Lena Reindl

Ulrike Bacher

Frank Dicker

Tamara Weiss

Wolfgang Kern

Susanne Schnittger

Torsten Haferlach

Claudia Haferlach
email: claudia.haferlach@mll-online.com

Dr. med Claudia Pd

M.D. Haferlach

Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms

Keywords: LYMPHOMAS, CHROMOSOME STRUCTURE, DIAGNOSTIC HAEMATOLOGY 14q deletion, chronic lymphocytic leukemia (CLL), mature B-cell neoplasms, interphase fluorescence in situ hybridization (FISH), chromosome banding analysis

14q-deletions were recurrently described in mature B-cell neoplasms, but not yet characterized in a larger cohort. With chromosome banding analysis, we identified 47 del(14q) cases in 3054 mature B-cell neoplasms (1.5%) (frequencies in CLL: 1.9%; CLL/PL: 9.0%; others: 0.2%). Interphase FISH was performed with probes for 14q22. 1, 14q24.1, 14q32.33, and IgH (14q32.3). The del(14q) had heteregeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IgH locus (14q32.3) between IgH3'-flanking and IgVH probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. 81% of del(14q) cases showed 1-3 additional cytogenetic alterations (in 45%, +12), and 56% were IgVH-unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B-CLL immunophenotype was found. Clinical follow-up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3-year-OS did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21.0 months vs. 80.1 months, p=0.015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B-cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment.

Introduction

Rearrangements of the IgH locus on chromosome 14q32 are identified in various mature Bcell lymphomas, which is illustrated by the t(11;14)(q13;q32)/IGH-CCND1 rearrangement in mantle cell lymphoma, or the t(14;18)(q32;q21)/IGH-BCL2 in follicular lymphoma. Even in Bcell chronic lymphocytic leukemia (B-CLL), reciprocal 14q32/IGH rearrangements are observed with low frequencies [START_REF] Haferlach | Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping[END_REF]. However, it has been shown that the long arm of chromosome 14q is not only involved in reciprocal rearrangements in the mature B-cell neoplasms, but can as well be affected by deletions [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF].

In 1990, Kristofferson et al. published a case series of six patients with different mature B-cell neoplasms who were affected by a 14q deletion [START_REF] Kristoffersson | Deletion of 14q in non-Hodgkin's lymphoma[END_REF]. In the Mitelman Database, a total of 413 cases with a del(14q) have been collected so far [START_REF] Mitelman | Mitelman Database of Chromosome Aberrations in Cancer[END_REF]. Of those, two thirds were classified as hematologic malignancies, while 30% were characterized as B-CLL or other mature B-cell neoplasms (Figure 1) [START_REF] Mitelman | Mitelman Database of Chromosome Aberrations in Cancer[END_REF].

The only larger study of a cohort of 23 patients with different B-cell malignancies and evidence of the del(14q) has been performed by Pospisilova et al. Using array CGH (arraycomparative genomic hybridization) and FISH (fluorescence in situ hybridization), the authors were able to separate cases with a 14q deletion in two subgroups which either showed involvement of the IgH locus on 14q32.33 or did not. Apparently, the size of deletion was variable but clustering at specific chromosomal bands was observed [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF].

It was suggested that the exact localization of the 14q deletion is biologically relevant: IgVH deletions being localized at the telomeric end of chromosome 14q probably have no oncogenic potential and reflect more likely physiological events accompanying somatic V-D-J assembly [START_REF] Quintero-Rivera | Frequency of 5'IGH deletions in B-cell chronic lymphocytic leukemia[END_REF]Wlodarska et al, 2007a). In contrast, 14q deletions with an interstitial localization seem to be oncogenic. Based on the hypothesis that interstitial 14q deletions may operate in a translocation-like manner and activate an unknown oncogene, Pospisilova et al. performed quantitative PCR (qPCR) and microarray analysis to identify the potentially affected gene. However, no gene could be identified which was overexpressed due to rearrangement of the IgH locus [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF].

Considering the low proliferation rates of metaphases in cases with mature B-cell neoplasms [START_REF] Juliusson | Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities[END_REF], diagnosis of the cytogenetic alterations has mainly been based on interphase FISH in these entities. With specific cultivation techniques using CD40 ligand (a B-cell mitogen) or a combination of CpG-oligodeoxynucleotides and the interleukin IL-2 for stimulation of cell division, generation of metaphases has been considerably improved in B-CLL and other mature B-cell neoplasms. With these novel metaphase cultivation techniques, chromosomal alteration rates similar to the results of interphase FISH were being observed in mature B-cell neoplasms [START_REF] Buhmann | CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B-cell chronic lymphocytic leukaemia cells[END_REF][START_REF] Dicker | Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression[END_REF][START_REF] Haferlach | Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping[END_REF][START_REF] Mayr | Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia[END_REF]. These approaches revealed new insights in the genetic diversity of B-cell neoplasms and allowed a comprehensive picture of the cytogenetic alterations in these heterogeneous malignancies.

So far, a limited number of studies only focused on the frequency and clinical impact of 14q deletions in the mature B-cell neoplasms, the exact characterization of the deletion, and the patterns of additional cytogenetic alterations. To further clarify these aspects, and to determine the frequency of 14q deletions in a large cohort, we here evaluated the incidence of this cytogenetic alteration in 3054 patients with various mature B-cell neoplasms with chromosome banding analysis by use of specific metaphases cultivation protocols [START_REF] Dicker | Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression[END_REF][START_REF] Haferlach | Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping[END_REF]. Those 47 lymphoma cases with 14q deletions were studied in detail with interphase FISH. In addition, immunophenotypes, cytomorphology, additional cytogenetic abnormalities, the IgVH mutation status, and clinical outcomes were analyzed.

Patients

In the period of August 2005 to March 2009, a total of 3054 cases with mature B-cell neoplasms were investigated by chromosome banding analysis in the Munich Leukemia Laboratory (MLL). Peripheral blood or bone marrow samples had been sent from diverse hematologic centers and clinical practices for diagnostic work-up. When patients agreed with the performance of genetic analysis, they gave as well informed written consent to the use of laboratory results for scientific studies. The study design adhered to the declaration of Helsinki.

In these 3054 cases, we were able to identify 47 cases with a 14q deletion by chromosome banding analysis. There were 31 male and 16 female patients corresponding to a male/female ratio of 1.9. Median age was 67.5 years (range, 50.3 to 85.2 years). In addition, patients were analyzed by interphase FISH combined with immunophenotyping and cytomorphology. Following the laboratory results, and the clinical data obtained from the patients physicians, the 47 cases were categorized according to the WHO classification (2008) as follows: 35/47 patients with a 14q deletion (75%) had B-CLL, 10 (21%) had CLL/PL (atypical CLL), and 2 (4%) were diagnosed with other mature B-cell neoplasms.

Methods

Metaphase Cytogenetics

For metaphase induction, 10 7 peripheral blood or bone marrow cells were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) with 20% fetal calf serum (FCS) in the presence of the immunostimulatory CpG-oligonucleotide DSP30 (2 µM) (TIBMolBiol, Berlin, Germany) and interleukin 2 (IL-2) (200 U/ml) (PreproTech Inc., Rocky Hill, USA) following a previously established protocol for karyotyping in mature B-cell neoplasms [START_REF] Dicker | Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression[END_REF]. Briefly, after 48 hours incubation of metaphases at 37°C, colcemid (Sigma, Munich, Germany) was added at a concentration of 0.15µg/ml for another 24 hours before chromosome preparation. In addition, a parallel culture with 12-O-tetradecanoylphorbol-13acetate (TPA) was started. After 72 hours, colcemid (0.15µg/ml) was added to the TPA stimulated culture for another 2 hours before chromosome preparation. Chromosome preparation and staining was done according to standard protocols (Schoch et al, 2002b).

Cases with complex aberrations underwent 24-color FISH procedure (Schoch et al, 2002a).

Chromosomes were classified according to the International System for Human Cytogenetic Nomenclature [START_REF] Iscn ; Shaffer | Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature[END_REF].

Fluorescence in situ hybridization

Interphase FISH was done as described previously [START_REF] Dicker | Complex aberrant karyotypes and unbalanced translocations in CLL correlate with an unmutated IgVH status: a study on 133 patients studied with chromosome banding analysis, interphase FISH, IgVH mutation status, ZAP-70 RNA exrpession and immunophenotyping[END_REF]. Probes were used for the 14q22.1 (BAC RP11-218E20; BlueGnome, Cambridge, UK), for 14q24.1 (BACs PR11-35D12, RP11-226F19; BlueGnome), and 14q32.33 (BAC RP11-521B24, BlueGnome) regions, combined with a probe for the IgH locus (14q32.3, flanking IgH3'-region, marking the IgVH region) (Abbott, Wiesbaden, Germany). The exact localization of the BACS is shown in Table 1 and Figure 2. The localization and hybridization efficiency was tested on metaphases obtained from PHA-stimulated lymphocytes.

Cytomorphology

Cases with less than 10% prolymphocytes were classified as typical CLL, those with 10-55% of prolymphocytes were interpreted as CLL/PL (atypical CLL), while presence of ≥55% of prolymphocytes qualified for the "PLL category".

Immunophenotyping

Immunophenotyping with multiparameter flow cytometry (MFC) followed previously described procedures for mature B-cell neoplasms [START_REF] Kern | Correlation of flow cytometrically determined expression of ZAP-70 using the SBZAP antibody with IgVH mutation status and cytogenetics in 1,229 patients with chronic lymphocytic leukemia[END_REF].

