all patients who received HDMTX/IFO for CNS involvement of malignant lymphoma at our hospital. Therapy consisted of 4 g/m 2 of MTX (4 h infusion on day 1) and 1.5-2 g/m 2 /day of IFO (3 h infusion on days 3-5). The study included 20 patients with a median age of 65 years (range, 30-83) and CNS relapse of a malignant lymphoma. Seventeen patients had been pretreated with up to two chemotherapy regimens. The objective response rate was 90% with 12 complete or unconfirmed complete (CR and CRu) and six partial remissions. All patients had at least stabilization of their neurological symptoms. Myelosuppression was the most common toxicity. The median follow-up time was 14.9 months. The median time to neurological progression was 8.9 months. Twelve patients received subsequent therapy, including high-dose chemotherapy with autologous stem cell transplantation in five cases. The median overall survival was not reached. Systemic chemotherapy with HDMTX/IFO is a feasible and promising treatment modality for CNS relapse of a malignant lymphoma.

Introduction

Central nervous system (CNS) involvement is a serious complication of hematologic malignancies. Standard therapy has not been established for these patients. Patients with leptomeningeal involvement usually receive intrathecal (IT) chemotherapy consisting of methotrexate (MTX) or cytarabine, which is sometimes combined with radiotherapy for nodular tumor masses [START_REF] Bokstein | Leptomeningeal metastases from solid tumors: a comparison of two prospective series treated with and without intra-cerebrospinal fluid chemotherapy[END_REF][START_REF] Glantz | Randomized trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis[END_REF][START_REF] Glantz | A randomized controlled trial comparing intrathecal sustained-release cytarabine (DepoCyt) to intrathecal methotrexate in patients with neoplastic meningitis from solid tumors[END_REF][START_REF] Grossman | Randomized prospective comparison of intraventricular methotrexate and thiotepa in patients with previously untreated neoplastic meningitis. Eastern Cooperative Oncology Group[END_REF]. Patients presenting with parenchymal brain involvement are usually submitted to whole-brain irradiation (WBI). With this therapy, patients with malignant CNS involvement generally have a poor prognosis with a median survival of usually less than 6 months. Systemic disease is a frequent cause of death in these patients [START_REF] Bokstein | Central nervous system relapse of systemic non-Hodgkin's lymphoma: results of treatment based on high-dose methotrexate combination chemotherapy[END_REF][START_REF] Bollen | Central nervous system relapse in non-Hodgkin lymphoma. A single-center study of 532 patients[END_REF][START_REF] Lagerwaard | Identification of prognostic factors in patients with brain metastases: a review of 1292 patients[END_REF][START_REF] Van Besien | Risk factors, treatment, and outcome of central nervous system recurrence in adults with intermediate-grade and immunoblastic lymphoma[END_REF].

In the management of CNS malignancies, systemic therapy offers several potential advantages over local treatment modalities like IT chemotherapy or radiation, the most important one being simultaneous treatment of the systemic disease [START_REF] Bokstein | Central nervous system relapse of systemic non-Hodgkin's lymphoma: results of treatment based on high-dose methotrexate combination chemotherapy[END_REF][START_REF] Van Besien | Risk factors, treatment, and outcome of central nervous system recurrence in adults with intermediate-grade and immunoblastic lymphoma[END_REF][START_REF] Blaney | Pharmacologic strategies for the treatment of meningeal malignancy[END_REF]. Furthermore, systemic application achieves a more uniform distribution of the drug throughout the neuroaxis and obviates ventricular reservoir insertion or multiple lumbar punctures (LP) for drug instillation with all the potential acute and late complications. Finally, a much wider array of chemotherapeutic agents can be administered by intravenous (IV) than by IT application. On the other hand, considerable systemic toxicity is associated with IV chemotherapy, whereas IT therapy poses potential infectious and neurotoxic risks.

