The genetic basis of early T-cell precursor acute lymphoblastic leukaemia
Jinghui ZhangLi DingLinda HolmfeldtGang WuSusan L. HeatleyDebbie Payne-TurnerJohn EastonXiang ChenJianmin WangMichael RuschCharles LuShann-Ching ChenLei WeiJ. Racquel Collins-UnderwoodJing MaKathryn G. RobertsStanley PoundsAnatoly UlyanovJared BecksfortPankaj GuptaRobert HuetherRichard W. KriwackiMatthew ParkerDaniel McGoldrickDavid ZhaoDaniel P. AlfordStephen EspyKiran Chand BobbaGuangchun SongDeqing PeiCheng ChengStefan RobertsMichael I. BarbatoDario CampanaElaine Coustan‐SmithSheila ShurtleffSusana C. RaimondiMaria KleppeJan CoolsKristin A. ShimanoMichelle L. HermistonSergei DoulatovKolja EppertElisa LaurentiFaiyaz NottaJohn E. DickGiuseppe BassoStephen P. HungerMignon L. LohMeenakshi DevidasBrent L. WoodStuart S. WinterKimberly P. DunsmoreRobert S. FultonLucinda FultonXin HongChristopher HarrisDavid J. DoolingKerri OchoaKimberly JohnsonJohn C. ObenauerWilliam E. EvansChing‐Hon PuiClayton W. NaeveTimothy J. LeyElaine R. MardisRichard K. WilsonJames R. DowningCharles G. Mullighan
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Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL. This work shows that treatments used for acute myeloid leukaemia and targeted therapies could be used for early T-cell precursor acute lymphoblastic leukaemia. The early T-cell precursor (ETP) subtype of childhood acute lymphoblastic leukaemia (ALL) has a poor prognosis when treated with standard chemotherapy. Whole genome sequencing is used here to gain insights into the genetic basis of the condition. The results reveal a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling, lesions that disrupt haemopoiesis (many of which arise from chromosomal rearrangements that generate novel chimeric in-frame fusion genes), and inactivating mutations in histone modifying genes. This mutation pattern resembles that of myeloid malignancies, suggesting that myeloid-directed therapies such as high-dose cytarabine, or targeted therapies that inhibit cytokine receptor and JAK signalling, might be effective in ETP ALL.Keywords:
RUNX1
ETV6
Abstract Background A constitutively active RAS protein in the absence of stimulation of the epidermal growth factor receptor (EGFR) is the result of mutations in KRAS and NRAS genes. Mutations in the KRAS exon 2 and outside exon 2 have been found to predict the resistance to anti-EGFR monoclonal therapy. A substantial proportion of metastatic colorectal cancer cases (mCRC) exhibit RAS mutations outside KRAS exon 2, particularly in KRAS exon 3 and 4 and NRAS exons 2, 3. No data about RAS mutations outside KRAS exon 2 are available for Jordanian patients with mCRC. We aim to study the molecular spectrum, frequency, and distribution pattern of KRAS and NRAS mutations in Jordanian patients with mCRC. Methods A cohort of 190 Jordanian metastatic colorectal cancer patients were enrolled in the trial. We detected mutations in exon 2 of the KRAS and NRAS gene as well as mutations outside of exon 2 using the StripAssay technique. The KRAS StripAssay covered 29 mutations and 22 NRAS mutations. Results Mutations were observed in 92 (48.42%) cases, and KRAS exon 2 accounted for 76 cases (83.69%). KRAS G12D was the most common mutation, occurring in 18 cases, followed by KRAS G12A in 16 cases, and G12T in 13 cases. Mutations outside of KRAS exon 2 represented 16.3% of the mutated cases. Among those, 6 cases (6.48%) carried mutations in NRAS exon 2, 3 and 10 cases (10.87%) in KRAS exon 3 and 4. Conclusion The frequency of NRAS and KRAS mutations outside of exon 2 appears to be higher in Jordanian patients in comparison with patients from western countries. KRAS mutations outside of exon 2 should be tested routinely to identify patients who should not be treated with anti-EGFR antibodies.
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Determination of the mutations' status in the KRAS and NRAS genes is a necessary requirement in the treatment of patients with colorectal cancer (CRC). Patients with certain mutations in the KRAS and NRAS genes are resistant to anti-EGFR drug therapy and have a lower median survival rate than those with WT (wild type) genotypes, that indicates a negative prognosis in the case when mutations are present. Currently, there are no registered targeted drugs for carriers of the KRAS and NRAS genes mutations, however, preparations based on small molecules are under way. The gold standard for detecting mutations in the KRAS and NRAS genes is the analysis of the biopsy material in paraffin blocks. However, this method has significant limitations that can be circumvented by the analysis of circulating tumor DNA a promising new method in the diagnosis of colorectal cancer.
