Chromosome aberrations in atypical chronic lymphocytic ... - Nature

Leukemia (1997) 11, 1933–1940  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Chromosome aberrations in atypical chronic lymphocytic leukemia: a cytogenetic and interphase cytogenetic study R Bigoni1, A Cuneo1, MG Roberti1, A Bardi1, GM Rigolin1, N Piva1, G Scapoli1, R Spanedda1, M Negrini2, F Bullrich2, ML Veronese2, CM Croce2 and G Castoldi1 Dipartimento di Scienze Biomediche e Terapie Avanzate, Sezione di Ematologia, Universita` di Ferrara, Ferrara, Italy; and 2Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA

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To define better the chromosomal profile of atypical chronic lymphocytic leukemia (aCLL), cytogenetic and interphase cytogenetic studies were performed in 43 cases, using mitogenstimulated cultures and DNA probes detecting the two most frequently occurring aberrations in CLL, ie 112 and 13q14 deletions. All cases showed monoclonal CD5/CD19-positive lymphocytosis, with more than 10% large lymphocytes and/or prolymphocytes in peripheral blood smears and reactivity with FMC7, or bright expression of surface immunoglobulins in a fraction of the cases. Karyotype aberrations were detected in 27 of 43 cases (62.8%). Recurrent chromosome changes were 112 (nine cases), 13q14 aberrations (five cases), 11q anomalies (three cases), 6q21-q23 abnormalities and 4q anomalies with different breakpoints (two cases each). Additional chromosome changes were seen in four cases with 112, in three cases with 13q14 anomalies, in two cases with 11q anomalies, in one case with 6q and 4q anomalies. Trisomy 12 was associated with 13q14 anomalies in three cases, one of which also had an 11q abnormality; other associations, found in one case each, were: 13q14 deletion with a 6q anomaly, 11q anomaly with 13q− and 7q2, a 6q anomaly with 7q2 and 112. Interphase cytogenetics confirmed the results of chromosome banding analysis and showed that six patients with normal karyotype or no mitosis in fact had concomitant 112 and 13q14 deletion in four cases and isolated 112 or 13q14 deletion in one case each, with a resultant 76% overall incidence of cytogenetic abnormalities. The presence of 112, 13q14 deletions, 11q, and 6q21-q23 anomalies in 19 cases was associated with a 2-month median interval between diagnosis and start of treatment, as compared with a 24-month median interval in 14 cases with normal karyotype or non-recurrent chromosome changes (P 5 0.003). We conclude that aCLL is characterized by a relatively high incidence of chromosome anomalies, with recurrent chromosome changes, involving chromosomes 12, 13q14, 6q21q23, 11q, and, possibly, 4q. The presence of complex karyotypes, with concomitant abnormalities of 13q, 112, 6q, 11q, suggests that the development of sequential chromosome changes, rather than any single specific anomaly, may underlie leukemogenesis in this cytologic subset of CLL, partially accounting for the relatively aggressive clinical course. Keywords: atypical CLL; cytogenetics; FISH

Introduction The importance of cytogenetics of B cell chronic lymphocytic leukemia (CLL) has been highlighted over the past 10 years in a number of studies, reviewed by Juliusson and Gahrton,1 documenting that some chromosome changes, such as trisomy 12, del (11q) or 14q anomalies are associated with therapy-demanding disease and, ultimately, with shorter survival.2 More recently, introduction of fluorescence in situ hybridization (FISH), detecting specific chromosome anomalies in interphase cells, has shown that some patients with normal

Correspondence: A Cuneo, Dipartimento di Scienze Biomediche e Terapie Avanzate, Sezione di Ematologia, University of Ferrara, via Savonarola 9, 44100 Ferrara, Italy Received 22 May 1997; accepted 3 July 1997

karyotype may in fact carry chromosomally abnormal clones escaping detection at metaphase analysis due to their low mitotic index.3,4 Meanwhile, increasing awareness of cytologic heterogeneity of CLL,5 prompted the FAB group to propose standardized criteria for the recognition of two related forms, namely typical CLL and atypical CLL (aCLL), and for their distinction from other chronic (mature) B lymphoid leukemias,6 including prolymphocytic leukemia and non-Hodgkin’s lymphomas in leukemic phase. The idea has gradually emerged that aCLL may sometimes run a relatively aggressive clinical course6 and some hematologic features, that may predict disease evolution, have been identified.7 Although knowledge of the cytogenetic profile of aCLL is incomplete, the association of trisomy 12 and atypical morphology has been recently demonstrated8–10 and chromosome changes, such as 6q− and t(11;14), that may have prognostic significance in lymphoid neoplasias, have been found to be frequently associated with cytologically atypical CLL.11,12 Thus, chromosome anomalies may contribute towards a refinement of the classification of CLL13 and may potentially provide a biologic argument supporting the observation that a relatively poor prognosis may be associated with aCLL. To define better the cytogenetic profile of aCLL, this study of 43 cases was carried out combining conventional chromosome analysis (CCA) and FISH, using probes detecting the two most frequent anomalies in CLL, ie +12 and del(13q14). Materials and methods

