Gain or amplification of the short arm of chromosome 2 (2p) is a recurrent cytogenetic abnormality in B-lymphoid disorders including chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL) [1,2]. 2p gain can extend across the whole chromosome arm, or encompass only a minimally gained region of approximately 1.3 Mb [3]. A number of genes in the 2p region are important to B-cell development as well as oncogenesis, and have been postulated as important targets within this gain. These include the proto-oncogene MYCN in chromosome band 2p24.3, and the NFKB subunit REL, nuclear transporter XPO1, and proto-oncogene BCL11A within the minimally gained chromosomal region of 2p15–16.1 [3,4].
Reports on frequency of 2p gain in CLL range from 3–28% in untreated patients [4,5] and can be acquired during clonal evolution [6]. 2p gain in CLL has been previously associated with transformation to DLBCL (Richter’s transformation) [7], where it has been detected in CLL prior to transformation and afterwards within the transformed lymphoma cells [6]. Similarly, 2p gain has also been described in FL prior to and after transformation to DLBCL, as well as in de novo DLBCL, indicating that this alteration may be contributing to an aggressive B-cell phenotype [8]. 2p gain has been associated with a poor overall survival in CLL [7]; however, this association has not been consistently observed [9]. Importantly, previous studies were limited by either small numbers of patients with 2p gain or were performed when treatment for CLL predominately involved chemotherapy or chemo-immunotherapy.
In recent years, the treatment paradigm for CLL has evolved. Bruton’s tyrosine kinase (BTK) inhibitors, such as ibrutinib, have become a standard therapy. Progression on ibrutinib typically occurs due to progressive CLL via development of a drug resistant clone or through Richter’s transformation [10]. 2p gain has been associated with increased drug resistance [3,11], and in vitro studies in primary CLL cells have shown less cytotoxicity with ibrutinib in samples with 2p gain [3]. However, data regarding the prognostic significance of 2p gain in a uniformly treated population is currently lacking. Therefore, we examined 2p gain in patients enrolled at our institution across four ibrutinib clinical trials to assess if it is associated with progression on ibrutinib.
All patients consented to participate in the study in accordance with the Declaration of Helsinki and enrolled on protocols approved by the Ohio State University cancer IRB. The characteristics of this patient population and the four ibrutinib clinical trials (NCT01105247, NCT01217749, NCT01589302, NCT01578707) have been previously described [12–15]. Fluorescence in situ hybridization (FISH) analysis was performed on nuclei from mitogen-stimulated cultures from peripheral blood or bone marrow samples collected prior to receiving ibrutinib using standard laboratory procedures. The minimally gained region of 2p, previously characterized in the literature by array comparative genomic hybridization, includes the gene REL [3]. Therefore, to identify 2p gain we used a FISH probe targeting the REL (2p15) locus with a DIRC1 (2q32.1) probe as a control (Empire Genomics, Williamsville, New York, USA). Hybridization was according to the manufacturer’s directions. Two hundred cells were analyzed, 100 each by two independent observers.
Baseline variables were compared between patients with and without 2p gain using the Fisher exact test or the Wilcoxon rank-sum test. Cumulative incidence of ibrutinib discontinuation due to progression or Richter’s transformation was estimated, with discontinuation for other reasons treated as a competing risk. The Gray test was used to test for cumulative incidence differences between groups. Progression-free survival (PFS) and overall survival (OS) were measured from the date of first treatment with ibrutinib until the date of progression and/or death, censoring event-free patients at last follow-up. Estimates of PFS and OS were calculated by the Kaplan-Meier method, and differences between curves were tested with the log-rank test. All tests were 2-sided; statistical significance was declared at α = 0.05.
Of the 308 study-enrolled patients, 296 had sufficient sample available for analysis. Only 8 patients were treatment-naïve. 2p gain by FISH was observed in 44 (14.9%, 1 treatment-naïve) of the 296 patients. 2p gain was only apparent by banded metaphase analysis in 2 of 44 of the patients who were positive by FISH. Five patients had additional material of unknown origin present on 2p. Characteristics of the patient population are shown in Table 1. The proportion of deletion 17p was higher in patients with 2p gain compared with those without 2p gain (53% vs. 38%), but did not reach statistical significance (p=0.06). Previously reported associations of 2p gain with deletion 11q and unmutated IGHV [1,3,5] were not significant within our patient population (p=0.15 and p=0.21, respectively). Presence of 2p gain was, however, significantly associated with the occurrence of other adverse cytogenetics, including BCL6 (p=0.009) and MYC abnormalities (p= 0.004) by FISH, as well as complex karyotype (≥3 unrelated chromosome abnormalities, p=0.03).