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A constitutively active RAS protein in the absence of stimulation of the epidermal growth factor receptor (EGFR) is the result of mutations in KRAS and NRAS genes. Mutations in the KRAS exon 2 and outside exon 2 have been found to predict the resistance to anti-EGFR monoclonal therapy. A substantial proportion of metastatic colorectal cancer cases (mCRC) exhibit RAS mutations outside KRAS exon 2, particularly in KRAS exon 3 and 4 and NRAS exons 2 and 3. No data about RAS mutations outside KRAS exon 2 are available for Jordanian patients with mCRC. We aim to study the molecular spectrum, frequency, and distribution pattern of KRAS and NRAS mutations in Jordanian patients with mCRC.A cohort of 190 Jordanian metastatic colorectal cancer patients were enrolled in the trial. We detected mutations in exon 2 of the KRAS and NRAS gene as well as mutations outside of exon 2 using the StripAssay technique. The KRAS StripAssay covered 29 mutations and 22 NRAS mutations.Mutations were observed in 92 (48.42%) cases, and KRAS exon 2 mutations accounted for 76 cases (83.69%). KRAS G12D was the most common mutation, occurring in 18 cases, followed by KRAS G12A in 16 cases, and G12T in 13 cases. Mutations outside of KRAS exon 2 represented 16.3% of the mutated cases. Among those, 6 cases (6.48%) carried mutations in NRAS exon 2 and 3, and 10 cases (10.87%) in KRAS exon 3 and 4.The frequency of NRAS and KRAS mutations outside of exon 2 appears to be higher in Jordanian patients in comparison with patients from western countries. KRAS mutations outside of exon 2 should be tested routinely to identify patients who should not be treated with anti-EGFR antibodies.
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The epidermal growth factor receptor (EGFR) is an excellent candidate for targeted therapy in colorectal cancer. Recent studies have demonstrated that apart from wild-type KRAS, a wild-type BRAF and NRAS genotype is required for response to anti-EGFR therapy. This suggests that NRAS and BRAF genotype criteria should be used together with KRAS genotype to select patients who will likely benefit from anti-EGFR therapy. We investigated the prevalence of mutations in the KRAS, BRAF and NRAS genes and its correlation with demographic characteristics, tumor location and stage in 100 colorectal carcinoma patients from India. The frequency of KRAS, BRAF and NRAS mutations was found to be 23%, 17% and 2.0%, respectively. There was no significant difference in KRAS, NRAS and BRAF mutation with respect to gender, age, tumor location (colon vs rectum) and clinicopathological stage. In addition, we found a novel point variant (T20I) of unknown significance in NRAS exon 1 in addition to a KRAS codon 12 mutation in one of the rectal carcinoma patients. In the present study, combined evaluation of genetic biomarkers (KRAS, NRAS and BRAF) was able to classify 42% of colorectal cancer patients as likely non-responders to anti-EGFR therapy.
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e13115 Background: Mutations in Kras and Nras genes in primary colorectal tumors are the reasons for nonresponse to anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). Results of the molecular analysis guides the treatment decision-making in mCRC. The aim of this study was to evaluate the Kras and Nras mutation data from 720 patients with mCRC between 01 January 2015 and 31 January 2016 using minisequencing method. Methods: We performed a two round nested PCR for the amplification of Exons 1, 2, 3 of Kras and Nras genes harbouring codons 12, 13, 59, 61, 117, and 146 followed by a multiplex minisequencing reaction for the detection of potential mutations. Results: A total of 720 patients were analysed for the mutations in codons 12, 13, 59, 61, 117, and 146 of Kras and Nras genes. Results were obtained from 718 out of 720 (99,72%) patients and no results were obtained from 2 (0,28%) of the cases. 400/718 (55,71%) patients were wild-type for Kras mutations while 318 of them carried a Kras mutation (44,29%). 8 of 318 (2,52%) patients carried two mutations which made totally 326 Kras mutations. Kras mutation rates were 192/326 (58,90%), 98/326 (30,06%), 3/326 (0,92%), 8/326 (2,45%), 3/326 (0,92%), 22/326 (6,75%) in codons 12, 13, 59, 61, 117, 146 respectively. Additional Nras mutation testing was performed for 400 patients which were found to be wild-type for Kras mutations. 31/400 (7,75%) patients were found to carry Nras mutations while 369 (92,25%) of them were wild-type for Nras mutations. 1 of 31 (3,23%) patients carried two mutations which made totally 32 Nras mutations. Mutation distribution was 10/32 (31,25%), 4/32 (12,50%), 4/32 (12,50%), 11/32 (34,38%), 2/32 (6,25%), 1/32 (3,13%) in codons 12, 13, 59, 61, 117, 146 respectively. Conclusions: High rate (349/718, 48,61%) of mutation finding in Kras and Nras testing makes mutation profiling in Kras and Nras genes an effective approach to select patients who are more likely to respond to anti-EGFR immunotherapy or rule out this treatment for those who are not likely to respond, and predict patient outcomes using minisequencing reaction which is a highly sensitive and cost effective method.