Analysis of the IgVH mutation status

For analysis of the IgVH mutation status [START_REF] Dicker | Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression[END_REF], monoclonal immunoglobulin rearrangements were amplified by PCR from cDNA using 6 different consensus framework region 1 (FR1) V H forward primers and one consensus joining region J H reverse primer. PCR products were agarose gel purified and analyzed by direct sequencing. Sequences were aligned to immunoglobulin sequences in the IMGT (http://imgt.cines.fr/) database and evaluated according to ERIC recommendations [START_REF] Ghia | ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia[END_REF]. For comparison of dichotomous variables between different groups the two-sided Fisher´s exact test was used. Survival was defined as the time from diagnosis to death or date of the last follow-up. Time to treatment (TTT) was defined as the time from diagnosis to date of first treatment or date of the last follow-up. All tests were 2-sided, accepting p≤0.05 as indicating a statistically significant difference. Statistical analysis was performed with the SPSS software (version 14.0; SPSS, Chicago, USA).

Statistics

Results

Frequency of 14q deletions in the different lymphoma entities

A total of 3054 cases with diverse mature B-cell neoplasms (B-CLL: n=1865, CLL/PL: n=111, others: n=1078) were investigated by chromosome banding analysis. Thus, 47 cases with 14q deletion were identified corresponding to an overall frequency of 1.5%.

Regarding the distinct entities, 35 cases with 14q deletion were classified as B-CLL, 10 as CLL/PL, and 2 as other mature B-cell neoplasms (including one case of lymphoplasmocytic lymphoma and one case of marginal zone lymphoma). Therefore, the incidence of 14q deletions was 1.9% in CLL, 9.0% in CLL/PL and 0.2% in other mature B-cell neoplasms with a significant association between the 14q deletion and a CLL/PL immunophenotype (CLL vs. CLL/PL: p<0.001; CLL vs. other mature B-cell neoplasms: p<0.001; CLL/PL vs. other mature B-cell neoplasms: p<0.001). (Patient's characteristics are depicted in Table 2.)

Characterization of the breakpoints

The centromeric and telomeric breakpoints of the 14q deletions were investigated in all 47 cases by interphase FISH probes covering the centromeric side (14.q22.1; BAC RP11-218E20), the 14q24.1 locus (BACs RP11-35D12, RP11-226F19), and the (telomeric) 14q32.33 region (BAC RP11-521B24) combined with a probe for the IgH locus (14q32.33) (Table 3, Figure 3).

Concerning the centromeric side of the del(14q), a deletion of 14q22.1 was very rare as BAC RP11-218E20 was deleted in 3/47 cases (6%) only.

With regard to 14q24.1 (being covered by BACs RP11-35D12 and RP11-226F19), FISH revealed the following categories:

1. While no deletion of the region detected by BAC RP11-35D12 (at 14q.24.1) was found in 29/47 cases (62%), all but one case (46/47; 98%) showed loss of one signal for BAC RP11-226F19 (at 14q24.1). Thus, in 29 patients (62%), the centromeric breakpoint could be localized between the regions covered by BACs RP11-35D12 and RP11-226F19 at 14q24.1. 3. In one case (2%), both BACs on 14q24 (RP11-35D12 and RP11-226F19) failed to detect a deletion suggesting that the breakpoint was closer to the telomeric end of the chromosome.

At the telomeric side (14q32.33, being covered by BAC RP11-521B24 and the IgH locus probe), three different signal patterns were observed:

1. Deletion detected with the 3'flanking IGH probe, while the variable region of the IgH locus was not affected: n=21 (21/47; 45% of all cases).

2. Complete deletion of the region covered with both IGH probes: n=11 (23%).

3. No deletion detected with both IGH probes: n=15 (32%).

Clustering of breakpoints

In 16/47 cases (34% of all cases), breakpoints showed clustering at the centromeric side of the 14q deletion between the BACs RP11-35D12 and RP11-226F19 at 14q24.1 and at the telomeric side between the 3'flanking IGH-probe and the IgVH region at 14q32.3

[corresponding to a del(14)(q24q32)]. This was resulting in a deletion of 37.2 Mb size.

Analysis of additional cytogenetic alterations

In 9/47 (19%) cases, the del(14q) was the sole chromosomal abnormality. 16/47 (34%) and 5/47 (11%) showed one or two additional cytogenetic alterations, respectively. Complex aberrant karyotypes as defined by the simultaneous occurrence of at least three additional clonal cytogenetic alterations were detected in 17/47 (36%) of the patients with a del(14q) (Table 3).

When the 14q deletions were included in the analysis, patients showed a mean number of 4.1 cytogenetic alterations (range, 1-15). As the most frequent additional abnormality, we detected trisomy 12 (21/47; 45%). In 11/21 cases (52%), trisomy 12 (+12) was the only additional change.

In cases with one additional alteration accompanying the del(14q) (n=16), the most frequent alteration was a trisomy 12 (13/16; 81%). This was followed by additional 13q deletions (2/16; 13%). In the group with two additional alterations besides the 14q deletion, most frequent was as well trisomy 12 (3/5; 40%). In cases with a complex aberrant karyotype, 17p deletions were observed in 8/17 cases (47%), +12 in 5 (29%), and del(13q) in 4 (24%) (Figure 4). We then analyzed the additional alterations separately for each category: In CLL, 6/35 (17%) cases showed no additional alteration, while 14/35 (40%) had one, and 3/35 (9%) had two additional alterations. Three or more additional alterations (corresponding to a complex karyotype) were observed in 12/35 (34%) CLL cases. CLL/PL showed one additional alteration in 2/10 cases (20%), one additional alteration in 2/10 cases (20%), two additional alterations in 2/10 cases (20%), and additional complex alterations in 4 cases (4/10; 40%). In other mature B-cell neoplasms we had the following results: 2 additional alterations: n=1/2 (50%), ≥3 additional abnormalities: n=1/2 (50%). Trisomy 12 occurred with comparable frequency in CLL (16/35; 46%), CLL/PL (4/10; 40%), and other mature B-cell neoplasms (1/2; 50%).

Analysis of the IgVH mutation status

IgVH mutation analysis was performed in 27 patients with a del(14q) (from the original 47 patients; 57%) and in 1572 patients without a del(14q) (from an original amount of 3054 patients; 51%). An unmutated IgVH status was detected in 15/27 patients (56%) carrying a 14q deletion. In comparison, an unmutated IgVH mutation status was detected in 635 out of 1572 patients without a del(14q) (40.0%). Thus, an unmutated IgVH status was slightly more frequent in del(14q) patients (p=0.09).

In more detail, in the 21/35 CLL patients with del(14q) in whom IgVH mutation analysis was performed, 12 cases showed an unmutated IgVH mutation status (57%) and 9 cases showed a mutated IgVH mutation status (42%). For the patients with CLL/PL, IgVH mutation analysis was performed in 6/10 cases with del(14q) (3/6 unmutated and 3/6 mutated IgVH mutation status). In both cases with other mature B-cell neoplasms no data on the IgVH mutation status was available.

Characterization of patients with breakpoints in 14q24 and in 14q32

The above described subgroup (n=16) with clustering of breakpoints and a del(14)(q24q32) due to breakpoints between BAC RP11-35D12 and RP11-226F19 (at 14q24.1) and between the 3'flanking IGH probe and the IgVH region (at 14q32.3) was associated with the immunophenotype of a B-CLL. In this specific subgroup, All cases (16/16) with a del(14q) compared to 1863/3054 (61%) of all investigated cases were classified as CLL (p=0.1; n.s.). This subgroup -being outlined by a del(14)(q24q32) -showed in 4/16 of cases (25%) the 14q deletion as sole chromosomal alteration, 13/16 (19%) showed one, and 1/16 (6%) showed two additional alterations. In 8/16 cases (50%), the del(14)(q24q32) appeared within a In 11 of these cases with a del(14)(q24q32), IgVH mutation status analysis was performed.

An unmutated IgVH mutation status was present in 9/11 cases (82%) which was significantly more frequent as compared to the del(14q) negative cohort (635/1572; 40%, p=0.023).

Prognostic impact of 14q deletions

Clinical follow-up data were available in 32 patients with 14q deletions and CLL and CLL/PL. These were compared to 383 CLL and CLL/PL cases without 14q deletion and available follow-up data. The 3-year overall survival (OS) did not differ significantly between both groups (14q deletion: 86.4%; no 14q deletion: 88.7%, p=0.195), but time to treatment (TTT) was significantly shorter in the del(14q) cases (21.0 months vs. 80.1 months, p=0.015, Figure 5).

Next, all 415 cases with CLL or CLL/PL were assigned to five subgroups with known prognostic relevance in addition to cases with 14q-deletion: del(14q): n=32, del(17p): n=26, del(11q): n=42, +12: n=43, del(13q): n=180, or none of the mentioned abnormalities: n=92.

The assignment was hierarchical in the order as mentioned above. OS at 3 years was 86.4%, 31.4%, 77.5%, 96.9%, 93.4%, and 93.9%, respectively (Figure 6a). For time to treatment (TTT), results were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months, and 147.0 months (Figure 6b). In addition, Cox regression analyses were performed. In univariate analysis testing the above mentioned genetic categories and IgVH mutation status only 17p deletion and a mutated IgVH status were significantly associated with OS (for del(17p): p<0.001, Hazard ratio (HR) 8.3 (95% confidence interval (CI): 3.5 -18.9); for mutated IgVH status: p=0.009, HR 0.3 (95% CI 0,1-0.8)). In multivariate analysis both parameters retained significance (for del(17p): p<0.001, Hazard ratio (HR) 8.7 (95% confidence interval (CI): 3.6 -21.4); for mutated IgVH status: p=0.026, HR 0.4 (95% CI 0.2-0.9). In the respective univariate analyses for TTT, 13q deletions as the sole abnormality and a mutated IgVH status were significantly associated with a longer TTT (p<0.001, HR: 0.4, 95% CI: 0.3-0.7 and p<0.0001, HR: 0.2, 95% CI: 0.1-0.3), while 14q deletion (p=0.018, HR: 2.1, 95% CI: 1.1-4.0) and 11q deletions were significantly associated with a shorter TTT (p<0.001, HR: 3.1, 95% CI: 2.0-5.0). In multivariate analysis only a mutated IgVH status retained a significant association with longer TTT (p<0.001, HR: 0.2, 95% CI: 0.2-0.4).