Patient selection Fifty-seven patients with a diagnosis of atypical B-CLL were selected on the basis of the presence of more than 10% large lymphocytes and/or prolymphocytes (see below) among approximately 260 B-CLLs, seen at our Institution over a 9year period. Diagnoses were made in all cases according to standard clinical, cytologic and immunologic criteria. Histologic studies were performed for diagnostic purposes in 27 cases (bone biopsy in 19 cases and superficial lymph node biopsy in eight cases). Transformation of aCLL into prolymphocytic leukemia (PLL) was not an exclusion criteria, whereas those patients presenting with ‘de novo’ PLL were not considered in this study. Staging procedures according to Rai’s classification included physical examination, a routine laboratory profile, chest X-ray film and abdomen ultrasonography.

Morphologic and immunologic studies Cytologic diagnoses were made in all B-CLLs on peripheral blood (PB) and bone marrow (BM) smears. The percentage of

Chromosomes/FISH in atypical CLL R Bigoni et al

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small lymphocytes (SL) (ie cells less than 14 mm in diameter without detectable nucleoli), large lymphocytes (LL) (ie cells greater than 14 mm in diameter, having inconspicuous nucleoli) and prolymphocytes (PL) (cells usually greater than 14 mm with a prominent central nucleolus) was recorded. Patients were classified according to the FAB criteria6 as CLL in the presence of less than 10% LL and PL, as aCLL when PL and LL comprised between 10% and 55% of all lymphocytes and as PLL when more than 55% PL were present on PB smears. Immunophenotyping was performed as routine diagnostic work-up. Cytofluorimetric study of PB cells, gating primarily on lymphocytes was performed on a FACScan analyser (Becton Dickinson, San Jose, CA, USA). Commercially available reagents detecting the following surface markers were used: B cell lineage: CD10, CD19, CD22, CD23; T cell lineage: CD2, CD3, CD5. Expression of the CD11c and CD25 antigens was also assessed, as well as reactivity with the monoclonal antibody FMC7 (Silenius Lab, Hawthorn, Australia). Double labeling with monoclonal antibodies detecting the CD19 and CD5 antigens was performed. The cut-off point for positivity was set at 30% cells showing fluorescence above controls. Surface immunoglobulins (slg) were detected using rabbit antihuman antibodies against Ig heavy and light chain. sIg expression of the leukemic cells was interpreted as ‘weak’ if the mean intensity of fluorescence was ,256 and ‘bright’ if .256 (logarithmic acquisition, 0–1024 channel range).

Cytogenetic studies Cytogenetic techniques employed in our laboratory were described previously.14 After separation over a Ficoll gradient, PB lymphocytes were cultured in RPMI 1640 with 10% fetal calf serum. Two or more of the following mitogens were employed: phorbol myristate acetate (PMA) (50 ng/ml) lipopolysaccaride (LPS) (0.1 mg/ml); phytohemagglutinin (PHA) (0.1 mg/ml), pokeweed mitogen (0.1 mg/ml). Karyotypic analyses were performed within the first 2 years of diagnosis in all patients. At least 10 mitoses were karyotyped and chromosome anomalies were described according to the ISCN.15

FISH studies Detection of trisomy 12: The presence of trisomy 12 was assessed by using a chromosome 12-specific pericentromeric probe (Oncor, Gaithersburg, MD, USA). Experimental conditions, according to the manufacturer’s instructions, were employed as previously described.16 Detection of del 13q: Deletions involving the 13q14 region in interphase cells were studied with the biotin-labeled C21 cosmid. The C21 cosmid clone was isolated by PCR screening, with GCT16C05 microsatellite specific primers, of a cosmid library constructed from CEPHB YAC clone 745E3. The GCT16C05 marker is located between Rb and the D13S25 marker and is homozygously or hemizygously lost in more than 40% of B-CLL.17 To prevent false positive results, dual color hybridization was performed by adding a digoxigenin-labeled probe (control probe), recognizing DNA sequences at the telomeres of chromosome 13q (Oncor). Sig-

nal screening was performed on those slides with high hybridization efficiency, indicated by the presence of more than 80% interphase cells with two 13q telomere signals. Hybridization conditions are described below.