Both systemic ifosfamide and high-dose MTX (doses of 1.0 g/m 2 or more) reach cytostatic levels in the cerebrospinal fluid (CSF) and are effective for treating various malignancies [START_REF] Hiraga | Rapid infusion of highdose methotrexate resulting in enhanced penetration into cerebrospinal fluid and intensified tumor response in primary central nervous system lymphomas[END_REF][START_REF] Tetef | Pharmacokinetics and toxicity of high-dose intravenous methotrexate in the treatment of leptomeningeal carcinomatosis[END_REF][START_REF] Yule | Cyclophosphamide and ifosfamide metabolites in the cerebrospinal fluid of children[END_REF]. This monocenter study retrospectively analyzed the activity and toxicity of combination chemotherapy with high-dose methotrexate and ifosfamide (HDMTX/IFO) for CNS manifestations of non-Hodgkin's lymphoma.

Materials and methods

Patients

The study included all patients treated with HDMTX/IFO for CNS lymphoma (including the retina) at our institution from July 2002 to August 2007. CNS disease was diagnosed radiologically [brain lesions and/or meningeal enhancement on cranial and spinal magnetic resonance imaging (MRI) or computed tomography (CT)] or by CSF cytomorphology or immunocytology. Intraocular malignancy was diagnosed by slit-lamp examination and aqueous cytology or chorioretinal biopsy. All patients underwent a staging procedure for systemic tumor manifestations with bone marrow biopsy and contrast-enhanced CT of the thorax and abdomen.

Exclusion criteria were creatinine clearance <50 ml/min, liver disease with elevation of bilirubin >2 mg/dl, previous WBI, and active systemic infection. All patients gave written informed consent before starting treatment.

Treatment

Patients received an IV infusion of MTX 4 g/m 2 over 4 h on day 1 and IFO 1.5 or 2 g/m 2 /day over 3 h on days 3 to 5. The MTX dose was adjusted for creatinine clearance (CC) <100 ml/min as follows: CC/100 × 4g/m 2 . Supportive therapy included antiemetics, intravenous hydration, urine alkalization, mesna, and a leucovorin rescue beginning 24 h after the start of MTX infusion. Systemic corticosteroids were applied only for symptom control at the beginning of therapy. Treatment was continued for a maximum of eight cycles and stopped in cases where further therapy was prevented by radiologic or CSF progression, clinical deterioration, or toxicity.

Response and toxicity

In patients treated after August 2005, response of parenchymal lesions was evaluated according to the then published criteria of the International Primary CNS Lymphoma Collaborative Group (IPCG) [START_REF] Abrey | Report of an international workshop to standardize baseline evaluation and response criteria for primary CNS lymphoma[END_REF]. Those included the specification of an unconfirmed complete remission (CRu) for cases with small but persisting enhancing abnormalities on brain imaging or patients fulfilling the criteria for complete remission (CR) but still receiving corticosteroids. In three patients treated before 2005, IPCG guidelines were used for a retrospective evaluation of parenchymal brain lesions. These patients had CR according to both Cheson criteria [START_REF] Cheson | Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group[END_REF] used initially and IPCG criteria used for the present analysis.

In leptomeningeal disease, CR was defined as a negative CSF cytomorphology in two consecutive LP and disappearance of meningeal enhancement on gadolinium MRI, if present at first presentation. Progressive disease (PD) was defined as an increase of CSF tumor cells, while stable disease (SD) was considered to be any other condition. Response and SD were only documented if neurological symptoms had improved or remained stable; patients were otherwise assessed as having PD [START_REF] Glantz | Randomized trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis[END_REF][START_REF] Grossman | Randomized prospective comparison of intraventricular methotrexate and thiotepa in patients with previously untreated neoplastic meningitis. Eastern Cooperative Oncology Group[END_REF]. Response of measurable systemic disease was documented according to Cheson's criteria [START_REF] Cheson | Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group[END_REF].

Neurological response was classified as complete response, improvement, stable disease, or worsening. Neurological complete response was defined as complete resolution of all neurological symptoms and signs attributable to the malignant disease, improvement as regression of symptoms with residual deficits, worsening as neurological deterioration, and stable disease as no improvement in the absence of new symptoms.

Response evaluation of all CNS tumor sites using the initial method (MRI, CT, CSF cytomorphology/immunocytology, and ophthalmologic examination) was performed at least after every second chemotherapy cycle and every 3 months after therapy. A full clinical and neurological assessment, including toxicity evaluation, was performed before each chemotherapy course.

Adverse events were graded according to the Common Terminology Criteria (CTC) of the National Cancer Institute, version 3.0, and the highest observed grade was recorded for each patient.

