Isochromosome 17q in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), presence of acquired cytogenetic abnormalities may help to estimate prognosis. However, deletion of TP53 gene, which is associated with an aggressive course of the disease and poor prognosis along with a lack of response to treatment, is one of the alterations which may escape cytogenetic diagnoses in CLL. Thus, other techniques have emerged such as interphase fluorescence in situ hybridization (iFISH). Deletion of TP53 may but must not go together with the formation of an isochromosome i(17q); surprisingly this subgroup of patients was not in the focus of CLL studies yet. This study was about if presence of i(17q) could be indicative for a new subgroup in CLL with more adverse prognosis. As a result, TP53 deletion was detected in 18 out of 150 (12%) here studied CLL cases. Six of those cases (~33%) had the TP53 deletion accompanied by an i(17q). Interestingly, the cases with i(17q) showed a tendency towards more associated chromosomal aberrations. These findings may be the bases for follow-up studies in CLL patients with TP53 deletion with and without i(17q); it may be suggested that the i(17q) presents an even more adverse prognostic marker than TP53 deletion alone.


Introduction
Chronic lymphocytic leukemia (CLL) is a relatively frequently observable acquired disease in men and women of >50 years of age [1]. Also CLL is a heterogeneous malignancy, as the survival of CLL patients can be in the range of months to decades according to the underlying genetic abnormalities [2]. The most frequent cytogenetic aberrations in CLL are involving chromosomal subbands 13q14 (50-60%), 14q32 (12-15%), 11q22 (10-20%), and 17p13 (5-10%) as well as trisomy 12 (15-25%); each group has different prognoses and survival rates [1,3]. Deletion of TP53 gene, which is located in the short arm of chromosome 17 towards the telomeric region in 17p13.1, is associated with poor prognosis and lack of response to fludarabine-based regimens.
TP53 deletion in CLL can be associated with isochromosome formation of the long arm of one chromosome 17 leading at the same time to partial monosomy 17p and partial trisomy 17q. In general, isochromosome i(17q) is the most frequently observed isochromosome in hematological malignancies and it can be present as primary or secondary aberration; that is, it may play roles during development as well as progression of the malignancy. Presence of i(17q) as a sole abnormality is associated with a high risk of progression and an aggressive clinical course, but i(17q) can also be found as part of a complex karyotype [4][5][6]. In solid tumors, i(17q) is reported predominantly in medulloblastoma [7], there often associated with c-myc amplification [8].
Overall, detection of acquired chromosomal abnormalities such as i(17q) just based on GTG-banding may be limited due to low mitotic potential of CLL bone marrow cells. Thus, nowadays other techniques are applied to overcome this problem, by name interphase fluorescence in situ hybridization (iFISH), multiplex ligation dependent probe amplification (MLPA), and array-comparative genomic hybridization (aCGH) [2,9,10]. Here we studied 150 CLL samples and concentrated on the questions (i) if i(17q) can be detected reliably by MLPA and (ii) if i(17q) presence in patients with TP53 deletion is associated with more complex cytogenetic aberrations. An association with the clinical outcome would have been favorable as well; unfortunately this was not possible due to lack of necessary clinical data.

Patients and Sample
Preparation. The present study included 150 CLL patients, which were diagnosed according to standard criteria [11]. The samples were obtained under informed consent of the corresponding patients and according to institutional ethical committee guidelines (Ethical Committee of the Friedrich Schiller University Jena).
DNA was extracted from lymphocytes of 85 CLL cases by a commercial kit (Qiagen) according to manufacturer's instructions. DNA was derived from different sources: 2 samples from heparinized bone marrow, 8 samples from heparinized blood, and 75 samples from cytogenetically prepared cells fixed in methanol/acetic acid (3 : 1)-48 of them derived from bone marrow and 27 from blood. Details on the studied patients can be found in the paper by Alhourani et al. (2014): the 10 here in more detail studied patients with TP53 deletion (Table 1)  In the previous study, no special attention was given to the here treated i(17q) problem, and additional studies, esp. FISH experiments, and reinterpretation of MLPA and aCGH data were performed here.
For further investigation of i(17q) status, additional 65 CLL patients were included in this study with special focus on 8 cases (86 to 93) with TP53 (Table 1).
For iFISH, the following probes were used: For each iFISH analysis, 100-200 interphase nuclei were examined per case and probe.

Multiplex Ligation Dependent Probe Amplification (MLPA)
Analysis. Multiplex ligation dependent probe amplification (MLPA) was performed on 85 CLL cases using SALSA MLPA probemix P377-A1 for Hematological Malignancies Kit from (MRC-Holland, Amsterdam, Netherlands). The P377-A1 probemix kit contains 52 probes for overall 37 genes; TP53 which is located on the short arm of chromosome 17 is covered by 4 probes; likewise UNC13D and IKZF3 on q arm were covered by one probe for each of them [1].