Detection of t(11;14)(q13;q32): All cases were tested with a BCL1 yeast artificial chromosome (YAC) probe, spanning a 390 kb area in the BCL1 locus, including the major translocation cluster, as previously reported.4 The presence of trisomy/monosomy 11 was excluded using a chromosome 11specific pericentromeric probe (Oncor). Hybridization conditions, signal amplification and detecCells for FISH studies were derived from the same tion: samples that had been used for cytogenetic analysis. Usually, samples with more than 90% clonal CD5+/CD19+ cells were obtained by separation over Ficoll gradient. Ten normal control samples were tested in order to calculate the cut-off point for positivity using the chromosome-12-specific and 13q14 probe. The slides were incubated for 60 min with RNAse (100 mg/ml; Boehringer Mannheim, Germany), washed twice in 2 × SSC, dehydrated in ethanol alcohol series (75, 85 and 100%) and air dried. The denaturation was performed by immersion of the slides in a 70% formamide/2 × SSC solution at 70°C for 2 min and dehydrated in the same alcohol series. The 13q14 cosmid probe and the chromosome 12-specific pericentromeric probe were biotinylated with a BioNick Labelling System Kit (GIBCO BRL, Gaithersburg, MD, USA) according to the manufacturer’s instructions and precipitated with Human Cot-1 DNA (GIBCO BRL) to suppress non-specific signals due to repetitive sequences. The DNA probe was resuspended in a hybridization mixture with 50% formamide, 10% dextran sulphate in 12.5 × SSPE, 5 × Denhardt’s solution. Sixty microliters of the mixture were added to each slide and incubated overnight. Post-hybridization washes included 50% formamide/2 × SSC, 1 × SSC and 0.1 × SSC baths at 45°C each, with intermittent agitation. Detection was performed with alternating layers of fluoresceinated avidin (FITC-avidin) and biotinylated goat anti-avidin (5 ng/ml; Vector Laboratories, Burlingame, CA, USA), until two layers of avidin were applied; each treatment was followed by 2 × SSC washes 2 min each. Rhodamine-conjugated monoclonal antibodies (red) were employed for the detection of digoxigenin-labeled probes, using a commercially available detection kit (Oncor). A DAPI or propidium iodide fluorescent antifade solution was applied on to the slides as counter-stain, as appropriate. The evaluation was performed on more than 200 interphase cells with well-delineated signals on a Leitz Wetzlar fluorescence equipped microscope; the FISH experiment was repeated when more than 20% cells showed no signal. Images for illustration purposes were captured using a cooled charge-coupled device (CCD) camera (Princeton Instruments, Princeton, NJ, USA). Results Forty-three patients, corresponding to 16% of all CLLs seen at our Institution during the study period, fulfilled the above criteria for the diagnosis of aCLL. Fourteen ‘atypical CLLs’ with BCL1 translocation, deriving from the t(11;14)(q13;q32), shared cytoimmunologic and cytogenetic features with leu-


kemic mantle cell lymphoma,4 and were excluded from this analysis.

Hematologic features By definition, all cases had .10% large lymphocytes and/or prolymphocytes in PB smears (see Figure 1). Globally, prolymphocytes with prominent central nucleoli were seen infrequently, these cells representing 3% of all lymphocytes (median value in 43 cases, range 0–50%). Cells with indented or irregular nuclear outlines were only observed occasionally. In 30 cases, an admixture of small lymphocytes and of larger lymphocytes with round nuclei and ,10% prolymphocytes was observed, whereas in 13 cases .10% prolymphocytes were seen in PB smears, corresponding to the CLL/PL category of the FAB classification.6 These 43 patients will be globally referred to as aCLL. In 19 cases, bone biopsies showed a prevailing small lymphocyte infiltrate, with a minority of larger lymphoid cells or prolymphocytes. The infiltration pattern was nodular in two cases, interstitial in seven cases, mixed in three cases and diffuse in seven cases. In eight cases, histologic findings on lymph node specimen showed a diffuse infiltrate consisting of small lymphocytes, intermingled with some paraimmunoblasts and prolymphocytes, frequently forming pseudoproliferation centres. All cases were CD5+/CD19+. The results of immunophenotyping are presented in Table 1. Hematologic features are summarized in Table 2, in correlation with cytogenetic findings. Twenty-six patients are alive at 16–120 months (median follow-up 46 months); 13 patients died at 8–144 months (median 48 months). Four CLL-unrelated deaths were observed at 6– 120 months. Nineteen patients with recurrent karyotypic anomalies (+12, 13q14 deletions, 11q, 6q21-q23 anomalies) required earlier cytotoxic therapy (median: 2 months from diagnosis, range 0–20 months) than 14 cases with normal karyotype, or non-recurrent chromosome changes (median: 24 months, range 0–72+ months) (P = 0.0031, log-rank test).