According to Döhner's hierarchical model, patients with trisomy 12 without 11q deletion and without 17p deletion were assigned to an intermediate prognostic category. As 14q deletions are frequently associated with trisomy 12, we evaluated the impact of 14q deletion in the cohort of patients with trisomy 12 (without 11q or 17p deletion). 43 cases with trisomy 12 included in this analysis. Cox regression analysis revealed that the occurrence of 14q deletions in addition to trisomy 12 was significantly associated with shorter OS (p=0.036, HR: 13.9, 95% CI: 1.2-160.9) and shorter TTT (p=0.007, HR: 7.2, 95% CI: 1.7-30.0). Interestingly, in this cohort with trisomy 12 the IgVH mutation status had no significant impact on OS and TTT.

Discussion

In CLL, cytogenetic results are highly relevant to perform a biological subcategorization and to define the prognosis. Examples are the prognostically adverse 17p or 11q deletions which can be detected with interphase FISH [START_REF] Döhner | Genomic aberrations and survival in chronic lymphocytic leukemia[END_REF], but a comprehensive view of the complex cytogenetic patterns of CLL and other B-cell neoplasms requires illustration of the complete karyotypes by chromosome banding analysis. Following the development of specific protocols for cultivation of metaphases [START_REF] Buhmann | CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B-cell chronic lymphocytic leukaemia cells[END_REF][START_REF] Dicker | Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression[END_REF][START_REF] Mayr | Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia[END_REF], mature B-cell neoplasms can now be investigated in more detail, e.g. for occurrence of 14q deletions.

The most detailed analysis focusing on 14q deletions in mature B-cell neoplasms has been published by Pospisilova et al. who studied 23 affected patients with array CGH. They categorized the different breakpoints into two separate groups in dependence on the involvement of 14q32.33 (where IgH is localized) and were able to demonstrate specific breakpoint clusters [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF]. However, so far no study has evaluated the frequency of the del(14q) in a larger cohort of patients with mature B-cell neoplasms, and analysis of the associated cytogenetic patterns as of the potential clinical impact of this specific alteration were missing. Thus, we investigated the frequency of 14q deletions in a large cohort of 3054 cases with different mature B-cell neoplasms.

First, we detected 14q deletions in a 1.5% frequency with chromosome banding analysis, which suggests that the del(14q) is a rare recurrent alteration in CLL, CLL/PL, and other lymphoma entities, e.g. lymphoplasmocytic lymphoma. Similar, Put et al. revealed one case with a 14q deletion performing interphase FISH in 217 cases with CLL (resulting in a 0.5% frequency) [START_REF] Put | Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study[END_REF]. In plasma cell myeloma (PCM), Wlodarska et al. described occurrence of 14q deletions in 1.4% of cases with FISH analyses (Wlodarska et al, 2007b).

In our study, the highest frequency of 14q deletions was observed in CLL/PL with 9.0% of all cases (compared to 1.9% in typical CLL and to 0.2% in other mature B-cell neoplasms) which suggests that the del(14q) shows variable occurrence in different lymphoma subentities. However, in our cohort, most patients had a diagnosis of CLL or CLL/PL, while only two patients had other lymphoma entities. Thus, it seems that the frequency of 14q deletions in other lymphoma entities has to be further evaluated.

Investigation with FISH probes covering the 14q22.1, 14q24.1, 14q32.33, and 14q32.3/IgH regions demonstrated heterogeneous size of the 14q deletions in our cohort. Considering the centromeric side of the deletion, we identified a breakpoint cluster at 14q24.1 (in 62% of cases), whereas at the telomeric side, the most frequent breakpoint was localized within the IgH locus (14q32.3) between the IgH3'-flanking probe and the IgVH probe (in 45% of cases). Thus, we found the most frequent telomeric breakpoint of the del(14q) proximal of the IgVH region in 14q32, which was corresponding to the results of Pospisilova et al. (Pospisilova et al, 2007). Although the size of the deletions was variable, we were able to define a particular breakpoint pattern in 16 cases: The centromeric breakpoint was characterized by deletion of BAC RP11-226F19 but a remaining BAC RP11-35D12, while the telomeric breakpoint was deleting the IgH3'-flanking sequence without involvement of the IgVH sequence. This breakpoint cluster had as well been described by Pospisilova et al. [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF].

In the current study, only 5 of those 21 patients with a distal breakpoint in the IgH gene but a remaining variable region of IgH showed a different proximal breakpoint.

The finding of a breakpoint cluster for interstitial 14q deletions supports the hypothesis that the respective alteration results in a rearrangement of the proximal IgH region and an unknown oncogene at 14q24 [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF]. Using compiled FISH and qPCR, Pospisilova et al. identified a heterogeneous pattern of the 14q24.1 breakpoints: At the centromeric side, they were either localized proximal or distal of the ZFP36L1 gene or within the respective gene. The 14q32.33 breakpoints were found centromeric or telomeric to sequences flanking Eµ (the 5´enhancer of IgH), or were localized within the region coding Eµ. According to the authors, the involvement of IgH in this particular del(14)(q24.1q32.33) was suggestive for function of this alteration in a translocation-like manner and activation of an unknown oncogene at 14q due to juxtaposition of IgH to the Eµ enhancer. Gene rearrangements due to interstitial deletions are rare but have been described e.g. with respect to the FIPL1-PDGFRA in chronic eosinophilic leukemia (CEL) [START_REF] Cools | A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome[END_REF] or MLL-CBL rearrangements in acute myeloid leukemia (AML) [START_REF] Fu | Identification of CBL, a proto-oncogene at 11q23.3, as a novel MLL fusion partner in a patient with de novo acute myeloid leukemia[END_REF].

Otherwise, the interstitial deletion was discussed to result in loss of a potential tumor suppressor gene. However, qPCR studies from Pospisilova et al. for analysis of the expression patterns of ZFP36L1 and 14 other genes (e.g. RAD51L1) within the proximal 3 Mb from the del(14)(q24.1q32.33) breakpoint revealed no consistent overexpression of any gene analyzed [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF]. Nevertheless, loss of function of one or both alleles of a putative tumor-suppressor gene is still possible. The interstitial deletion might also cause haploinsufficiency of a proto-oncogene. A specific unstable DNA sequence at chromosome bands 14q24.1 and 14q32.3 might explain clustering of the breakpoints in 35% of the cases with 14q deletions. In this study, the minimal deleted region was beginning at band 14q24.1 between BACs RP11-35D12 and RP11-226F19 and ended proximally of the IgH locus at band 14q32.3. No homozygous deletion of 14q was detected by using the mentioned BACs in this analysis as in the study of [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF].

It might be interesting to investigate the loss of both alleles for distinctive genes with the microarray technique. This would allow to clarify whether cryptic deletions exist on the homologous chromosome 14q and would facilitate the identification of the relevant genes.

Very recently, a homozygous deletion of TRAF3 was detected in 2 out of 31 cases with CLL and other mature B-cell neoplasms with 14q deletions [START_REF] Nagel | Biallelic inactivation of TRAF3 in a subset of B-cell lymphomas with interstitial del(14)(q24.1q32.33)[END_REF]. The TRAF3 (TNF receptor-associated factor 3)-gene is located at 14q32.32 within the deleted region. The TRAF3 protein negatively regulates the NF-kB pathway and has been shown to be recurrently affected by biallelic TRAF3 deletion and inactivating mutation in plasma cell myeloma and Waldenström's macroglobulinemia [START_REF] Annunziata | Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma[END_REF][START_REF] Braggio | Identification of copy number abnormalities and inactivating mutations in two negative regulators of nuclear factor-kappaB signaling pathways in Waldenstrom's macroglobulinemia[END_REF][START_REF] Keats | Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma[END_REF]. These results suggest that TRAF3 is a tumor suppressor gene that might be involved in the pathogenesis of lymphoma with 14q deletions.

Similar to the del(14q), different breakpoint patterns have as well been described for 13q deletions [START_REF] Ouillette | Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14[END_REF]. Ouilette et al. performed SNP array analysis in 171 CLL patients with a del(13q) and were able to demonstrate variablility of sizes of 13q-deletions but that centromeric and telomeric breakpoints showed clustering in different regions. The del(13q) cases were separated in two groups: 60% of cases showed no involvement of the retinoblastoma gene (RB), while 40% showed involvement of the RB gene. In the first group, a further subgroup was determined which was characterized by a relatively uniform length of the del(13q) extending from breakpoints close to miR15q/16 locus to a telomeric breakpoint cluster within region 50.2 Mb to 50.5 Mb.

The most frequent additional cytogenetic alteration was trisomy 12 in 45% of all patients with a del(14q) in this study. Similar, Pospisilova et al. reported an additional trisomy 12 in 47% of cases [START_REF] Pospisilova | Interstitial del(14)(q) involving IGH: a novel recurrent aberration in B-NHL[END_REF]. Numerical gains of chromosome 12 are frequent in mature B-cell neoplasms, with a specific association to cases with IgH rearrangements such as the t(14;18) or the t(14;19). A query in the Mitelman Database [START_REF] Mitelman | Mitelman Database of Chromosome Aberrations in Cancer[END_REF] revealed that 15% of lymphoma cases with a t(14;18)(q32;q21), 35% of cases with t(14;19)(q32;q13), 6% of patients with t(8;14)(q24;q32), and 3% of cases with t(11;14)(q13;q32) showed an additional trisomy 12. Martin-Subero et al. described trisomy 12 as typical additional alteration in lymphoma cases with a t(14;19) and BCL3 rearrangement [START_REF] Martin-Subero | A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation[END_REF]. Although no significant differences regarding overall survival of patients with a del(14q) in comparison to those without was detected, the time to treatment was significantly shorter in cases with a del(14q). The comparison with other prognostically relevant subgroups in the CLL patients showed that the clinical impact was comparable to other high risk cytogenetic karyotypes such as deletions of 11q or 17p. However, since clinical data were available only in part of the patients from this study, additional cases for confirmation of these data is needed. This would allow final conclusions whether inclusion of the del(14q) in the risk categorization of CLL and CLL/PL is being justified. Again, it should be addressed that the overrepresentation of CLL patients in this cohort requires analysis of additional cases from other lymphoma entities to evaluate the prognostic impact of 14q deletions as well in these subgroups.