Table 1:
Baseline Characteristics of CLL cohort
| Characteristic | Total (n = 296) | No 2p gain (n = 252) | 2p gain (n = 44) | P-value |
|---|---|---|---|---|
| Monotherapy with ibrutinib | ||||
| No | 67 (23) | 55 (22) | 12 (27) | 0.44 |
| Yes | 229 (77) | 197 (78) | 32 (73) | |
| Age | ||||
| Median | 65 | 65 | 62 | 0.21 |
| Range | 26–91 | 26–91 | 50–79 | |
| Sex, no. (%) | ||||
| Male | 209 (71) | 183 (73) | 26 (59) | 0.08 |
| Female | 87 (29) | 69 (27) | 18 (41) | |
| Rai Stage, no. (%) | ||||
| 0 | 11 (4) | 10 (4) | 1 (2) | |
| I | 67 (23) | 55 (22) | 12 (27) | 0.86 |
| II | 22 (7) | 20 (8) | 2 (5) | |
| III | 43 (15) | 38 (15) | 5 (11) | |
| IV | 153 (52) | 129 (51) | 24 (55) | |
| Elevated LDH | ||||
| No | 110 (38) | 94 (38) | 16 (37) | 1.00 |
| Yes | 178 (62) | 151 (62) | 27 (63) | |
| LDH | ||||
| Median | 219 | 217 | 221 | 0.92 |
| Range | 96–1485 | 96–1485 | 100–495 | |
| Number of Prior Therapies | ||||
| Median | 3 | 3 | 3.5 | 0.74 |
| Range | 0–16 | 0–16 | 0–12 | |
| BCL6 | ||||
| No | 266 (91) | 232 (93) | 34 (79) | 0.009 |
| Yes | 27 (9) | 18 (7) | 9 (21) | |
| MYC | ||||
| No | 230 (79) | 204 (82) | 26 (60) | 0.004 |
| Yes | 63 (22) | 46 (18) | 17 (40) | |
| Trisomy 12 | ||||
| No | 241 (82) | 207 (83) | 34 (79) | 0.52 |
| Yes | 52 (18) | 43 (17) | 9 (21) | |
| Del(13q) | ||||
| No | 141 (48) | 121 (48) | 20 (47) | 0.87 |
| Yes | 152 (52) | 129 (52) | 23 (53) | |
| Del(11q) | ||||
| No | 210 (72) | 175 (70) | 35 (81) | 0.15 |
| Yes | 83 (28) | 75 (30) | 8 (19) | |
| Del(17p) | ||||
| No | 176 (60) | 156 (62) | 20 (47) | 0.06 |
| Yes | 117 (40) | 94 (38) | 23 (53) | |
| Del(11q)/Del(17p) | ||||
| No | 109 (37) | 94 (38) | 15 (35) | 0.86 |
| Yes | 184 (63) | 156 (62) | 28 (65) | |
| Complex Cytogenetics | ||||
| No | 119 (41) | 108 (44) | 11 (26) | 0.03 |
| Yes | 170 (59) | 138 (56) | 32 (74) | |
| IGHV | ||||
| Mutated | 53 (20) | 48 (22) | 5 (12) | 0.21 |
| Unmutated | 211 (80) | 174 (78) | 37 (88) |
Abbreviations: no, number; LDH, serum lactate dehydrogenase; del, deletion; IGHV, immunoglobulin variable heavy chain
With a median follow-up of 60 months, 36% (n=16) of patients with 2p gain progressed with CLL, 11% (n=5) progressed with Richter’s transformation, 18% (n=8) discontinued therapy for reasons unrelated to progression, and 30% (n=13) were still on treatment. Outcomes for patients with no 2p gain were similar: 21% (n=52) progressed with CLL, 9% (n=23) progressed with Richter’s transformation, 33% (n=82) discontinued therapy for reasons unrelated to progression, and 33% (n=82) were still on treatment. Cumulative incidence of ibrutinib discontinuation for CLL progression and Richter’s transformation was not significantly different between patients with 2p gain compared to those without (p=0.10 and p=0.66, respectively), nor was PFS (p=0.94) or OS (p=0.81) (Figure 1). Furthermore, we examined the impact of proportion of nuclei with 2p gain as a continuous variable and found no statistically significant prognostic effect on PFS (p=0.19) and OS (p=0.20).