Clonal chromosome changes were detected in 27 of 43 cases (62.8%), normal karyotype was seen in six cases and no mitoses were obtained in 10 cases. Trisomy 12 was seen in 10 cases (one of which had partial trisomy 12q) (Nos 1–9, 18), deletions or translocations 13q in nine cases (Nos 4, 7, 9–15), abnormalities of 11q, 1q and 10q in four cases each (Nos 7, 15–17; Nos 4, 16, 18, 23, and 4, 11, 22, 27, respectively), aberrations of 3q, 6q, 7q and 17p in three cases each (Nos 4, 14, 23; Nos 10, 18, 19; Nos 15, 18, 22; Nos 7, 14, 18, respectively). Two cases each had 3p translocations and structural abnormalities of 4q (one interstitial deletion and one 4q+ chromosome, with distinct breakpoints) (Nos 4, 7 and 20, 21). Possible recurrent ‘primary’ anomalies (ie those aberrations present in all abnormal karyotypes in a given patient) were: total or partial trisomy 12 in nine cases, one of which also had a 13q− chromosome in all abnormal metaphases (Nos 1–9); 13q abnormalities in five cases (Nos 10–14), 11q abnormalities in three cases (Nos 15–17) 6q21-q23 abnormalities in two cases, one of which also had +12 in all abnormal metaphases. Aberrations of 4q were seen in two cases, showing different breakpoints (see Table 1). The following associations were noted (see Table 3): trisomy 12 with 13q14 abnormalities in three cases (Nos 4, 7 and 9), one of which also had an 11q abnormality; 13q14 aberrations with a 6q abnormality in one case (No. 10), 11q aberrations with 13q− and 7q− in one case (No. 15); 6q abnormalities with 7q− and +12 in one case (No. 18).

FISH analysis

Cytogenetic findings

Control samples: Hybridization of the chromosome 12pericentromeric probe to normal peripheral blood cells (10 control cases) yielded two signals in more than 98% interphase cells, with three signals present in 0.7% interphase cells (mean value). In our controls, more than 95% normal interphase cells had two signals when hybridized with the 13q14 C21 cosmid, whereas 3.8% and 3.1% interphase cells (mean values) had none or one signal, respectively. The cutoff point for positivity was therefore set at 3% for trisomy 12 and at 10% for 13q14 deletions.

Karyotypes are described in Table 3, together with the results of FISH analysis.

Trisomy 12:

Interphase cytogenetic analysis could detect

Figure 1 Small lymphocytes and large lymphocytes with diameter greater than 14 mm (ie greater than two red blood cells) are shown (arrowed: case Nos 9, 17 and 19).

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Table 1

Immunologic findings in 43 atypical B-CLL according to the cytogenetic findingsa

Antigen

No. of positive cases/Total tested % positive cells (median value, range in parentheses) +12

Abn 13q

Abn 11q

Abn 6q

Other abn

Normal

Failures

Total

CD5/CD19+

9/9 71% (31–98)

5/5 83% (31–97)

3/3 86.5% (82–93)

2/2 89.5% (89–90)

8/8 71% (36–82)

6/6 52% (30–97)

10/10 80.5% (31–97)

43/43

CD23

5/7 73.5% (45–93)

3/4 90% (35–93)

2/3

1/2 93%

5/5 71% (50–97)

9/10 83% (70–97)

30/38

78–86

5/7 72% (40–91)

7/8 75% (60–92)

5/5 85% (46–91)

3/3 91% (90–94)

2/2

6/7 68% (48–93)

4/4 71.5% (57–88)

9/10 87% (65–98)

36/39

4/7 70% (56–79)

2/4

1/3 57%

0/5

2/4

13/29

(32–78)

4/5 70% (43–92)

3/9

3/5

3/8

2/6

CD22

FMC7

bright sIg

(88–90) 0/1

1/3

1/2

b

(77–91) 2/10

15/43 b

a The patients with multiple abnormalities are included in one cytogenetic category only, corresponding to the possible ‘primary’ chromosome change in the karyotype. b Nine cases were FMC7+ and bright sIg+. Abn, abnormal; sIg, surface immunoglobulins.