In conclusion, 14q deletions are recurrent abnormalities with a low frequency in CLL and other mature B-cell neoplasms. The size of the del(14q) is variable, being similar to previous observations in cases with a del(13q) [START_REF] Ouillette | Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14[END_REF]. However, recurrent breakpoints can be identified in 14q24.1 and 14q32.3. Further, 14q deletions are frequently accompanied by a CLL immunophenotype, an additional trisomy 12, an unmutated IgVH status, and seem to have an adverse prognostic impact which should be confirmed in additional studies. It might be discussed to add FISH probes for 14q24.1 and for the IgH locus to the standard panel in CLL and other mature B-cell neoplasms. This would allow to identify additional cases and to explore further the clinical impact of this rare cytogenetic alteration. cases with a del(14q) (http://www.cydas.org) [START_REF] Hiller | CyDAS: a cytogenetic data analysis system[END_REF]. deletion as the sole abnormality: n=180, and none of the mentioned abnormalities: n=92 (G6). OS at 3 years was 86.4%, 31.4%, 77.5%, 96.9%, 93.4% and 93.9%, respectively (Figure 6a). For time to treatment (TTT), results were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months and 147.0 months (Figure 6b). Table 1: Position, size and labelling of the different BACs being used for FISH analysis in the 47 lymphoma cases with a del(14q) (bp: base pairs).

International ID Chromosome

  cases (36%) showed a deletion as detected by BACs RP11-35D12 and RP11-226F19.

  karyotype. Trisomy 12 was the most frequent additional alteration (5/16; 31%), in 3/5 (60%) being seen as sole secondary alteration.

  and 12 cases with trisomy 12 accompanied by a 14q deletion were

Figure 1 :

 1 Figure 1: Incidence of 14q deletions in the "Mitelman Database of Chromosome Alterations in Cancer" (Mitelman et al, 2009); left: in all hematologic neoplasms; right: in lymphatic neoplasms (CLL: chronic lymphocyte leukemia; mature B-NHL: mature B-cell neoplasms; DLBCL: diffuse large B-cell lymphoma; AL: acute leukemia; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; CML: chronic myeloid leukemia; MPN: myeloproliferative neoplasms; MDS: myelodysplastic syndromes; T-NHL: mature T-cell neoplasms; PCM: plasma cell myeloma; HL: Hodgkin lymphoma; HCL: hairy cell leukemia; FL: follicular lymphoma; MZL: marginal zone lymphoma; MCL: mantle cell lymphoma).

Figure 2 :

 2 Figure 2: Chromosomal localization of the different BACs being used for FISH analysis.

Figure 3 :

 3 Figure 3: Breakpoints of BACs and IgH probe of all 47 cases with a del(14q) analyzed: breakpoint in 14q24.1: no deletion detected with BAC RP11-35D12 and deletion detected with BAC RP11-226F19; breakpoint in 14q32.3: deletion detected with the 3'flanking IgH probe and no deletion detected with the probe in the variable region of the IgH locus.

Figure 4 :

 4 Figure 4: Illustration of all cytogenetic losses and gains with the support of CyDAS in the 47

Figure 5 :

 5 Figure 5: Kaplan Meier plot depicting time to treatment (TTT of CLL and CLL/PL patients with 14q deletions (n=32) compared to CLL and CLL/PL patients without a del(14q) (n=383) (21.0 months vs. 80.1 months).

Figure 6 :

 6 Figure 6: Kaplan Meier plot depicting a) OS of CLL and CLL/PL patients and b) time to treatment (TTT) of the CLL and CLL/PL cohort (n=415) in the different cytogenetic categories: del(14q): n=32 (G1), del(17p): n=26 (G2), del(11q): n=42, +12: n=43, del(13q)

F

  Figure2

  Figure 6a

Table 2 :

 2 Patients´ characteristics.

		without 14q deletion	with 14q deletion
	Disease (number of patients)
	CLL (n=1865)	1830	35 (1.9%)
	CLL/PL (n=111)	101	10 (9.0%)
	other B-cell neoplasms (n=1078)	1076	2 (0.2%)
	Median age (years)
	CLL	66.3	65.0
	CLL/PL	67.9	69.1
	other B-cell neoplasms	67.3	70.5
	Sex ratio (male: female)
	CLL	1181:649 (1.8)	24:11 (2.2)
	CLL/PL	68:33 (2.1)	6:4 (1.5)
	other mature B-cell neoplasms	621:455 (1.4)	1:1 (1.0)

Table 3 :

 3 Genetic characterization of the 47 B-cell lymphoma cases with 14q deletion. FISH

	probes: 14q22.1: BAC RP11-218E20 (green); 14q24.1: BAC RP11-35D12 (red) and BAC
	RP11-226F19(green); 14q32.33: BAC RP11-521B24 (red); G: green signal; R: red signal; F:
	fusion signal (existing of one red and one green signal); IGH probe: IgH3' (red) and IgVH
	(green); UM: unmutated; M: mutated; LPL: lymphoplasmocytic lymphoma; MZL: marginal
	zone lymphoma.

t(3;17)(q27;q23),+12,del(14)(q22q32)[2]/47,XY,der(1)t(1;15)(p13;q24),t(8;8)(q22;q24),+der(12)t(12;17)(q13;q23),del(14)(q22q32),der(14)t(1;14)(q11;p11), der(15)t(1;15)(p13;q24),der(

17

)del(17)(p12)del(17)(q21)[10]/46,XY[,XY,del(3)(p21p25),del(4)(q21q35),der(8)del(8)(p11)ins(8;17)(q22;??)t(4;8)(q?;q24)t(4;17)(q?;?)ins(10;13)(q22;q14q21),der(10)t(10;19)(q22;?),del(14)(q22q32), -17 [

British Journal of Haematology


Meriem Mechri

Ralph Epaud

Sophie Emond

Aurore Coulomb

Francis Jaubert

Delphine Feldmann

Annick ; Clément

Ailbhe Tarrant

Jacques De

F Flamein

A Clement

J De Blic

R Abou Taam

F Brunelle

H Ducou Le Pointe
email: hubert.ducou-le-pointe@trs.aphp.fr

Pr Hubert

Ducou Le Pointe

(HRCT) Findings and its Relation to Histological Analysis

Keywords: High-Resolution Computed Tomography surfactant, radiological, Histological, HRCT surfactant protein C, HRCT, Histological surfactant protein C, HRCT, Histological

come

Introduction:

Idiopathic forms of interstitial lung disease (ILD) in children are associated with a wide spectrum of underlying pathologies. Until recently, a single classification system has been used, regardless of age, and children with ILD were forced into published adult disease classification systems 1 . In 2007, a multi-disciplinary working group of pediatric pathologists, clinicians and radiologist (the Children's Interstitial Lung Disease (chILD) Research Cooperative) designed and conducted a retrospective clinical, radiographic and histopathologic review and proposed a new classification system for children with diffuse lung disease (chILD syndrome) 2 . In this classification, surfactant genetic abnormalities account for more than 10 % of the patients reviewed. Pulmonary surfactant is a mixture of phospholipids and specific proteins secreted by type II epithelial cell to reduce the alveolar surface tension at the air-liquid interface and prevent end-expiratory atelectasis 3 . The hydrophobic surfactant protein SP-C, play an important role in the spreading and stabilization of phospholipids films in the alveolus 3 highlighted by lung disease caused by inherited abnormalities in its genes 4,5 . Since the first description by Nogee et al., more than 35 dominantly expressed mutations in the SFTPC gene have been identified in association with acute or chronic lung disease in patients ranging in age from newborn to adults [6][7][8][9][10] . These mutations, whether inherited or spontaneous are always found in an autosomal dominant form, the most frequently observed (nearly 50 %) being p.[Ile73Thr] or I73T). The phenotype associated with SFTPC mutations is eminently variable. Indeed, neonatal forms leading to death in the first years of life as well as childhood and adult forms with chronic respiratory disease have been observed. The effects of SFTPC mutations on surfactant homeostasis have not been clearly elucidated. In an in vitro study off cells with a SFTPC deletion, accumulation of misfolded proSP-C in the Endoplasmic reticulum (ER) causes disruption of the ubiquitin/proteasome system 11 , activates apoptosis and interferes with non mutated protein Although consensus exists on the need to consider early open lung in children with ILD, genetic testing for SFTPC mutation has become widespread and may be performed relatively quickly. In addition, recent advances in imaging techniques have contributed to more efficiently predict histological findings. Indeed, HRCT has been proposed for either diagnosis or following ILD precluding the need to obtain a surgical biopsy 13,14 . Thus, the aim of the present study was to determine HRCT features of children with SFTPC mutation and to bring new insight in the correlation between HRCT and histological findings. To address these questions HRCT finding of a cohort of 15 children with interstitial chronic lung disease related to SFTPC gene mutations were retrospectively reviewed and subsequently correlated with histological analysis when lung biopsies were available.