Statistical analysis

A descriptive analysis of qualitative and quantitative variables was done. Frequencies, median values, and ranges were specified when applicable. Estimates of the overall survival (OAS) and time to neurological progression (TTP) were calculated for all patients using the Kaplan-Meier method. The OAS was calculated from the start of therapy to the last follow-up or to death from any cause. The time to neurological progression was measured from the start of therapy until neurological deterioration, death from CNS disease, or the last follow-up. Responding or stable patients who continued with other treatment (due to toxicity or the attending physician's decision) were censored for calculation of the TTP.

Results

Patient characteristics

Twenty patients with a median age of 65 years (Table 1) were treated with at least one HDMTX/IFO course. Meningeal involvement was confirmed by cytomorphologic CSF evaluation in all patients. Parenchymal brain involvement was confirmed by biopsy in all but two patients who had developed parenchymal brain lesions during treatment for histologically proven systemic lymphoma. Seventeen patients had diffused large B-cell lymphoma (DLBCL) in the CNS. Secondary CNS involvement of systemic DLBCL was present in eight of them and a relapsed PCNSL/PIOL in seven, while two had an aggressive large-cell lymphoma in the CNS in the presence of systemic indolent B-cell lymphoma (Richter's transformation of Waldenstrom's macroglobulinemia). Two patients had simultaneous systemic and CNS involvement of mantle cell lymphoma and marginal zone lymphoma, respectively, and one patient had a T-cell lymphoma.

Three patients with systemic indolent B-cell lymphoma already had CNS disease at first presentation and were therapy-naive. All others had a CNS relapse and were pretreated with up to two different chemotherapy regimens. Systemic pretreatment regimens were CHOP-like (with or without rituximab). CNS-directed pretreatment (one or two therapy lines) had been given to ten patients and consisted of IT chemotherapy with MTX or IV chemotherapy with HDMTX, high-dose cytarabine, ifosfamide, or topotecan. Nine patients (45%) had concomitant systemic disease.

Therapy

Patients were treated with a median of five HDMTX/IFO cycles (range, 1 to 8). Nine patients received the full MTX dose, while 11 had a dose adjustment to 50-90% based on their creatinine clearance. Dose adjustment was necessary in nine (64%) patients >60 years but only in two (33%) younger ones (p=0.28).

HDMTX/IFO was terminated prematurely in ten patients for the following reasons: creatinine clearance <50 ml/min (n=3, all with grade 2 nephrotoxicity), the doctor's decision to intensify treatment by high-dose chemotherapy with autologous stem cell transplantation (SCT; n=3), disease progression (n=2), the patient's request (n=1), and toxic death (n=1). In three other patients, therapy was modified as follows: An individual decision was made to stop HDMTX after one to five cycles due to grade 1, 2, or 3 nephrotoxicity, and therapy was continued with IFO alone. Therapy had to be modified or terminated due to toxicity or renal function impairment in five (36%) patients over 60 and in one (25%) younger patient (p=0.4).

No concurrent lymphoma-directed therapy was given except to one patient with CNS relapse of a mantle cell lymphoma, who was additionally treated with IT liposomal cytarabine.

Granulocyte colony-stimulating factor (G-CSF)-supported mobilization of peripheral blood stem cells after HDMTX/ IFO in six patients was successful in all cases. Five of them proceeded to high-dose chemotherapy with carmustine or melphalan in combination with thiotepa followed by autologous stem cell transplantation (three for consolidation and two at relapse), and eight patients received various other consolidation or salvage treatments (systemic therapy with topotecan, high-dose cytarabine or zevaline, WBI, or IT chemotherapy).

Response

Eighteen patients (90%) responded to HDMTX/IFO with a CR in ten cases, a CRu in two, and a PR in six (Fig. 1). Two patients had SD for 1.9 and 5.1 months. Neurological symptoms resolved completely in eight patients, improved in nine, and remained stable over 1.3 to 5.5 months in three (two with an objective response and one with stable CNS disease). Pretreatment with HDMTX led to a CNS response rate of 85.7% (six of seven patients) as compared to 92.3% (12 of 13 patients) with other regimens or without pretreatment (p=0.6).