Table 2

Salient hematologic features according to cytogenetic findings in 43 CLL mixed cell type

+12

13q

11q

6q

Other

Normal

Failures

Total

63 (49–78)

65 (54–78)

54 (48–58)

(32–74)

51 (50–77)

65 (56–77)

68 (42–83)

61 (32–83)

28 (13–120)

15 (10–31)

28 (25–670)

(28–225)

25.8 (14–50)

14.5 (11–230)

31 (10–220)

24 (10–670)

Lymphadenopathy (No. of cases/total)

8/9

4/5

3/3

1/2

4/8

4/6

6/10

30/43

Splenomegaly (No. of cases/total)

5/9

2/5

2/3

2/2

1/8

4/6

3/10

19/43

0 (1); I (2); III (4); IV (2)

0 (1); I (2); III (2)

I (1); III (1); IV (1)

III (2)

0 (3); I (2); II (2); III (1)

0 (1); I (1); II (1); III (3)

0 (3); I (3); III (2); IV (2)

0 (9); II (11); II (3); III (15); IV (5)

Months from diagnosis to progression to Rai stage III/IVa

12/60

48/72

—

—

58/72

48

36/48

—

Months between diagnosis and start of therapy (median and range)

0 (0–20)

20 (2–26)

0 (0–24)

(0–2)

26 (2–72+)

15 (0–48)

20 (0–72)

8 (0–72+)

Median age (range) WBC at diagnosis (×109/l) median (range)

Rai stage (No. of cases)

Months from diagnosis to progression to Rai stage III/IV are indicated for each stage 0–II patient. One patient, in the +12, 13q−, 11q− groups, four patients in the group ‘others’ and five patients in the group ‘failure’ did not show disease evolution.

a

a clone with three signals using the chromosome 12-pericentromeric probe in all nine cases with +12 in karyotypes. As expected, two signals were seen in patient No. 6, having trisomy 12q13-qter. In addition, five patients with normal karyotype (Nos 28 and 29) and with inadequate mitotic yield (Nos 34–36) were found to carry trisomy 12 in interphase cells. The percentage of trisomic cells ranged between 21 and 78%, median value 54%.

13q14 deletions: Seventeen of 43 patients in this series were shown to carry deletions of the 13q14 DNA target region

in 28–90% of interphase cells, median value 80%. In 15 cases monoalleleic deletions (ie one signal in interphase cells) were observed, whereas in two cases (Nos 28 and 29), a mixture of interphase cells with monoallelic and biallelic 13q14 deletions (ie no signal in interphase cells) was detected. All nine cases having karyotypes with 13q abnormalities were shown to carry 13q14 deletions in interphase cells by FISH. The involvement of the 13q14 band was cytogenetically detectable in only five cases, whereas a more distal breakpoint was recognized in four cases. Three additional cases (Nos 16, 17 and 22) with abnormal karyotypes without involvement of chromosome 13, had a 13q14 deletion as detected by FISH.


Table 3

Case

Karyotypes (No. of metaphases in parentheses)