Patients and Methods

This study is a retrospective study performed in 2 university hospital-based pediatric pulmonology practice (Hôpital Armand Trousseau and Hôpital Necker enfants-malades, Paris, France) for a 15-year period (1993-2008). The protocol was accepted by the Committee for the protection of individuals in Biochemical research as required by French legislation and written informed consent was obtained from the patients or their next of kin included in this study.

Patients

The characteristics of patients are summarized in Table 1. Fifteen children, 7 females and 8 males with a definitive diagnosis of SFTPC gene mutation were included. All the patients were immunocompetent and screening for metabolic, infectious, and immunological etiologies was negative. Genetic and clinical characteristics of all these patients have been described in detail in previous reports 8,15 . Three patients, members, or relatives of a same family, have also been reported previously 6 . Median age of onset was 2 (range 0-18) months.

Three patients had neonatal respiratory distress, and symptom onset was associated with acute bronchiolitis in 7 (47 %) patients. Cough, tachypnea, and gastroesophageal reflux and failure to thrive were initially noticed in all the children. Physical examination at presentation revealed tachypnea and retraction in all patients. Low oxygen saturation (<95%) was observed initially in all patients. Treatment are summarized in table 1 and included methylprednisolone pulse (n=15), hydroxychloroquin (n=8) and azithromycin (n=5). Median follow-up was 3.1 tracheal carina; intermediate was considered between the carina and inferior pulmonary veins, and lower zone predominance was considered present when disease extent was greatest below this level. Specific involvement or sparing of the subpleural region (<1cm from the pleura) was assessed. Ground-glass opacities describe lung that is higher in attenuation than a normal lung on inspiration but lower than the attenuation of bronchial walls and blood vessels so that these lung markings remain visible 16 . The term lung cyst is used to refer to a well-defined, rounded, and circumscribed lesion, with a wall that may be uniform or varied in thickness but which is usually thin (with thickness of less than 3 mm) 17 . Because this study was retrospective, the intervals of follow-up HRCT scanners were highly centre-dependent and based on the decision of the physician in charge of the child. Only HRCT scanners available ware taking into consideration for analysis. Control HRCT scans were obtained from 6 agematched patients with a definitive diagnosis of ABCA3 (ATP-binding cassette subfamily A, member 3) mutations, a gene expressed in lamellar bodies of alveolar type II cells which has a critical role in surfactant storage and homeostasis 18 . Among them, 2 patients carried homozygous missense mutations, 3 patients carried compound heterozygous missense mutations and 1 child had compound heterozygous missense and nonsense mutations.

Median age at HRCT scans was 2 months (range 1 month to 12 years).

Lung biopsies and their analysis

Surgical lung biopsies ranging from 0.

Initial HRCT findings

The median age at initial HRCT scanner was 4 (range 1-40) months (one child had the scanner performed 40 months after the diagnosis for technical reasons). High-resolution CT findings are summarized in Table 2. The most frequent abnormalities identified on initial HRCT were ground-glass opacities (93 %) and lung cysts (40 %). Other abnormalities noted included interlobular septal thickening (7 %), air space consolidation (7 %), paraseptal emphysema (13 %), and pulmonary nodules (7 %). In comparison, ground-glass opacities were constantly observed in all of the 6 patients with ABCA3 mutations. However, opacities were more prominent with presence in some cases of alveolar condensations.

Bilateral ground-glass opacities were shown in 14 children (Table 3), either as an isolated finding (60 %) or as part of a mixed pattern with lung cysts (33 %), air space consolidation (7 %), paraseptal emphysema, and centrolobular nodules (7 %). They were homogeneously diffuse in 9 cases (60 %) (Figure 1A). When a patchy distribution was present, this was noted to have basal subpleural predominance in 1 case (7 %), predominance for the lower lobes in 2 cases (13 %) (Figure 1B), a central predominance in 1 case (7 %), and a predominance for the posterior portions of the lung in 1 case (7 %).

Thin-walled (<1mm) lung cysts of varying sizes were seen in 6 infants (40 %) (Table 4), either as an isolated finding (7 %) (Figure 2A), or as a mixed pattern with areas of groundglass opacities (33 %), paraseptal emphysema (13 %) (Figure 2B), septal thickening (7 %), and non-septal thickening (7 %). A predominantly subpleural distribution was seen in 1 child (7 %); diffuse cysts with random distribution was seen in 4 cases (27 %) and upper lung zone predominance was seen in 1 case (7 %). follow-up HRCT scans (3 and 9 months and 4 and 9 months, respectively), and 1 patient had 3 follow-up HRCT scans (3, 6 and 12 months). Evaluation of the follow-up HRCT scans (Table 5) revealed the development of lung cysts in 5 patients (45 %) who had previously only had ground-glass opacities (Figures 3A and3B). These new cystic changes were associated with a stable pattern of ground-glass opacities in 2 cases, decrease in the degree of ground-glass opacities in 1 case, and an aggravation of this pattern in 2 cases. These cysts were diffuse in all cases but showed a predilection for different regions of the lung: upper lobes (n=1), basal subpleural (n=1), peripheral (n=1), and central (n=1). In 1 case, lung cysts were homogenous and diffuse. Patients who had previously documented cysts on their initial HRCT scans also demonstrated change when follow-up HRCT was performed. In 5 patients (45 %), we noted an increase in the number or size of preexisting lung cysts (Figures 4A and4B), which were associated with stable ground-glass opacities (n=4), aggravated ground-glass opacities (n=1), and progressive pulmonary fibrosis (n=1). The HRCT findings of pulmonary fibrosis consisted of bilateral and diffuse honeycombing, septal and non-septal thickening, and thickening of bronchovascular bundles and distortion of pulmonary fissures. In 1 patient, we observed persistent isolated ground-glass opacities.

Follow-up HRCT scan

Pathology findings

Eight of the 15 patients (53 %) underwent lung biopsy the median age at biopsy being 4 (range 3-7) months. Histological analysis revealed 2 main types of lesions. The first type occurred in the alveolar lumen, were we observed the following changes: accumulation of macrophages intermingled with few inflammatory cells and cholesterol cleft, type II pneumocyte hyperplasia, and alveolar septal thickening by primitive mesenchymal cells and lymphocytes either isolated or clustered (Figure 5A). These lesions were diffuse in 7 cases (90 F o r P e e r R e v i e w 10 %) and patchy in 1 case (13 %). The second type of lesion corresponds to dilatation of the respiratory bronchiole and alveolar duct in 7 cases (88 %) associated with muscular hyperplasia in 2 patients (25%) (Figure 6A). There was no interstitial pulmonary fibrosis or alveolar proteinosis observed.

Relation between HRCT and pathology findings

Radiological-pathological confrontation is summarized in Table 6. Ground-glass opacities on HRCT were observed in all the patients except 1 in which the CT scan was performed late (40 months). All these children also have diffuse alveolar septal thickening by primitive mesenchymal cells and lymphocytes, intra-alveolar accumulation of macrophages, and type II pneumocyte hyperplasia. Small lung cysts were observed initially in half of the children but appeared during follow-up in all the cases. They were related with dilatation of the terminal bronchioles and alveolar duct which were noticed in all but one biopsy.

Discussion:

In the present study, we have shown for the first time in a large cohort of children with SFTPC mutation associated lung diseases that HRCT pattern were characterized with 2 main abnormalities: ground-glass opacities that were predominately diffuse but can be patchy in some cases and lung cysts with thin wall and different sizes.

Ground-glass opacities have been commonly described in previous reports 6,19,20 . They are predominately diffuse but can be patchy in some cases. In our series, they were present in all the patients except one in whom the HRCT scan was done late. We also note in our study the high frequency of cystic lesions on HRCT scan. They appeared in 45% of the children during follow-up and increased in number in 45%. Similar change during follow-up is typical of surfactant abnormality and was noted by Brody 16 . In our series, ground-glass attenuation This HRCT presentation, namely the ground-glass opacities and cystic patterns is, nonetheless, not specific of SFTPC associated disorders. We have shown that this presentation may also be observed in children with ABCA3 mutations as previously described 21 .However, the high frequency of SFTPC associated disorders in children ILD 2,7 , the feasibility of SFTPC genetic analysis as well as the difficulty to perform lung biopsy in patients with fragile respiratory condition strongly encourage to perform SFTPC genetic screening before lung biopsy in children with ground-glass opacities associated with cystic lung changes on HRCT scanner.

In the literature, there are histological studies of patients with SFTPC gene mutations showing 4 histological patterns: desquamative interstitial pneumonitis (DIP), nonspecific interstitial pneumonitis(NSIP), (UIP) and pulmonary alveolar proteinosis 7,16,22 in children and usual interstitial pneumonitis (UIP) in adults. In children, DIP and NSIP have also been described in patients with mutations in the ATP-binding cassette transporter A3 (ABCA3) gene 21 . Chibbar et al. showed that both children and adults with NSIP and UIP patterns, respectively, carried a common mutation within the SFTPC gene 23 . Thomas et al. described a mutation in SFTPC gene associated with large kindred with familial form of pulmonary fibrosis, including adults with the most common pathological subset of pulmonary fibrosis, UIP, and children with cellular NSIP 24 . Brasch reported histological patterns of NSIP and pulmonary alveolar proteinosis in a 13-month-old infant with a de novo heterozygous missense mutation of the SFTPC gene 20 . The histological findings were classified as DIP because of an increased number of macrophages in the alveolar lumen 25 and as NSIP and UIP because of the numerous inflammatory cells in the alveolar septum 26,27 . In this study, we have related ground-glass opacities on HRCT to diffuse alveolar septal thickening by primitive 12 mesenchymal cells and lymphocytes, intra-alveolar accumulation of macrophages, and type II pneumocytes hyperplasia. Small lung cysts were related to dilatation of the respiratory bronchioli and alveolar duct. On initial HRCT scan analysis, large cystic lung changes in the periseptal and subpleural regions were that of subpleural emphysema. On lung biopsies, millimetric small cysts corresponding to a dilatation of respiratory bronchioli and alveolar ducts, sometimes in subpleural area and periseptal regions, were observed, whereas histological emphysema was not present. Large cyst (>1mm) probably corresponds to emphysema, whereas small cyst may be in part due to cystic dilatation of the proximal part of the acini.

