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Altered RAS-BRAF-MAPK-ERK pathway in CLL
genicity of the mutations, five mutations in the 3’ untrans- lated region (cases 1, 3, 11, 28 and 30) and one missense mutation (case 4, SOS2 gene) were discarded as not being pathogenic. We were able to demonstrate that the muta- tion in the 3’ untranslated region of KITLG (case 1) was functional as we detected high levels of phosphorylated ERK, a surrogate marker of RAS-BRAF-MAPK-ERK path- way activation (Figure 3A). Due to the absence of cryo- preserved material, we could not analyze the functionality of these mutations in the remaining cases. Therefore, con- sidering only the putative functional mutations, a total of 26 functional mutations affecting genes of the RAS-BRAF- MAPK-ERK pathway were observed in 25 of 452 CLL patients (5.5%). In 11 of the 25 patients (44%) these muta- tions were clonal (VAF ≥0.40) and in the other 14 patients (56%) they were subclonal (VAF <0.40). Mutations were detected in genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) in 12/452 patients (2.6%), in BRAF in 9/452 patients (2.0%), and in genes downstream of BRAF (MAP2K1 alias MEK1, MAP2K2 alias MEK2) in 5/452 patients (1.1%). The most frequent single mutated gene was BRAF (n=9/26, 34.6%) followed by PTPN11 (n=5/26, 19.2%), MAP2K2 (n=3/26, 11.5%), KRAS (n=3/26, 11.5%), and MAP2K1, (2/26 cases, 7.7%); mutations of GNB1, NRAS, KIT, and KITLG were each found in one patient. One patient had concomitant muta- tions of PTPN11 and KRAS. Interestingly, BRAF mutations were localized between exons 11 to 15 and most of them occurred in the activation loop (A-loop) near the V600 position or near the phosphate-binding loop (P-loop) at residues 464-469. Only in one case did the BRAF mutation correspond to V600E, the most common mutation described in a variety of human malignancies including HCL.17
Association of mutations in the RAS-BRAF-MAPK-ERK pathway with clinical and biological features
The main clinical and biological characteristics of the 25 patients with functional mutations in the RAS-BRAF- MAPK-ERK pathway are listed in Table 2.
The age, sex and clinical stage of the patients with mutations in the RAS-BRAF-MAPK-ERK pathway were similar to those of the patients without mutations. However, patients with mutations in RAS-BRAF-MAPK- ERK pathway genes more frequently had abnormal values of lactate dehydrogenase, high expression of ZAP-70, CD38 and CD49d, trisomy 12 and most of them had U- IGHV (21/24, 87%) (P≤0.05 in all comparisons) (Table 2). Patients with mutations in the RAS-BRAF-MAPK-ERK pathway more frequently had three or more driver muta- tions than patients without mutations in the pathway, but no differences were observed in the genes most frequently mutated in CLL (NOTCH1, SF3B1, BIRC3, TP53 or ATM) (Table 2). Six cases contemporaneously carried mutations in TP53, ATM or BIRC3. As most patients with mutations in the RAS-BRAF-MAPK-ERK pathway had U-IGHV, we conducted a similar analysis including only the subgroup of U-IGHV patients. As seen in Table 3, only lactate dehy- drogenase and trisomy 12 maintained statistical signifi- cance. Figure 1 shows a brick-plot of concomitant gene mutations/cytogenetic aberrations for cases with RAS- BRAF-MAPK-ERK pathway mutations.
Patients with mutations in the RAS-BRAF-MAPK-ERK pathway required treatment more frequently, considering both the whole group (88% versus 43%; P<0.001) and
within the U-IGHV subgroup (95% versus 75%; P<0.048). There were no differences in the type of treatment received or the response achieved according to the pres- ence or absence of mutations in the pathway (Table 2). Five-year TTFT of patients with Binet A or B disease was 82% [95% confidence interval (95% CI): 66-98%] in patients with mutations in the RAS-BRAF-MAPK-ERK pathway versus 50% (95% CI: 42-58%) in the unmutated group; P<0.001]. The comparison between clonal and sub- clonal mutated cases showed that the 5-year TTFT was
Table 2. Main clinical and biological characteristics of patients according to mutations in the RAS-BRAF-MAPK-ERK pathway.
Parameter
Gender
Age (years), median (range) Binet stage
Rai stage
Lymphocytes count (x109/L), median (range)
Absolute CLL cell count (x109/L),
Mutated P-value (n=25)
19 (76%) ns
61 (44-84) ns
21 (88%)
1 (4%) ns 2 (8%)
13 (54%)
9 (38%) ns
2 (8%)
11 (1-75) ns
6 (0.7-83) ns
147 (125-159) ns
170 (99-315) ns
7/18 (39%) ns
median (range)
Hemoglobin (g/L), median (range)
Platelets (x109/L),
median (range)
B2 microglobulin Lacate dehydrogenase IGHV
CD49d
CD38
ZAP-70
Genetics
Driver mutations
NOTCH1
SF3B1
TP53
BIRC3
ATM
Treated
Response to treatment*
5-yearTTFT(95%CI)*
5-year OS (95% CI)
5-year t-DLBCL
Category
Male (%)
A B C
0 I-II III-IV
UNV*
UNV*
Unmutated
>30%
Unmutated (n=427)
257 (60%)
61 (18-93)
366 (87%) 47 (11%) 8 (2%)
278 (66%) 130 (31%) 12 (3%)
11 (1-203)
8 (0.4-192)
141 (45-177)
204 (49-791)
119/373 (32%)
26/407 (6%)
145/421 (34%)
92/290 (32%)
96/403 (24%)
98/394 (25%)
148/308(48%) 48/308 (16%) 26/307 (8%) 11/308 (4%)
159/427(37%) 52/427 (12%) 38/427(9%) 21/397 (5%) 38/427 (9%) 47/427 (11%) 22/25 (88%)
102 (55%) 48 (26%) 13 (7%)
50%(42-58)
80% (74-86)
2% (1-3)
6/19 (32%) 21/24 (87%) 9/13 (69%) 10/23 (43%) 14/21 (67%)
0.002 <0.001 0.012 0.046 <0.001
>30%
≥20%
del(13q)(q14.3) Trisomy 12 del(11q)(q22.3) del(17p)(p13.1)
3/13 (23%) ns
≥3 Mutated Mutated Disrupted Disrupted Disrupted 184/427 (43%)
17/25 (68%)
5/25 (20%) ns
1/25 (4%) ns 2/23 (9%) ns 2/25 (8%) ns 3/25 (12%) ns
<0.001
12 (57%)
4 (19%) ns 3 (14%)
11% (0-25)
6/13 (46%)
0/13 (0%) ns 1/13 (8%) ns
0.011
0.003
CR
PR Failure
A&B
All
All
82%(66-98)
78% (60-96) ns
<0.001 0.080
*It was not possible to assess the response to treatment in 21/184 (11%) of the unmutated patients and in 2/21 (9%) of the mutated patients. CLL: chronic lymphocytic leukemia; UNV: above normal value; CR: complete response, PR: partial response, TTFT: time to first treatment; OS: overall survival; 95% CI: 95% confidence interval; t-DLBCL: transformation into diffuse large B-cell lymphoma (Richter syndrome); ns: not significant.
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