Results
Deletion of TP53 has been detected in 9/85 cases by MLPA. Besides a screening for TP53 deletion was done by iFISH in all of the studied 85 CLL cases to detect mosaic cases with low percentage of aberrant cells as well. Accordingly, TP53 deletion was detected in one additional CLL case, being present there in only 11.5% of the studied cells (case 10).
The overall detected 10 cases with TP53 deletion (Table 2) were further studied by iFISH using probes IKZF3 in 17q12, UNC13D in 17q25.1, and subtelomeric probes (17pter and 17qter; Figure 1(a)); furthermore iFISH-probes for the most frequent aberrations in CLL and, in part, aCGH (case 3; Figure 1(b)) have been applied in those cases, as specified by Alhourani et al. (2014). So, overall 3/85 (∼3.5%) of here studied CLL cases had the loss of TP53 due to formation of an i(17q) which is equal to 30% of these patients.
Further 8 cases with TP53 deletion were found in additional 65 studied CLL patients by iFISH-probe. Here, subtelomeric (17pter and 17qter) probes were applied to identify the three among them cases with i(17q). A probe for 17p11.2 and 17q12 confirmed the isochromosome status in those cases (Table 3).
In the here studied cases with i(17q), this alteration was accompanied by additional chromosomal aberrations (Table 4). For all of them, amplification of c-myc was excluded. While in the first 85 CLL patients, cases 1 and 2 were accompanied by five additional acquired chromosomal rearrangements and case 3 had only one additional change. In cases 1 and 2, at least one of these additional changes was correlated with an adverse prognosis; in case 3 the del(13) is considered to be a favorable prognostic factor. Cases 4-10, which showed just deletion of TP53 without isochromosome formation, had either no further aberrations (cases 4 and 9) or just one additional chromosomal alteration associated with good prognosis (cases 5, 7, and 10). Only case 8 showed two additional chromosomal alterations with known adverse prognostic meaning.
Among the 8 cases with TP53 deletion studied only by iFISH, three cases revealed i(17q). While case 88 showed 8 additional chromosomal rearrangements (two of them were associated with good prognosis), the other two cases, 86 and 87, had only one additional chromosomal alteration.

Discussion
Generally, isochromosome formation is characterized by the loss of the entire short arm with subsequent duplication of the entire long arm, resulting in two homologous arms attached to a single centromere as mirror images [4,7,13]. There are two hypotheses to explain the formation of isochromosome, either by transverse instead of longitudinal division of the centromere or by chromatid exchange involving two homologous chromosomes. The rate of the appearance of isochromosomes is different among the various types of tumors, with the highest occurrence in germ cell neoplasms (60%) and the lowest in chronic myeloproliferative disorders (2.3%) [14]. Surprisingly, although i(17q) appeared in 6/150 (4%) here studied CLL patients, that is, and 6/18 (∼33%) of CLL patients with a deletion of TP53, this chromosomal aberration has not been studied in detail yet in this patient group. Still, there is one study including 2 CLL patients with i(17q) which showed that such isochromosome most likely forms due to clustered breakpoints in 17q11 and is not associated with TP53 mutations of the intact chromosome 17 [4]. In 2006, i(17q) was found to be present in 4/16 (25%) CLL patients with TP53  n n n.a n.a 94 to 150 n n.a n.a n.a n.a n = no aberration, d = deletion, a = amplification, n.a. = not tested, and [] = percentage of cells with aberration.
gene loss [15]; that is, the here reported frequency is within the same range. However, the initial finding of an i(17q) in 2/21 (9.5%) CLL cases seems to be overestimated due to small sample size [16]. Even though here only 6 cases with i(17q) could be studied, the results summarized in Table 4 show a clear tendency: cases with i(17q) are associated with more aberrations compared to those which have just deletion of TP53. Cases 3 and 8 do not exactly fit into this suggestion. However, case 3 had only 25% of the cells with an i(17q) indicating an early phase of the disease; in case 8 cytogenetics provided a hint on an ongoing karyotypic evolution and already advanced stage of the disease.
Whereas both Baliakas et al. and Rigolin et al. reported that complex karyotype predicts a worse overall survival, also Baliakas et al. demonstrated that complex karyotype is identified as an independent prognostic factor for shorter time-to-first-treatment [17,18].

UNC13D
ATM TP53 D17Z1 Subtel. 17pter Subtel. 17qter  karyotype have shorter overall survival than those with only del(17p) [19]. Due to lack of clinical data, the clinical impact of i(17q) could not be followed up, but in spite of that the present study gives first hints that i(17q) presence may be an indicator for more aggressive course of CLL disease than just TP53 deletion without i(17q) formation. Similar findings were reported for other hematological neoplasia, like acute lymphocytic leukemia [20], acute promyelocytic leukemia [21], chronic myeloid leukemia [5], or other myeloid leukemia [22][23][24].
As previously outlined by us and others, MLPA is a quick and inexpensive screening tool for CLL diagnostics [1,25]. However, its inability to detect low level mosaics needs to be considered and thus a diagnostic scheme combining cytogenetics, iFISH, and MLPA needs to be considered for reliable testing of CLL cases in diagnostics [1]. Thus, in Figure 2 we suggest a scheme of how to detect i(17q) reliably.
In conclusion, i(17q) presence in CLL cases with TP53 deletion should be considered as a potentially adverse marker for more aggressive course of the disease than monosomy of 17p13.1 alone; it needs to be kept in mind that MLPA alone may be not sufficient to pick up all corresponding cases and a combination with iFISH may be considered additionally.