Patients with total or partial +12 1 47,XY,+12 2 47,XX,+12 3 47,XX+12 4 43-47,XY, t(1;4)(q32;p16), inv(1)(p11q31), t(2;11)(q13;p15), t(3;17)(q29;q21), del(3)(p21), −6, −8, +add(9)(p23), t(10;13)(q23;q14), +12 [cp18] 5 47,XX, +12 6 47,XY,+der(18?)t(12;18?)(q13;q12?) 7 44-46,XY, +der(3)t(3;15)(p12;p11), −11, −12. −14, −15, +12,+der(17)t(11;17)(q25;p13),t(13;19)(q14;q13), [cp15] 8 47,XX,+12/47,idem,del(20)(q12) (1) 9 47,XY, +12, del(13)(q22q32) Patients with 13q abnormalities 10 46,XX,del(13)(q14q32) (2)/46, idem, t(1;6)(p12;q27) (14) 11 46,XY, del(9)(q11q13), del(10)(q22q23), del(13)(q14q32) 12 46,XY, del(13)(q22) 13 46,XX, del(13)(q22q32) 14 46,XX, add(3)(q?), del(13)(q14q22), add(17)(p11), +mar [cp14] Patients with 11q abnormalities 15 46-48,XY del(7)(q32), +add(11)(q21?), del(13)(q22q32), add(17)(q23), +mar [cp16] 16 46-47,XX,t(1;1)(q32;p32), add(2)(p?),), +6,+add(8)(p12?), +del(11)(q21q2?2),add(16)(q21?)+ mar [cp16] 17 46,XY, add(11)(q21) Patients with 6q21–q23 abnormalities 18 48,XY, +add(6)(q23), del(7)(q32), +12,−19,+mar (13)/48, idem, add(1)(q?), add(14)(p11), add(17)(p12) (2) 19 46,XY, del(6)(q2?1) Others 20 AL 21 22

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Karyotypes and interphase cytogenetic findings in 43 patients with CLL mixed cell typea

Abnormal/ Total

FISH 13q− (yes/no) (% cells with 1 signal in positive cases)

+12 (yes/no) (% cells with 3 signals in positive cases)

4/10

no

yes (45)

2/12

no

yes (61)

7/15

no

yes (73)

18/24

yes (44)

yes (55)

3/14 5/14

no no

yes (78) no

15/20

yes (85)

yes (72)

4/11

no

yes (72)

7/13

yes (70)

yes (75)

16/16

yes (65)

no

10/15

yes (80)

no

8/8

yes (80)

no

3/10

yes (80)

no

14/18

yes (28)

no

16/21

yes (78)

no

16/20

no

no

7/10

yes (50)

no

15/15

yes (85)

yes (53)

11/11

no

no

46,XX, del(4)(q11q21)

3/10

no

n0

46,XY, add(4)(q31) (4)/47,idem, +mar (3) 44-48,XY,del(1)(p12), del(7)(q32), −8, del(10)(q24), +13, +19, +21, +mar1, +mar2 [cp16]

7/15 16/19

no no

no no

23

46-47,XY, add(1)(q?), add(3)(q25?),−10, add(14) (q22?), add(18)(q21?), +mar [cp12]

12/16

yes (80)

no

24

46, XY, del(2)(p14)

3/12

no

no

25

47, XY,+3

10/12

no

no

26

47,XY, +mar

2/13

no

no

27

46,XY, del(10)(q11q21)

3/15

no

no

0/16

yes (90)b

yes (43)

0/10 0/15 0/14

yes 90b yes (75) no

yes (32) no no

0/12 0/18

no no

no no

yes (85) no

yes (21) yes (37)

Normal and failures 28 46,XX 29 46,XX 30 46,XY 31 46,XY 32 46,XY 33 46,XY 34 no mitosis 35 no mitosis


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Table 3

(Continued)

36 37 38 39 40 41 42 43

no no no no no no no no

mitosis mitosis mitosis mitosis mitosis mitosis mitosis mitosis

yes (67) no no no no no no no

yes (41) no no no no no no no

a The patients are grouped under different cytogenetic categories ie +12, abnormalities of 13 q, etc, according to the anomaly that was identified as the possible primary chromosome change in karyotypes. Patient No. 9 had +12 and del(13q) in all abnormal cells; patient No. 18 had a 6q abnormality and +12 in all abnormal cells: because FISH detected only 53% interphase cells with +12, he was included in the ’6q21-q23 anomalies’ group. b Mixture of cells with monoallelic and biallelic deletion in the same patient.

Interphase cytogenetic analysis could detect deletions at 13q14 in three cases with normal karyotypes (Nos 28–30) and in two cases without analyzable mitoses (Nos 34 and 36). Eight cases (Nos 4, 7, 9, 18, 28, 29, 34, 36) had both 13q14 deletions and +12 in interphase cells.