After natural exposure, CT scan is the greatest contributor to total radiation exposure and Brenner et al. notes that the evidence is "convincing" that the radiation dose from CT scans can lead to cancer induction in adults and "very convincing" in the case of children 28 . Hence, it is of importance to adjust protocols in order to reduce unnecessary radiation exposure from CT. Exposure parameters have considerably decreased during the study, but these efforts must be sustained. In this context, we also recommend to use a sequential technique during followup, including for multi detector CT.

With this report as our basis, the presence of bilateral ground-glass opacity or a combination of ground-glass opacities and cysts suggests the diagnosis of SFTPC mutation associated disorders and strongly encourage to perform genetic screening. We believe that this study is of value in highlighting this rare condition, and future prospective studies in children and adults are needed to further characterize CT findings in surfactant dysfunction disorders.

Figures:

Fig. 1: Four-year-old boy with SFTPC mutation. HRCT section through the carina shows diffuse and uniform ground-glass opacities (A).Two-year-old girl with SFTPC mutation. Initial HRCT (at 3 months) shows homogenous ground-glass opacity in the lower lobes (B). One month old girl with compound heterozygous missense ABCA3 mutations. Initial HRCT shows ground-glass opacity in the lower lobes (C). Fig. 2: Two-year-old girl with SFTPC mutation. Initial HRCT (at 16 months) shows paraseptal emphysema (curved arrow) associated with widespread cystic airspaces and diffuse ground-glass opacities (A). Seventeen-year-old boy with SFTPC mutation. The HRCT done at 14 -year -old showed isolated lung cysts with thin wall and variable sizes (B). One month old girl with compound heterozygous missense ABCA3 mutations. Initial HRCT shows association of ground-glass opacity, alveolar condensations and lung cysts (C). Fig. 3: A one-year-old boy with SFTPC mutation. The initial computed tomography shows diffuse ground-glass opacities (A). The follow-up HRCT scan done 14 months later revealed the appearance of new lung cysts in lower lobes surimposed on diffuse ground-glass opacities (B). Fig. 4: Two-year-old girl with SFTPC mutation. Initial HRCT scan diffuse shows lung cysts with subpleural predominance associated with diffuse ground-glass opacities (A). The followup HRCT scan done 6 months later, revealed an increase in the number and size of lung cysts associated with stable ground-glass opacities (B). OW ( 9) OW ( 9) OW ( 7)

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High-Resolution Computed Tomography (HRCT) Findings and its Relation to Histological Analysis

M. Mechri *1, 2, 3 , R. Epaud *2, 3, 4, 5 , S. Emond 1, 6, 7 , A. Coulomb 2, 3, 8 , F. Jaubert 1, 6, 8 , A. Tarrant 1, 2, 3 , D.

Feldmann 2, 3, 9 , F. Flamein 2, 4 , A. Clement 2, 3, 4, 5 J. de Blic 5, 6, 7 , R. Abou Taam 5, 6, 7 , F. Brunelle

Abstract:

Aim of the study

Determine high-resolution tomography (HRCT) scan characteristics in children with SFTPC mutation and correlate them to histological findings.

Patients and Methods

This retrospective multicenter study included 15 children (7 females and 8 males) with SFTPC mutations. HRCT scans have been performed in all the children and lung biopsies in 8 children.

Results

From all signs assessed on initial HRCT scans, ground-glass opacities (n=14, 93 %) and lung cysts (n=6, 40 %) were predominant. Interlobular septal thickening (n=1, 7 %), air space consolidation (n=1, 7 %), paraseptal emphysema (n=2, 13 %), and pulmonary nodules (n=1, 7 %) were also found. Histological analysis revealed accumulation of macrophages in the alveolar lumen, type II pneumocyte hyperplasia, and alveolar septal thickening. Dilatation of the respiratory bronchiole and alveolar duct associated with muscular hyperplasia were also described. Interestingly, lung cysts on HRCT scans were associated with dilatation of terminal bronchioli and alveolar duct in lung biopsies.

Conclusion

In children with SFTPC mutations, HRCT scan finding were highly correlated to the histological findings and, as such, represent a useful tool to identify patients that may require SFTPC gene sequencing. Although consensus exists on the need to consider early open lung in children with ILD, genetic testing for SFTPC mutation has become widespread and may be performed relatively quickly. In addition, recent advances in imaging techniques have contributed to more efficiently predict histological findings. Indeed, HRCT has been proposed for either diagnosis or following ILD precluding the need to obtain a surgical biopsy 13,14 . Thus, the aim of the present study was to determine HRCT features of children with SFTPC mutation and to bring new insight in the correlation between HRCT and histological findings. To address these questions HRCT finding of a cohort of 15 children with interstitial chronic lung disease related to SFTPC gene mutations were retrospectively reviewed and subsequently correlated with histological analysis when lung biopsies were available.

Patients

The characteristics of patients are summarized in Table 1. Fifteen children, 7 females and 8 males with a definitive diagnosis of SFTPC gene mutation were included. All the patients were immunocompetent and screening for metabolic, infectious, and immunological etiologies was negative. Genetic and clinical characteristics of all these patients have been described in detail in previous reports 8,15 . Three patients, members, or relatives of a same family, have also been reported previously 6 . Median age of onset was 2 (range 0-18) months.

Three patients had neonatal respiratory distress, and symptom onset was associated with acute bronchiolitis in 7 (47 %) patients. Cough, tachypnea, and gastroesophageal reflux and failure to thrive were initially noticed in all the children. Physical examination at presentation revealed tachypnea and retraction in all patients. Low oxygen saturation (<95%) was observed initially in all patients. Treatment are summarized in table 1 and included methylprednisolone pulse (n=15), hydroxychloroquin (n=8) and azithromycin (n=5). Median follow-up was 3.1

(1-17.3) years. At the end of the study, all the children were alive but 11 patients (73 %) were still symptomatic with cough, dyspnea or exercise intolerance. and lower zone predominance was considered present when disease extent was greatest below this level. Specific involvement or sparing of the subpleural region (<1cm from the pleura) was assessed. Ground-glass opacities describe lung that is higher in attenuation than a normal lung on inspiration but lower than the attenuation of bronchial walls and blood vessels so that these lung markings remain visible 16 . The term lung cyst is used to refer to a well-defined, rounded, and circumscribed lesion, with a wall that may be uniform or varied in thickness but which is usually thin (with thickness of less than 3 mm) 17 . Because this study was retrospective, the intervals of follow-up HRCT scanners were highly centre-dependent and based on the decision of the physician in charge of the child. Only HRCT scanners available ware taking into consideration for analysis. Control HRCT scans were obtained from 6 agematched patients with a definitive diagnosis of ABCA3 (ATP-binding cassette subfamily A, member 3) mutations, a gene expressed in lamellar bodies of alveolar type II cells which has a critical role in surfactant storage and homeostasis 18 . Among them, 2 patients carried homozygous missense mutations, 3 patients carried compound heterozygous missense mutations and 1 child had compound heterozygous missense and nonsense mutations.

Median age at HRCT scans was 2 months (range 1 month to 12 years).

Lung biopsies and their analysis

Surgical lung biopsies ranging from 0. Results:

Initial HRCT findings

The median age at initial HRCT scanner was 4 (range 1-40) months (one child had the scanner performed 40 months after the diagnosis for technical reasons). High-resolution CT findings are summarized in Table 2. The most frequent abnormalities identified on initial HRCT were ground-glass opacities (93 %) and lung cysts (40 %). Other abnormalities noted included interlobular septal thickening (7 %), air space consolidation (7 %), paraseptal emphysema (13 %), and pulmonary nodules (7 %). In comparison, ground-glass opacities were constantly observed in all of the 6 patients with ABCA3 mutations. However, opacities were more prominent with presence in some cases of alveolar condensations.

Bilateral ground-glass opacities were shown in 14 children (Table 3), either as an isolated finding (60 %) or as part of a mixed pattern with lung cysts (33 %), air space consolidation (7 %), paraseptal emphysema, and centrolobular nodules (7 %). They were homogeneously diffuse in 9 cases (60 %) (Figure 1A). When a patchy distribution was present, this was noted to have basal subpleural predominance in 1 case (7 %), predominance for the lower lobes in 2 cases (13 %) (Figure 1B), a central predominance in 1 case (7 %), and a predominance for the posterior portions of the lung in 1 case (7 %).

Thin-walled (<1mm) lung cysts of varying sizes were seen in 6 infants (40 %) (Table 4), either as an isolated finding (7 %) (Figure 2A), or as a mixed pattern with areas of groundglass opacities (33 %), paraseptal emphysema (13 %) (Figure 2B), septal thickening (7 %), and non-septal thickening (7 %). A predominantly subpleural distribution was seen in 1 child (7 %); diffuse cysts with random distribution was seen in 4 cases (27 %) and upper lung zone predominance was seen in 1 case (7 %). 5) revealed the development of lung cysts in 5 patients (45 %) who had previously only had ground-glass opacities (Figures 3A and3B). These new cystic changes were associated with a stable pattern of ground-glass opacities in 2 cases, decrease in the degree of ground-glass opacities in 1 case, and an aggravation of this pattern in 2 cases. These cysts were diffuse in all cases but showed a predilection for different regions of the lung: upper lobes (n=1), basal subpleural (n=1), peripheral (n=1), and central (n=1). In 1 case, lung cysts were homogenous and diffuse. Patients who had previously documented cysts on their initial HRCT scans also demonstrated change when follow-up HRCT was performed. In 5 patients (45 %), we noted an increase in the number or size of preexisting lung cysts (Figures 4A and4B), which were associated with stable ground-glass opacities (n=4), aggravated ground-glass opacities (n=1), and progressive pulmonary fibrosis (n=1). The HRCT findings of pulmonary fibrosis consisted of bilateral and diffuse honeycombing, septal and non-septal thickening, and thickening of bronchovascular bundles and distortion of pulmonary fissures. In 1 patient, we observed persistent isolated ground-glass opacities.