Systemic manifestations responded in four patients: axillary lymph node enlargement in a thyroid non-Hodgkin's lymphoma disappeared completely, as did intrapulmonary nodules in a relapsed PCNSL and bone marrow infiltration in a marginal zone lymphoma after six to eight cycles of HDMTX/IFO, while pulmonary manifestations in one systemic DLBCL were in partial remission after four cycles.

One patient with Waldenstrom's macroglobulinemia remained stable according to his paraprotein levels. In four patients, systemic disease (bone marrow involvement of DLBCL or indolent lymphoma) was not reevaluated due either to the doctor's decision or to death before systemic response evaluation.

Toxicity

Myelosuppression was the most frequent toxicity and involved grades 3 and 4 leukopenia in 13 patients (65%), grade 3 and 4 thrombopenia in six (30%), and grade 3 anemia in four (20%). There were no significant differences in the frequency of thrombopenia and anemia between patients over 60 years and younger ones. However, grade 4 leukopenia occurred only in patients over 60. Grades 3, 2, and 1 infection was observed in one patient each. Grade 3 nephrotoxicity occurred in one patient and grades 1 and 2 in 11 patients, leading to omission of HDMTX in three of them. In three patients, HDMTX/IFO was terminated prematurely due to nephrotoxicity grade 2. One patient developed acute ifosfamide-related grade 2 encephalopathy, which reversed spontaneously after 3 days. Other toxicities were nausea (grade 3 in one patient and grades 1 and 2 in nine patients) and fatigue (grade 3 in one patient and grades 1 and 2 in seven patients).

There were two therapy-related deaths, both due to sepsis in neutropenia: An 83-year-old patient with DLBCL and bone marrow infiltration died after two chemotherapy cycles, and a 73-year-old patient with stage IV mantle cell lymphoma died after six chemotherapy courses.

Survival

After a median follow-up of 14.9 months, 13 patients were alive, and seven had died (five from tumor progression and two of toxicity). The site of relapse/progression was the CNS in all patients. For the whole patient population, the median TTP was 8.9 months (range 1.3 to 30 months), and Fig. 1 Patient with CNS and pulmonary relapse of DLBCL responding to therapy (a-c cranial MRI, d-e chest CT scan): a, d after partial surgical removal of CNS tumor and before chemotherapy; b, d after four courses of HDMTX/IFO; c 3 months after high-dose carmustine/thiotepa and autologous SCT the median OAS was not reached (range 1.8 to 50+ months; Fig. 2).

There were no significant differences in outcome with respect to age (≤60 versus >60 years), meningeal involvement, or lymphoma type (Table 2); however, a trend toward longer survival was seen in patients without meningeal involvement and with PCNSL. The estimated survival at 12 months was 55% for older and 83% for younger patients (p=0.2).

Seven patients survived longer than 12 months; four of them received high-dose therapy with autologous stem-cell transplantation after HDMTX/IFO.

Discussion

Data supporting the utility of systemic chemotherapy for secondary CNS involvement in malignant disease are mostly limited to small phase II studies or retrospective analyses of heterogeneous patient populations. Some analyses suggested a survival advantage for CNS-directed systemic chemotherapy and found no evidence for a superiority of IT therapy [START_REF] Bokstein | Leptomeningeal metastases from solid tumors: a comparison of two prospective series treated with and without intra-cerebrospinal fluid chemotherapy[END_REF][START_REF] Boogerd | Meningeal carcinomatosis in breast cancer. Prognostic factors and influence of treatment[END_REF][16][START_REF] Siegal | Leptomeningeal metastases: analysis of 31 patients with sustained off-therapy response following combined-modality therapy[END_REF].

Two studies have reported on systemic HDMTX monotherapy for CNS manifestations of lymphoma and solid tumors [START_REF] Glantz | High-dose intravenous methotrexate for patients with nonleukemic leptomeningeal cancer: is intrathecal chemotherapy necessary?[END_REF][START_REF] Lassman | Systemic highdose intravenous methotrexate for central nervous system metastases[END_REF]. Glantz et al. found that treatment with 8 g/m 2 of MTX led to a high-objective response rate of 81% and a median survival of 13.8 months in 16 patients with lymphoma and solid tumor neoplastic meningitis. The study suggested a superiority of systemic HDMTX over IT MTX applied in a historic control group [START_REF] Glantz | High-dose intravenous methotrexate for patients with nonleukemic leptomeningeal cancer: is intrathecal chemotherapy necessary?[END_REF]. Lassmann et al. administered 3.5 g/m 2 of MTX to 23 solid tumor patients, 44% of whom had isolated meningeal metastases, and reported a PR in 33% and a median OAS of 20 weeks [START_REF] Lassman | Systemic highdose intravenous methotrexate for central nervous system metastases[END_REF]. In a prospective study, the combination of 3.5 g/m 2 MTX, procarbazine, and IT cytarabine in 23 patients with secondary CNS lymphoma achieved a response rate of 100% with a 33% CR rate for leptomeningeal disease. The median OAS of the whole group was 6 months [START_REF] Bokstein | Central nervous system relapse of systemic non-Hodgkin's lymphoma: results of treatment based on high-dose methotrexate combination chemotherapy[END_REF].