t(11;14)(q13;q32): Two signals in .95% interphase cells were detected by FISH in all 43 cases, using the BCL1 YAC probe. No patient was found with monosomy 11. Discussion In this study, a systematic approach to the definition of the cytogenetic profile of aCLL was used, with conventional chromosome analysis and detection by FISH of the two most frequent changes in CLL, ie +12 and 13q14 deletions. Stimulation by phorbol esters may improve the detection of chromosome anomalies, especially del(13q), in CLL2,18 and interphase FISH is useful in this disorder,816,19 in which in vitro divisions may frequently occur in normal T lymphocytes.20 A preliminary methodological problem in the analysis of the cytogenetic profile of aCLL is represented by the difficulty in recognizing ‘primary’ chromosome changes, due to the complexity of the karyotypes and to the lack of information about the underlying molecular defects. To be able to analyze the correlation of cytogenetic and clinicobiologic features, we subdivided our patients into different groups according to the presence of some recurrent chromosome changes, which were present in all abnormal cells in a given patient (see Tables 1–3). Although these selected abnormalities may potentially represent ‘primary’ changes, their precise role in the transformation process remains poorly understood. Our patients denied history of antecedent lymphoproliferative disorder and showed monoclonal CD5+/CD19+ lymphocytosis with the morphologic features of aCLL by FAB criteria. Bone marrow and lymph node histology were consistent with the diagnosis of aCLL, including 19 cases having an atypical immunologic profile, consisting of FMC7 positivity and/or bright expression of sIg (see Table 1). The CD23 marker was expressed by the majority of our cases and no patient was found to have a mantle cell phenotype, as defined by the combination of CD5+/CD19+, CD23−, FMC7+ and bright sIg expression. Thus, the overall surface marker profile in our series is in line with results of previous immunologic studies in aCLL, showing variable proportions of cases with either bright sIg expression and/or FMC7 positivity.6,7,11,21 FMC7 expression was more frequently found among patients with

chromosome abnormalities than with normal karyotype (11/20 vs 0/5, P = 0.027).22 The evolutive phases of CLL, including prolymphocytoid transformation, were noted to be associated with a high incidence of karyotype anomalies, including +12 and a 14q+ chromosome, as early as 1984.23 Later on, after the FAB group proposed objective criteria for the distinction of typical CLL from allied disorders, convincing evidence was provided that trisomy 12 may characterize a subset of CLL with atypical morphologic features21 and few patients were reported with aCLL and 14q translocations,22 deletions (6q) and del (11q), as summarized in Table 4. The percentage of patients with chromosome changes was higher in this series (62.8%) than in 433 patients with cytologically unselected CLLs, collected in a multicentre study by Juliusson and co-workers,2 who found a 50% incidence of clonal abnormalities. In this report, the percentage of abnormal cases was even higher when considering that FISH detected +12 and/or 13q14 deletions in six patients with apparently normal karyotype or with no mitosis, with a resultant 76.7% overall incidence of cytogenetic abnormalities. A higher incidence of advanced Rai stages (III/IV) was noted in this series as compared with other studies of unselected CLL,24 possibly reflecting a more rapid disease evolution in aCLL. No correlation was found, in this (see Table 2) and other studies,22 between clinical stage and rate of karyotypically abnormal cases. Interestingly, 76% of aCLL were found to have abnormal karyotypes in a recent analysis of 90 cases10 and a 45% rate of clonal aberrations was detected by Hernandez and coworkers11 in 157 aCLL, a figure comparing favorably with 29% abnormal cases in 266 typical CLLs studied at the same institution. Our data confirm that +12 is frequently associated with aCLL (15/43 cases) and it is noteworthy that +12, when present, was always found in all abnormal metaphase cells. Although the pathogenetic role of +12 in CLL remains elusive, this finding shows that this numerical change may frequently be an early event in the history of the disease. In line with this view, the presence of trisomy 12 as a secondary karyotype anomaly was only rarely documented in two analyses of the nature of additional chromosome anomalies in CLL.25,26 Unbalanced abnormalities of 6q, leading to loss of genetic material at bands 6q21-23, were found in all abnormal cells in two patients, one of whom also had +12 in 53% of interphase cells. In our analysis and in a previous study,11 the 6q− chromosome frequently occurred as the sole anomaly, suggesting that it may be an important event in the genesis of CLL displaying atypical morphology. The findings by Offit et


Table 4 Recurrent primary chromosome changes in atypical CLL: review of the literature

Chromosome anomaly +12

No. of cases/ Total tested

Diagnosis

Ref.