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3589 Background: Mutations (mts) in RAS predict lack of response to anti-EGFR therapy in colorectal cancer. Outside the “typical RAS” mts ( KRAS/NRAS Codons 12, 13, 59, 61, 117, 146) cited in guidelines and anti-EGFR labeling, clinical impact of other “atypical RAS” mts is uncertain. Methods: Available literature and databases were surveyed to identify 80 KRAS/NRAS mts. We used the NovellusDx Functional Annotation for Cancer Treatment (FACT) to transfect these RAS mts (repeated a mean of 5.5 times/mt) in a cell-based assay that quantifies nuclear ERK localization as a measure of MAPK pathway activation, and normalized to wild type (WT) transfection. In 963 metastatic colorectal cancer patients (pts) with BRAF WT/ KRAS mutant tumors, overall survival (OS) was evaluated by level of RAS signaling activity. Results: Of the surveyed mutations,96% (45/47) of typical mts and 39% (13/33) of atypical mts increased MAPK pathway activation above WT (range: 107%-211% of WT activity). Within the typical RAS mts, mts in NRAS or exon 3, 4 of KRAS had higher activity than mts in exon 2 (codons 12/13) of KRAS, reaffirming the biologic relevance of expanded RAS testing (median activity of 130% vs 178%, P < 0.001). The median activity of atypical RAS mts was lower than typical RAS mts (110% vs 159%, P < 0.001). Several notable exceptions in atypical RAS mts with high activity levels were KRAS V14I, Q22K, D33E, N116S, and F156L (all > 165% of WT activity). Conversely, within the typical RAS mts in the guidelines, KRAS G13C and K117R were not shown to increase activity significantly above WT. Pts with any RAS mt with MAPK activity above the median of typical mts had a worse OS compared to pts below the median in univariate (HR 1.45, 95% CI 1.04-2.32, P = 0.033) and multivariate models (HR 1.96, 95% CI 1.13-3.42, P = 0.017) that controlled for age, gender, sidedness, and synchronous vs metachronous presentation. Conclusions: Functional characterization confirmed activity of RAS mts in the current guidelines, but also suggested that a subset of atypical RAS mutations have similar levels of activation of the MAPK pathway. Within the subset of pts with RAS mts, those mts resulting in high MAPK activity are associated with notably shorter OS.
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3617 Background: An exploratory biomarker analysis of the randomized, phase 3 monotherapy 20020408 study of pmab vs best supportive care (BSC) demonstrated that mutations in KRAS exon 3 and NRAS exons 2 and 3 appeared to be predictive of pmab response (Peeters et al, 2013). We expanded these results to determine whether mutations in exon 4 of the KRAS and NRAS genes are predictive for pmab treatment and to determine the treatment effect in the overall wild-type (WT) KRAS and NRAS population. Methods: Using a combination of Next Generation Sequencing, Sanger Sequencing, and WAVE-based SURVEYOR Scan Kits from Transgenomic, archival patient tumors were examined for mutations in KRAS and NRAS exon 4. These data were combined with previously presented data from KRAS and NRAS exon 2 and 3 analyses for evaluation of the comprehensive WT KRAS and NRAS subgroup. Results: 9/243 (3.7%) and 2/243 (0.8%) patient tumors with WT KRAS exon 2 status harbored a mutation in KRAS or NRAS exon 4, respectively. One tumor had mutations in both KRAS and NRAS exon 4. In the pmab arm, patients with WT KRAS and WT NRAS tumor status had an objective response rate (ORR) of 15% (11/72) whereas patients with mutant (MT) KRAS or MT NRAS tumor status had an ORR of 1% (1/95; 1 patient with MT KRAS exon 4 had a partial response). There were no responses in the BSC arm regardless of the tumor status. In this analysis set, the treatment hazard ratio (HR; pmab:BSC) for progression-free survival (PFS) in the WT KRAS and WT NRAS subgroup was 0.38 (95% CI: 0.27 - 0.56), and in the MT KRAS or MT NRAS subgroup was 0.98 (95% CI: 0.73 - 1.31). The original WT KRAS exon 2 subgroup PFS HR was 0.45 (95% CI: 0.34 - 0.59) (Amado et al, 2007). Conclusions: This exploratory analysis suggests that mutations in KRAS and NRAS exon 4 occur in a small, but meaningful percentage of patients with mCRC. Extending previous findings from this study in patients with MT KRAS and/or MT NRAS exon 2 and/or 3 tumors, patients with MT KRAS and/or MT NRAS exon 4 tumors do not appear to benefit from pmab therapy. Clinical trial information: NCT00113763.
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