Little data is available on IFO for treatment of malignant CNS disease. The drug was included in treatment protocols that proved effective in PCNSL and intraocular lymphoma, both at first diagnosis and at relapse [START_REF] Arellano-Rodrigo | Salvage treatment with etoposide (VP-16), ifosfamide and cytarabine (Ara-C) for patients with recurrent primary central nervous system lymphoma[END_REF][START_REF] Pels | Primary central nervous system lymphoma: results of a pilot and phase II study of systemic and intraventricular chemotherapy with deferred radiotherapy[END_REF][START_REF] Jahnke | Pharmacokinetics and efficacy of ifosfamide or trofosfamide in patients with intraocular lymphoma[END_REF].

The present study included a relatively old population with a high proportion of pretreated and potentially chemotherapy-resistant patients. An age of over 60 is a well-established risk factor in both PCNSL and systemic lymphoma [START_REF] Ferreri | Prognostic scoring system for primary CNS lymphomas: the International Extranodal Lymphoma Study Group experience[END_REF][START_REF] Shipp | Can we improve upon the international index?[END_REF]. Our study nevertheless achieved a high objective response rate of 90%. Moreover, the median TTP of 8.9 months compares favorably to the outcome reported in the literature. The study regimen proved effective for both parenchymal and meningeal involvement as well as concomitant systemic disease. Remarkably, a response was observed in nine of ten patients pretreated with up to two CNS-directed therapies, including HDMTX in six patients. Survival tended to be shorter in patients with meningeal disease than in those with brain parenchyma involvement. The reason for this difference remains speculative. These patient groups were comparable for age and pretreatment; however, four of six patients with meningeal disease had secondary CNS lymphoma. It may be speculated whether secondary CNS lymphoma has a generally worse prognosis than PCNSL.

Our results should be interpreted with caution because of some obvious study limitations such as the retrospective analysis and the relatively small number of patients. Moreover, our patient population was heterogeneous in terms of lymphoma subtype (including relapsed systemic DLBCL, PCNSL, and indolent CNS lymphoma), pretreatment (three patients were therapy-naive), and post-study treatment. In particular, subsequent treatment with highdose chemotherapy and autologous stem cell transplantation in five patients might have contributed to the favorable overall survival. High-dose chemotherapy with stem-cell transplantation is the only treatment associated with prolonged survival in patients with CNS involvement of aggressive lymphomas [START_REF] Alvarnas | High-dose therapy with hematopoietic cell transplantation for patients with central nervous system involvement by non-Hodgkin's lymphoma[END_REF][START_REF] Jahnke | Retrospective study of prognostic factors in non-Hodgkin lymphoma secondarily involving the central nervous system[END_REF]. WBI alone may represent an effective salvage treatment for many PCNSL patients [START_REF] Hottinger | Salvage whole brain radiotherapy for recurrent or refractory primary CNS lymphoma[END_REF]. However, there is growing evidence linking it to delayed neurotoxicity in these cases [START_REF] Fischer | Response of relapsed or refractory primary central nervous system lymphoma (PCNSL) to topotecan[END_REF][START_REF] Herrlinger | NOA-03 trial of highdose methotrexate in primary central nervous system lymphoma: final report[END_REF][START_REF] Omuro | Delayed neurotoxicity in primary central nervous system lymphoma[END_REF]. On the other hand, different salvage chemotherapy regimens have proven effective for PCNSL [START_REF] Reni | Therapeutic management of refractory or relapsed primary central nervous system lymphomas[END_REF]. Thus, delaying WBI in relapsed PCNSL seems a reasonable approach. Since the risk of late neurotoxicity appears to be particularly high when HDMTX is given after WBI [START_REF] Blay | High-dose methotrexate for the treatment of primary cerebral lymphomas: analysis of survival and late neurologic toxicity in a retrospective series[END_REF], patients pretreated with WBI do not receive HDMTX at our institution and were therefore excluded from this analysis. This might have introduced a selection bias, since relapse after WBI could entail a different prognosis than relapse after chemotherapy alone.