Figure 1:
Survival curves with and without 2p gain
Finally, we examined 2p gain longitudinally in this patient population. 153 of 296 patients had subsequent sample(s) submitted for REL FISH analyses with a median follow-up time of 3.1 years (range: 1 month-6.4 years), including 29 of the 44 patients positive for 2p gain prior to receiving ibrutinib. Of these 29 patients, 19 were negative for 2p gain in a subsequent follow-up sample (n=4 at relapse, n=2 after relapse and subsequent therapy, n=11 during continued response, and n=2 at ibrutinib discontinuation). Persistence of the 2p clone in subsequent samples was seen in 10 patients (n=4 at CLL relapse, n=2 at Richter’s transformation, n=2 during continued response at 1.5 and 1.6 years follow-up, n=2 at ibrutinib discontinuation at 1.8 and 1.9 years follow-up). Conversely, of the 124 patients negative for 2p gain prior to ibrutinib and with subsequent samples, only 4 acquired this abnormality in a follow-up sample (n=2 at CLL progression, n=2 at Richter’s transformation).
2p gain has historically been considered an adverse cytogenetic marker in CLL, including an association with Richter’s transformation [6,7]. In this study, 2p gain was not significantly associated with ibrutinib discontinuation for either progressive CLL or Richter’s transformation. Longitudinal data was available for 51.7% of our cohort, and within this sub-population, the majority of patients with 2p gain prior to ibrutinib no longer showed the abnormality in follow-up samples after therapy. Although 2p gain at baseline was not significantly associated with increased risk of transformation, presence of the abnormality near the time of transformation was seen in 4 patients. For these patients, we cannot exclude a role for 2p gain in transformation of CLL to lymphoma. As a limitation, our analyses for 2p gain was performed on bone marrow samples, therefore, it is not known if the 2p clone was present in lymphoma cells.
Our study differs from previous publications in a number of ways. Previous reports of 2p gain as a poor prognostic indicator occurred prior to tyrosine kinase inhibitors, and studies addressing the significance of 2p gain in the setting of tyrosine kinase inhibition have primarily occurred in vitro or as small case series [3,11]. Different methodologies have been used when assessing 2p gain, including multiple comparative genomic hybridization platforms, and well as various FISH probes. With our probe, we cannot differentiate small gains from large, which might include the MYCN gene that may be clinically relevant [4]. Our study was also enriched for patients later in the course of their disease, therefore we do not know if 2p gain was present at diagnosis or developed over time prior to the start of ibrutinib therapy. As has previously been reported [5], we found 2p gain predominately occurred with other cytogenetic abnormalities; 74% of patients with 2p gain had a complex karyotype. 2p gain frequently co-occurred with other adverse cytogenetic abnormalities, including BCL6 and MYC gain or rearrangement by FISH, which to our knowledge has not been previously described. Adverse outcomes and increased risk of Richter’s transformation previously associated with 2p gain may be attributable to 2p gain in combination with these additional aggressive genetic alterations in these patients. As we have seen with other adverse cytogenetic abnormalities [15], this study demonstrates the efficacy of ibrutinib in CLL patients with another high-risk genomic marker, 2p gain. Further research is needed to confirm these findings and evaluate the significance of this abnormality in newly diagnosed and with other targeted therapies.
Competing Interests:
KJM has received research support and has served as a consultant for Pharmacyclics. KAB has received research funding from Pharmacyclics and Janssen. SMJ has received research funding from Pharmacyclics. JAW has served as a consultant for Janssen, Pharmacyclics, Abbvie, AstraZeneca, and Arqule, and has received research funding from Abbvie and Loxo. The remaining authors declare no competing financial interests.
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