Follow-up HRCT scan

Pathology findings

Eight of the 15 patients (53 %) underwent lung biopsy the median age at biopsy being 4

(range 3-7) months. Histological analysis revealed 2 main types of lesions. The first type occurred in the alveolar lumen, were we observed the following changes: accumulation of macrophages intermingled with few inflammatory cells and cholesterol cleft, type II pneumocyte hyperplasia, and alveolar septal thickening by primitive mesenchymal cells and lymphocytes either isolated or clustered (Figure 5A). These lesions were diffuse in 7 cases (90 6A). There was no interstitial pulmonary fibrosis or alveolar proteinosis observed.

F

Relation between HRCT and pathology findings

Radiological-pathological confrontation is summarized in Table 6. Ground-glass opacities on HRCT were observed in all the patients except 1 in which the CT scan was performed late (40 months). All these children also have diffuse alveolar septal thickening by primitive mesenchymal cells and lymphocytes, intra-alveolar accumulation of macrophages, and type II pneumocyte hyperplasia. Small lung cysts were observed initially in half of the children but appeared during follow-up in all the cases. They were related with dilatation of the terminal bronchioles and alveolar duct which were noticed in all but one biopsy.

Discussion:

In the present study, we have shown for the first time in a large cohort of children with SFTPC mutation associated lung diseases that HRCT pattern were characterized with 2 main abnormalities: ground-glass opacities that were predominately diffuse but can be patchy in some cases and lung cysts with thin wall and different sizes.

Ground-glass opacities have been commonly described in previous reports 6,19,20 . They are predominately diffuse but can be patchy in some cases. In our series, they were present in all the patients except one in whom the HRCT scan was done late. We also note in our study the high frequency of cystic lesions on HRCT scan. They appeared in 45% of the children during follow-up and increased in number in 45%. Similar change during follow-up is typical of surfactant abnormality and was noted by Brody 16 . In our series, ground-glass attenuation This HRCT presentation, namely the ground-glass opacities and cystic patterns is, nonetheless, not specific of SFTPC associated disorders. We have shown that this presentation may also be observed in children with ABCA3 mutations as previously described 21 .However, the high frequency of SFTPC associated disorders in children ILD 2,7 , the feasibility of SFTPC genetic analysis as well as the difficulty to perform lung biopsy in patients with fragile respiratory condition strongly encourage to perform SFTPC genetic screening before lung biopsy in children with ground-glass opacities associated with cystic lung changes on HRCT scanner.

In the literature, there are histological studies of patients with SFTPC gene mutations showing 4 histological patterns: desquamative interstitial pneumonitis (DIP), nonspecific interstitial pneumonitis(NSIP), (UIP) and pulmonary alveolar proteinosis 7,16,22 in children and usual interstitial pneumonitis (UIP) in adults. In children, DIP and NSIP have also been described in patients with mutations in the ATP-binding cassette transporter A3 (ABCA3) gene 21 20 . The histological findings were classified as DIP because of an increased number of macrophages in the alveolar lumen 25 and as NSIP and UIP because of the numerous inflammatory cells in the alveolar septum 26,27 . In this study, we have related ground-glass opacities on HRCT to diffuse alveolar septal thickening by primitive 12 mesenchymal cells and lymphocytes, intra-alveolar accumulation of macrophages, and type II pneumocytes hyperplasia. Small lung cysts were related to dilatation of the respiratory bronchioli and alveolar duct. On initial HRCT scan analysis, large cystic lung changes in the periseptal and subpleural regions were that of subpleural emphysema. On lung biopsies, millimetric small cysts corresponding to a dilatation of respiratory bronchioli and alveolar ducts, sometimes in subpleural area and periseptal regions, were observed, whereas histological emphysema was not present. Large cyst (>1mm) probably corresponds to emphysema, whereas small cyst may be in part due to cystic dilatation of the proximal part of the acini.

After natural exposure, CT scan is the greatest contributor to total radiation exposure and Brenner et al. notes that the evidence is "convincing" that the radiation dose from CT scans can lead to cancer induction in adults and "very convincing" in the case of children 28 . Hence, it is of importance to adjust protocols in order to reduce unnecessary radiation exposure from CT. Exposure parameters have considerably decreased during the study, but these efforts must be sustained. In this context, we also recommend to use a sequential technique during followup, including for multi detector CT.

With this report as our basis, the presence of bilateral ground-glass opacity or a combination of ground-glass opacities and cysts suggests the diagnosis of SFTPC mutation associated disorders and strongly encourage to perform genetic screening. We believe that this study is of value in highlighting this rare condition, and future prospective studies in children and adults are needed to further characterize CT findings in surfactant dysfunction disorders. Fig. 1: Four-year-old boy with SFTPC mutation. HRCT section through the carina shows diffuse and uniform ground-glass opacities (A).Two-year-old girl with SFTPC mutation. Initial HRCT (at 3 months) shows homogenous ground-glass opacity in the lower lobes (B). One month old girl with compound heterozygous missense ABCA3 mutations. Initial HRCT shows ground-glass opacity in the lower lobes (C).

Fig. 2: Two-year-old girl with SFTPC mutation. Initial HRCT (at 16 months) shows paraseptal emphysema (curved arrow) associated with widespread cystic airspaces and diffuse ground-glass opacities (A). Seventeen-year-old boy with SFTPC mutation. The HRCT done at 14 -year -old showed isolated lung cysts with thin wall and variable sizes (B). One month old girl with compound heterozygous missense ABCA3 mutations. Initial HRCT shows association of ground-glass opacity, alveolar condensations and lung cysts (C).

Fig. 3: A one-year-old boy with SFTPC mutation. The initial computed tomography shows diffuse ground-glass opacities (A). The follow-up HRCT scan done 14 months later revealed the appearance of new lung cysts in lower lobes surimposed on diffuse ground-glass opacities (B).

Fig. 4: Two-year-old girl with SFTPC mutation. Initial HRCT scan diffuse shows lung cysts with subpleural predominance associated with diffuse ground-glass opacities (A). The followup HRCT scan done 6 months later, revealed an increase in the number and size of lung cysts associated with stable ground-glass opacities (B).

  results in the release of pro-inflammatory cytokines and enhances T-cell and fibroblast recruitment12 .

( 1 -

 1 17.3) years. At the end of the study, all the children were alive but 11 patients (73 %) were still symptomatic with cough, dyspnea or exercise intolerance.

6 HRCT

 6 scan and image analysis CT examinations were performed with two helical scanners and two multidetector CT scanners. In Armand Trousseau hospital, Elscint (CT Twin) with scan acquisition at 120 kV, 120 mAs and after 2003 a Philips (Mx 8000 IDT32) 16 slice multidetector CT was used with scan acquisition at the 90kV, 90mAs. In Necker Hospital, General Electric helical CT (Prospeed) with scan acquisition at 140kV, 130mAs, and after 2005 a General Electric (LightSpeed) 64 slice multidetector CT was used with scan acquisition at the 100kV, 100mA.All the patients underwent non-contrast-enhanced incremental (at intervals of 3 to 10 mm) or helical HRCT scan with a collimation of 16 X 1mm. Scans were obtained from the level of apices to the diaphragm. Image reconstruction was performed with a high special frequency algorithm. All CT scans were performed before the diagnosis, and none of the patients were receiving treatment at the time of first HRCT. The HRCT scans (initial and follow-up) were reviewed and interpreted by 2 experienced pediatric radiologists who arrived at their interpretations by consensus without knowledge of histological parameters. HRCT scan analysis was performed with reference to a conventional model in line with Austin's definition The scans were assessed for the presence, extent, and distribution of elementary lesions, such us areas of ground-glass opacity, lung cysts, lung consolidation, interlobular and intralobular septal thickening, thickening of bronchovascular bundles, and nodules (centrolobular or other).The anatomical distribution was classified as central (a predominance of abnormalities in the inner two-thirds of the lung), peripheral (a predominance of abnormalities in the outer one-third of the lung or along the interlobar fissures), or random (no central or peripheral predominance). The zonal predominance of abnormalities was assessed as being upper, intermediate lower, or random. Upper lung zone predominance was considered present when the extent of abnormalities was greatest above the level of the

9 Follow

 9 -up CT scans were performed on 11 of the 15 children. The median follow-up interval was 8 (range 3-12) months. Eight patients had 1 follow-up HRCT scan, 2 patients had 2

  with age, whereas small lung cysts usually became more prominent over time.

Fig. 5 :

 5 Fig. 5: A one-year old boy with SFTPC mutation who underwent lung biopsy in the middle lobe. High-power photomicrograph (original magnification × 200, hematoxylin eosine [H-E] stain) (A) shows numerous macrophages in alveoli (M) and type II pneumocytes hyperplasia (straight arrows) with septal thickening. HRCT section through the middle lobe (B) shows diffuse ground glass opacity.

Fig. 6 :

 6 Fig.6: A 3-year old girl with SFTPC mutation who underwent lung biopsy in the right upper lobe. High-power photomicrograph (original magnification × 200, hematoxylin eosine [H-E] stain) (A) shows cystic distension of respiratory ducts (curved arrow), numerous macrophages in alveoli (M) and type II pneumocytes hyperplasia (straight arrows). HRCT section through the upper lobes (B) shows diffuse lung cysts associated with diffuse ground glass opacities.