HDMTX/IFO had considerable hematotoxicity with a 70% incidence of CTC grades 3 and 4 leukopenia and an 8.7% treatment-related mortality rate. Prophylactic administration of G-CSF should therefore be considered in this protocol. Antibacterial and, in patients receiving corticosteroids, possibly antifungal prophylaxis might also reduce the rate of infectious complications, especially in older patients. Both HDMTX and IFO are potentially nephrotoxic, but renal function impairment was generally manageable with dose adjustment according to creatinine clearance. However, therapy had to be modified and terminated prematurely due to renal insufficiency in 30% of patients.

Systemic HDMTX/IFO seems to be a promising therapy option for CNS involvement of lymphoma. Our results justify evaluation of the MTX/IFO protocol in a prospective study.

Fig. 2

 2 Fig. 2 Overall survival (OAS) and time to neurological progression (TTP) for all patients

Table 1

 1 Patient characteristics at diagnosis of CNS disease

	Characteristics	Values
		n	Percent
	Age (years)	30-83 (median 65)
	Age >60	14
	Male/female	10/10
	Histologic diagnosis (CNS)
	DLBCL	17	85
	Mantle cell lymphoma	1	5
	Marginal zone lymphoma	1	5
	T-cell lymphoma	1	5
	CNS involvement
	Meningeal	3	15
	Parenchymal	13	65
	Meningeal+parenchymal	3	15
	Ocular	1	5
	Systemic disease	9	45
	Primary CNS involvement /CNS relapse	3/17	15/85
	Previous therapy
	1 regimen	13	65
	2 regimens	4	20
	Previous CNS directed therapy
	1 regimen	9	45
	2 regimens	1	5
	Symptoms a
	Cranial nerve palsy	5	25
	Sensorimotor deficits	4	20
	Nausea/Emesis	4	20
	Pain	4	20
	Cerebellar	5	20
	Neuropsychological	5	20
	Other	11	55

a More than one symptom per patient possible

Table 2

 2 Outcome by age, meningeal involvement, and lymphoma type

	Median TTP	Median OAS
	(months)	(months)


Noelia Cabrera

Pilar Casaña

Ana Rosa Cid

Saturnino Haya

Andrés Moret

Jose Antonio Aznar

Novel missense mutation c.2685G>C (p.Q895H) in VWF gene associated with very low levels of VWF mRNA

Keywords: VWF gene, von Willebrand disease, Missense mutation, Low expression, Type 2N, Factor VIII binding capacity

Homozygous patients for null alleles in VWF gene show a severe von Willebrand phenotype, whereas compound heterozygous patients only show the phenotype of the expressed allele. Five members of the same family were studied. The two patients showed borderline VWF levels, a mild factor VIII (FVIII) deficiency and a decrease of the binding of VWF to exogenous FVIII. The genetic analyses of the VWF gene confirmed that the patients were compound heterozygous for c.2561G>A (R854Q) and c.2685G>C (p.Q895H) mutations. The latter, is located in the 3′ extreme of exon 20, and it has not been previously described. Studies of the cDNA from platelet mRNA were performed to investigate the expression of p.H895 allele. The loss of heterozygosity at the cDNA level suggests a lack of expression of the p.H895 allele. The overall studies can explain the type 2N phenotype of the two patients, since the allele carrying the new missense mutation p.Q895H has shown a low expression of VWF gene.