51/90 12/21

CLL mixed cell types Atypical CLL by morphology (6 cases) or by immunologic features (15 cases) CLL mixed cell types (10 cases) CLL in prolymphocytic transformation (4 cases) atypical CLL CLL mixed cell types atypical CLL (morphology)

23 9

10/22 4/7 7/13 2/13 24/67

t(11;14)(q13;q32)

5/20 4/19a 2/10 2/100 5/179

8 32 33 11 39

atypical CLL, sharing immunologic features with MCL atypical CLL with CD11b+ CLL/PL atypical CLL/mantle cell leukemia? atypical CLL/mantle cell leukemia? CLL, rare

12 34 35 36 37

del(11q)

5/31 5/67

CLL mixed cell type atypical CLL (morphology)

11 39

t(14;19)(q32;q13)

3b

CLL mixed cell type

38

6q abnormal

4/31 2/67

CLL mixed cell type atypical CLL (morphology)

11 39

MCL, mantle cell lymphoma. a Bcl-1 rearrangement. b Total number of cases not available.

al,27 who described small lymphocytic lymphomas (ie the lymphomatous counterpart of CLL), with del(6)(q21q23), that was associated with circulating prolymphocytes/para immunoblasts in 14 out of 55 cases, support this argument. Translocations or deletions of 4q and 10q, were found either as the sole anomaly or as part of complex karyotypes in two and four cases, respectively. The breakpoints in these patients were heterogeneous at the cytogenetic level and study of more cases will be required to identify the commonly involved bands. In a detailed karyotypic study of 82 cytologically unselected CLLs,26 abnormalities of 4q and 10q were detected in seven and two cases, respectively, raising the possibility that involvement of these chromosome regions may represent a previously unrecognized non-random event in the genesis of CLL. An unexpected finding in this series was the 20.9% rate of 13q14 abnormalities in metaphase spreads. Although suboptimal banding resolution rendered recognition of 13q deletions difficult in some metaphases, the majority of interphase cells were shown to carry monoallelic deletions of the target DNA segment, located between the Rb gene and the D13S25 marker, in all cases with 13q14 deletions/translocations. In

addition, eight patients, without cytogenetic evidence of 13q anomaly, had 13q14 deletions in interphase cells, giving an overall 39.5% incidence for this abnormality. The simultaneous presence in two of our cases of monoallelic and biallelic deletions in interphase cells suggest that genetic damage occurred through discrete events targeting the 13q14 chromosome region. Deletions of 13q14 were recently detected by molecular genetic analysis in 33/75 B-CLL and in 4/16 NHL.28 Likewise, monosomy 13 or del(13q) are known to occur rather frequently in association with the t(11;14)(q13;q32).4,29 Several studies narrowed the smallest region of deletion involving 13q14 in lymphoid neoplasias to an approximately 500 kb segment,17,28 suggesting that a candidate tumor suppressor gene important in the pathogenesis or progression of CLL and other lymphoid neoplasias may be located in this region. It is reasonable, therefore, to assume that 13q14 deletions may play a role in the development of CLL as well as of other lymphoid malignancies and that additional genetic events may contribute to the heterogeneity of hematologic manifestations. Indeed, only two of our patients had del(13q) as the sole change and additional anomalies were +12 in three cases, and structural anomalies of 6q, 7q, 10q and 17p in one case each. Most of these abnormalities were previously found in association with atypical morphology and advanced/ progressive CLL.10,11,30 In a similar fashion, abnormalities of 11q, normally associated with morphologically typical CLL,31 were detected in this series in the context of complex karyotypes in 3/4 cases, suggesting that the sequential development of chromosome changes, rather than a specific cytogenetic event, may be regarded as the cytogenetic hallmark of aCLL. In conclusion, while confirming the presence of a high incidence of chromosome anomalies, including +12, this study extends our knowledge of the cytogenetic profile of aCLL, showing that: (1) the distinctive cytogenetic feature of aCLL may be represented by karyotypes displaying various combinations of chromosome anomalies, most of which represent recurrent cytogenetic changes in typical CLL; (2) aberrations involving 6q21-q23 and, possibly 4q and 10q, may be frequently found in aCLL; (3) abnormalities of 13q and 11q, normally associated with ‘typical’ CLL, are also found in aCLL, where they are almost invariably associated with additional chromosome aberrations; and (4) the sequential development of chromosome aberrations may underlie leukemogenesis in aCLL, and may partially account for the relatively unfavorable clinical outcome observed in some cases, requiring early cytotoxic therapy.

Acknowledgements This work was supported by CNR, ACRO Project, by BMH1, CA-CT 94-1703, by IIS, Italy–USA project on ‘Therapy of Tumors’ and by an Outstanding Investigator Award from the National Cancer Institute (CA 39860).

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