Fig. 1 :Fig. 2 :Fig. 4 :A 3 -

 1243 Fig. 1: Four-year-old boy with SFTPC mutation. HRCT section through the carina shows diffuse and uniform ground-glass opacities (A).Two-year-old girl with SFTPC mutation. Initial HRCT (at 3 months) shows homogenous ground-glass opacity in the lower lobes (B). One month old girl with compound heterozygous missense ABCA3 mutations. Initial HRCT shows ground-glass opacity in the lower lobes (C). 190x254mm (96 x 96 DPI)

  Idiopathic forms of interstitial lung disease (ILD) in children are associated with a wide spectrum of underlying pathologies. Until recently, a single classification system has been used, regardless of age, and children with ILD were forced into published adult disease classification systems1 . In 2007, a multi-disciplinary working group of pediatric pathologists, clinicians and radiologist (the Children's Interstitial Lung Disease (chILD) Research Cooperative) designed and conducted a retrospective clinical, radiographic and histopathologic review and proposed a new classification system for children with diffuse lung disease (chILD syndrome)2 . In this classification, surfactant genetic abnormalities account for more than 10 % of the patients reviewed. Pulmonary surfactant is a mixture of phospholipids and specific proteins secreted by type II epithelial cell to reduce the alveolar surface tension at the air-liquid interface and prevent end-expiratory atelectasis3 . The hydrophobic surfactant protein SP-C, play an important role in the spreading and stabilization of phospholipids films in the alveolus3 highlighted by lung disease caused by inherited abnormalities in its genes4,5 . Since the first description by Nogee et al., more than 35 dominantly expressed mutations in the SFTPC gene have been identified in association with acute or chronic lung disease in patients ranging in age from newborn to adults[6][7][8][9][10] . These mutations, whether inherited or spontaneous are always found in an autosomal dominant form, the most frequently observed (nearly 50 %) being p.[Ile73Thr] or I73T). The phenotype associated with SFTPC mutations is eminently variable. Indeed, neonatal forms leading to death in the first years of life as well as childhood and adult forms with chronic respiratory disease have been observed. The effects of SFTPC mutations on surfactant homeostasis have not been clearly elucidated. In an in vitro study off cells with a SFTPC deletion, accumulation of misfolded proSP-C in the Endoplasmic reticulum (ER) causes disruption of the ubiquitin/proteasome system11 , activates apoptosis and interferes with non mutated protein results in the release of pro-inflammatory cytokines and enhances T-cell and fibroblast recruitment12 .

  This study is a retrospective study performed in 2 university hospital-based pediatric pulmonology practice (Hôpital Armand Trousseau and Hôpital Necker enfants-malades, Paris, France) for a 15-year period(1993-2008). The protocol was accepted by the Committee for the protection of individuals in Biochemical research as required by French legislation and written informed consent was obtained from the patients or their next of kin included in this study.

6 HRCT

 6 scan and image analysis CT examinations were performed with two helical scanners and two multidetector CT scanners. In Armand Trousseau hospital, Elscint (CT Twin) with scan acquisition at 120 kV, 120 mAs and after 2003 a Philips (Mx 8000 IDT32) 16 slice multidetector CT was used with scan acquisition at the 90kV, 90mAs. In Necker Hospital, General Electric helical CT (Prospeed) with scan acquisition at 140kV, 130mAs, and after 2005 a General Electric (LightSpeed) 64 slice multidetector CT was used with scan acquisition at the 100kV, 100mA.All the patients underwent non-contrast-enhanced incremental (at intervals of 3 to 10 mm) or helical HRCT scan with a collimation of 16 X 1mm. Scans were obtained from the level of apices to the diaphragm. Image reconstruction was performed with a high special frequency algorithm. All CT scans were performed before the diagnosis, and none of the patients were receiving treatment at the time of first HRCT. The HRCT scans (initial and follow-up) were reviewed and interpreted by 2 experienced pediatric radiologists who arrived at their interpretations by consensus without knowledge of histological parameters. HRCT scan analysis was performed with reference to a conventional model in line with Austin's definition The scans were assessed for the presence, extent, and distribution of elementary lesions, such us areas of ground-glass opacity, lung cysts, lung consolidation, interlobular and intralobular septal thickening, thickening of bronchovascular bundles, and nodules (centrolobular or other).The anatomical distribution was classified as central (a predominance of abnormalities in the inner two-thirds of the lung), peripheral (a predominance of abnormalities in the outer one-third of the lung or along the interlobar fissures), or random (no central or peripheral predominance). The zonal predominance of abnormalities was assessed as being upper, intermediate lower, or random. Upper lung zone predominance was considered present when the extent of abnormalities was greatest above the level of the intermediate was considered between the carina and inferior pulmonary veins,

9 Follow

 9 -up CT scans were performed on 11 of the 15 children. The median follow-up interval was 8 (range 3-12) months. Eight patients had 1 follow-up HRCT scan, 2 patients had 2 follow-up HRCT scans (3 and 9 months and 4 and 9 months, respectively), and 1 patient had 3 follow-up HRCT scans(3, 6 and 12 months). Evaluation of the follow-up HRCT scans (Table

  with age, whereas small lung cysts usually became more prominent over time.

  Figures:

Fig. 5 :

 5 Fig. 5: A one-year old boy with SFTPC mutation who underwent lung biopsy in the middle lobe. High-power photomicrograph (original magnification × 200, hematoxylin eosine [H-E] stain) (A) shows numerous macrophages in alveoli (M) and type II pneumocytes hyperplasia (straight arrows) with septal thickening. HRCT section through the middle lobe (B) shows diffuse ground glass opacity.

Fig. 6 :

 6 Fig.6: A 3-year old girl with SFTPC mutation who underwent lung biopsy in the right upper lobe. High-power photomicrograph (original magnification × 200, hematoxylin eosine [H-E] stain) (A) shows cystic distension of respiratory ducts (curved arrow), numerous macrophages in alveoli (M) and type II pneumocytes hyperplasia (straight arrows). HRCT section through the upper lobes (B) shows diffuse lung cysts associated with diffuse ground glass opacities.

Table 1 : Characteristics of patients

 1

	Biopsy: LIL, left inferior lobe; LSL, left superior lobe; RIL, Right inferior lobe; RML, right medium lobe; RSL, right superior lobe.
	Treatment: MPP, methylprednisolone pulse; H, hydroxychloroquin; A, azithromycin. Oxygen status: OW, oxygen weaning; SOO, still on
	oxygen; duration of oxygen (months) in paracenteses

Table 2 : Initial HRCT signs of patients with SFTPC gene mutation

 2

			Page 18 of 42
	Initial HRCT findings	Patients	Controls (ABCA3 mutations)
		(n=15)	(n=6)
	Ground-glass opacities	14 (93)	6 (100)
	Lung cysts	6 (40)	3 (50)
	Interlobular septal thickening	1 (7)	2 (30)
	Consolidation	1 (7)	2 (30)
	Paraseptal emphysema	2 (13)	0 (0)
	Nodules	1 (7)	0 (0)
	Lobe retractions	0 (0)	1 (17)
	Percentage of total is indicated in parentheses.

Table 3 : Distribution of ground-glass opacities on initial HRCT in patients with SFTPC gene mutation

 3

		Pediatric Pulmonology
		All patients	Patients with lung biopsy
		(n=15)	(n=8)
	Homogenous diffuse	9 (60)	5 (63)
	Peripheral	1 (7)	1 (13)
	Central	1 (7)	0 (0)
	Posterior	1 (7)	0 (0)
	Lower	2 (13)	1 (13)
	Percentage of total is indicated in parentheses.

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Table 4 : Distribution of lung cysts on initial HRCT in patients with SFTPC gene mutation

 4

		Pediatric Pulmonology
		All patients	Patients with lung biopsy
		(n=15)	(n=8)
	Diffuse	4 (27)	3 (38)
	Upper lobe predominance	1 (7)	1 (13)
	Subpleural predominance	1 (7)	0 (0)
	Percentage of total is indicated in parentheses.

Page 20 of 42 John Wiley & Sons, Inc.

Table 5 : Findings on follow-up HRCT scans in patients with SFTPC gene mutation.

 5

			All patients (n=11)
		Stable	6 (55)
	Ground-glass opacities
	n = 10
		Increased	3 (27)
		Decreased	1 (9)
	Lung cyst	Appeared	5 (45)
	n = 10
		Increased	5 (45)
	Pulmonary fibrosis		1 (9)
	Percentage of total is indicated in parentheses.

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Table 6 : Radiological-pathological correlation.

 6

	Histology

  . Chibbar et al. showed that both children and adults with NSIP and UIP patterns, respectively, carried a common mutation within the SFTPC gene 23 . Thomas et al. described a mutation in SFTPC gene associated with large kindred with familial form of pulmonary fibrosis, including adults with the most common pathological subset of pulmonary fibrosis, UIP, and children with cellular NSIP 24 . Brasch reported histological patterns of NSIP and pulmonary alveolar proteinosis in a 13-month-old infant with a de novo heterozygous missense mutation of the SFTPC gene

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Acknowledgement

We thank all the members of the affected families and their referring clinicians for their collaborative participation in this study. This work was supported by Assistance Publique-Hôpitaux de Paris. "Surfactant Disorders and Chronic Lung Disease (APSE)," Clinical Trials gov. number, NCT00493363.

These authors contributed equally to this work and should be considered as first author.

The authors have no conflicts of interest to disclose.

Running title: HRCT and histological finding of SFTPC mutations

Acknowledgement

We thank all the members of the affected families and their referring clinicians for their collaborative participation in this study. This work was supported by Assistance Publique-Hôpitaux de Paris. "Surfactant Disorders and Chronic Lung Disease (APSE)," Clinical Trials gov. number, NCT00493363.