Introduction

Von Willebrand disease (VWD) is the most common inherited bleeding disorder in humans, is heterogeneous, and results from quantitative and/or qualitative defects in the von Willebrand factor (VWF). VWF is a large multimeric glycoprotein synthesized in the endothelial cells and in the megakaryocytes. It is required for platelet adhesion to the injured subendothelium and stabilizes coagulation factor VIII (FVIII) in the plasma [START_REF] Ruggeri | von Willebrand factor and von Willebrand disease[END_REF]. The VWF gene is localized on chromosome 12, spans 178 kb, contains 52 exons, and is transcribed into an 8.8-kb mRNA [START_REF] Mancuso | Structure of the gene for human von Willebrand factor[END_REF]. In recent years, many molecular defects of the VWF gene, associated with different VWD types, have been identified. (Sheffield University VWF http://www.vwf. group.shef.ac.uk/). VWD type 3 is the most severe variant and is characterized by extremely low or undetectable levels of VWF and by a recessive inherence. Different mutation types have been detected in severe VWD patients, such as frameshift, nonsense, splice site, and missense mutations. Large gene deletions are not very frequent and are usually associated with inhibitor development. Loss of heterozygosity in the cDNA VWF from the platelets indicates the presence of a null allele [START_REF] Nichols | Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: detection by exonic PCRrestriction fragment length polymorphism analysis[END_REF], although usually the specific molecular defect was unknown. Homozygotes for null alleles produce a total reduction of VWF levels in plasma, heterozygotes, generally, are asymptomatic, and compound heterozygotes for a null allele and other genetic defect show the phenotype of the expressed allele. Furthermore, the presence of null alleles could explain the variable penetrance among some families [START_REF] Eikenboom | Recessive inheritance of von Willebrand's disease type I[END_REF].

We now report one missense mutation c.2685G>C (p.Q895H), which produces a very low level of VWF mRNA. This mutation, combined with the common p.R854Q mutation, which in homozygosis causes type 2N VWD, allows the clinical expression of just the p.Q854 allele.

Materials and methods

Five members belonging to the same family were studied. They gave their consent after they were informed of the kind of study they were to take part in.

For diagnosis, determination of FVIII procoagulant activity (FVIII:C), VWF antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), VWF collagen-binding capacity (VWF:CB), and analysis of the multimeric structure were performed as previously described [START_REF] Casaña | Significant linkage and non-linkage of type 1 von Willebrand disease to the von Willebrand factor gene[END_REF]. The VWF-binding assay (VWF:FVIIIB) was carried out with minor changes to the method previously reported [START_REF] Casonato | The evaluation of factor VIII binding activity of von Willebrand factor by means of an ELISA method[END_REF].

The segregation polymorphism analyses were performed as previously described [START_REF] Casaña | Significant linkage and non-linkage of type 1 von Willebrand disease to the von Willebrand factor gene[END_REF]. The screening of the 52 exons and their boundary exon/intron of the VWF gene was carried out in the index case by single strand conformation polymorphism analyses (SSCPs), conformation-sensitive gel electrophoresis (CSGE), and sequencing of abnormal patterns.

To analyze the expression of VWF gene alleles, plateletrich plasma (PRP) was obtained by centrifugation at 150×g for 10 min at 20°C. Platelet pellets were immediately prepared to extract platelet RNA or frozen at -80°C. Platelet RNA was prepared from these pellets with RNeasy Total RNA kit. Platelet mRNA was reverse transcribed using the StrataScript First Strand cDNA Synthesis Kit. The cDNA was subsequently amplified by polymerase chain reaction to obtain a 540-bp fragment spanning from exon 17 to 21 of VWF gene. Restriction endonuclease assays with MspI, HpyCH4IV, and RsaI, were performed as recommended by the manufacturer and were analyzed by electrophoresis on 10% acrylamide/bis (29:1) gels and silver stain. The mean and SD are shown when three or more determinations were performed. n.a. Not available

Results and discussion

The proband (II:1) and one of her sisters (II:2), both blood group O, showed mild FVIII deficiency (15±3 and 19± 2 IU/dL respectively), below borderline VWF:Ag, VWF: RCo, and VWF:CB levels, and normal multimeric structure. The two patients were classified as VWD 2N based on marked decrease of the capacity of plasma VWF to bind exogenous FVIII. In addition, plasma VWF levels from parents were normal, and the father showed normal VWF: FVIIIB/VWF:Ag ratio (Table 1). Family segregation analyses of VWF gene markers showed that two of the sisters shared the same haplotypes (Fig. 1a). SSCP analyses showed an abnormal pattern in the exon 20 fragment. The DNA sequence analysis of this fragment revealed two mutations: c.2561G>A (p.R854Q) and c.2685G>C (p.Q895H). We did not find any other candidate mutations in the rest of the sequences of the VWF gene analysed. Family study of the p.R854Q mutation was performed by restriction enzyme digestion with MspI. To corroborate the new nucleotide substitution c.2685G>C (p.Q895H), we digested the 301-bp fragment corresponding to exon 20 with the restriction enzyme HpyCH4IV, since it generates a new restriction site.

The analyses performed confirmed that the father carried the c.2685G>C (p.Q895H) change, the mother carried the c.2561G>A (p.R854Q) mutation, whereas the two sisters were compound heterozygous for the two mutations. The HpyCh4IV restriction analysis was performed in at least 100 normal chromosomes in order to rule out the possibility of this new change being a polymorphism.

The two sisters with both mutations, [p.R854Q] + [p.Q895H], showed a marked decrease in the ability to bind exogenously added FVIII. Therefore, it might be thought that the new p.Q895H mutation on the second VWF allele may explain the observed type 2N VWD phenotype (Table 1). However, taking into account that the guanine 2685 is the last nucleotide in 3′ extreme of exon 20, a splice site prediction program was used in order to evaluate if this new mutation affects this donor splice site (http://www.fruitfly.org/seq_tools/splice.html). In the normal sequence, the donor site was predicted with a score of 0.86. For the changed c.2685G>C (p.Q895H), this score was reduced to 0.46. All this suggests that, in the p.H895 allele, an incorrect intron splicing could occur and that may affect the mRNA stability. To evaluate the expression of VWF gene in both alleles, we studied the platelet mRNA.

We performed restriction analyses with RsaI enzyme, since all the family members were heterozygotes for the polymorphism 15/292 A/G (p.T/A789). The two sisters (II:1, II:2) and their father (I:1) showed, at cDNA level, a loss of heterozygosity of the allele A which is associated with the changed c.2685G>C (p.Q895H) (Fig. 1b). This loss of heterozygosity suggests that the p.H895 allele is silent at mRNA level. The mother showed a heterozygous pattern both at gDNA as at cDNA level, which indicates that both alleles are expressed.

In summary, our patients are compound heterozygous for both missense mutations, p.Q895H and p.R854Q, as only the latter is expressed; their phenotype is characteristic of the recessive type 2N variant. Heterozygous family members for the p.Q895H mutation, as well as for p.R854Q mutation, result in asymptomatic phenotypes.

Furthermore, future in vitro expression studies with recombinant VWF could confirm whether the p.Q895H mutation is really the cause of low transcription of the VWF gene.

Fig. 1 a

 1 Fig. 1 a Pedigree from family with the segregation of polymorphisms and the p.R854Q and p.Q895H mutations in VWF gene. Solid symbols represent affected members, and half-filled symbols represent the heterozygous state. The arrow marks the index case. b Rsa I restriction analysis at gDNA and cDNA level. The resulting 322-bp

Table 1

 1 Phenotype and genotype data of the family

	Subject	FVIII:C (IU/dL)	VWF:Ag (IU/dL)	VWF:RCo (IU/dL)	VWF:FVIIIB/VWF:Ag	Mutation
	I:1	103±49	61±28	74±20	0.98	[p.Q895H]
	I:2	121	74	121	n.a.	[p.R854Q]
	II:1	15±3	31±6	32±2	0.1	[p.R854Q] + [p.Q895H]
	II:2	19±2	32±2	38±5	0.1	[p.R854Q] + [p.Q895H]
	II:3	84	70	114	1.05	none
	Normal	60-140	48-140	50-170	∼1

AnnHematol (2009) 88:245-247 DOI 10.1007/s00277-008-0576-7 N. Cabrera : P. Casaña (*) : A. R. Cid : S. Haya : A. Moret : J. A. Aznar Unidad de Coagulopatías Congénitas. Hospital Universitario La Fe., Av. Campanar 21, 46009 Valencia, Spain e-mail: casanya_pil@gva.es

Acknowledgments This work was partly supported in part by CSL Behring, and by FIS grant no. PI020612 (Spain). We wish to thank all the staff of the "Unidad de Coagulopatías Congénitas de la Comunidad Valenciana" for their technical and clinical assistance.