Characteristics of homologous recombination …...HRD ANALYSIS All tumors were analyzed using the...
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Figure 1. Correlation between HRD Scores in Primary and Recurrent Tumor Samples RESULTS Characteristics of homologous recombination deficiency (HRD) in paired primary and recurrent high-grade serous ovarian cancer (HGSOC) J. Patel 1 , J. Sehouli 2 , K. Timms 3 , C. Solimeno 3 , J. Reid 3 , J. Lanchbury 3 , I. Braicu 2 , S. DarbEsfahani 2 , M. Ganapathi 1 , R. Ganapathi 1 1. Department of Cancer Pharmacology, Levine Cancer Institute, Charlotte, NC, US 2. Department of Gynecology, Charité / University Hospital Berlin, Berlin, DE 3. Myriad Genetics Inc, Salt Lake City, UT, US METHODS COHORT ● Paired primary and recurrent HGSOC samples were obtained for 54 subjects. The majority of patients received platinum based therapy. ● A total of 116 samples were obtained. Samples from 2 individuals were excluded due to uncertainty regarding the tumor histology. HRD ANALYSIS ● All tumors were analyzed using the HRD assay, a next generation sequencing assay performed using DNA extracted from FFPE tumor tissue. 8 ● The HRD score is an algorithmic assessment of TAI (regions of allelic imbalance that extend to the subtelomere but do not cross the centromere), 1 LOH (number of LOH regions >15 Mb but less than the length of a whole chromosome), 2 and LST (breakpoints between regions of imbalance >10Mb after filtering out regions <3 Mb). 3 ● All tumor samples were also tested for BRCA1/2 mutations. ● An HRD threshold of 42 has been previously developed to detect 95% of tumors with BRCA1/2 mutations. 7 ● Tumor samples are categorized as having a high (≥ 42) or low (< 42) HRD score. CONCLUSIONS ● All markers of HRD examined, including mutations in BRCA1 and BRCA2, LOH in BRCA1 and BRCA2, and HRD score, were maintained between the primary and recurrent specimens. ● There was one possible reversion mutation in a recurrent sample; however, both the primary and recurrent tumors had high HRD scores and would be classified as HR deficient. ● High HRD scores (≥42) were observed in all specimens with mutations in BRCA1 or BRCA2; however, high HRD scores were more prevalent than mutations in BRCA1 or BRCA2 (60% versus 34%, respectively). ● These results suggest that characterization of HRD in primary or recurrent HGSOC specimens may be beneficial in selecting treatment strategies. BACKGROUND ● Defects in the homologous recombination (HR) pathway occur in up to 50% of epithelial ovarian tumors 1 and have a major impact on treatment response to DNA damaging agents. ● A homologous recombination deficiency (HRD) assay has been developed to assess 3 metrics of genomic instability: TAI (telomeric allelic imbalances) 2 , LOH (loss of heterozygosity) 3 , and LST (large-scale state transitions). 4 ● Previous studies have shown that the HRD score is significantly correlated with progression-free and overall survival in ovarian cancer 1,5 and predicts response to DNA-damaging agents in breast cancer. 6,7 ● Previous studies have been based on the HRD score in the primary, diagnostic tumor specimen. REFERENCES 1. Cancer Genome Atlas Network. Nature. 2011;474, 609. 2. Birkbak NJ, et al. Cancer Discov. 2012;2:366. 3. Abkevich V, et al. Br J Cancer. 2012;107:1776. 4. Popova T, et al. Cancer Res. 2012;72:5454. 5. Hennessy BT, et al. J Clin Oncol . 2010;28, 3570. 6. Isakoff SJ, et al. J Clin Oncol. 2015;33:1902. 7. Telli ML, et al. Clin Cancer Res. 2016;22:3764. 8. Timms KM, et al. Breast Cancer Res. 2014;16:475. Figure 3. Genomic Profiles of Primary and Recurrent Tumor Samples. Examples are shown for tumors with similar (Left) and very divergent (Right) primary and recurrent pairs. The yellow line indicates regions of LOH (allele dosage of 0) and non-LOH (allele dosage of 1). Areas of divergence (red arrows) and similarity (green arrows) are indicated. HRD scores are at the bottom right of each profile. 0 0.5 1 0 0.5 1 55 68 Allele Dosage 0 0.5 1 0 0.5 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 35 35 Allele Dosage Chromosome Location 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Chromosome Location KEY FINDINGS ● 17/52 (33%) primary-recurrent pairs had a mutation in BRCA1 (n=14) or BRCA2 (n=3) (Table 1). ● 10/38 (26%) primary-recurrent pairs had high HRD scores with intact BRCA1/2 (Figure 1). ● HRD scores between primary and recurrent specimens were highly correlated (ρ = 0.88) (Figure 1), despite evidence of additional acquired genomic rearrangements (Figure 3). ● HRD scores in specimens with BRCA1/2 mutations were higher in the recurrent than in the primary sample (p=0.064). ● A possible reversion mutation in BRCA1 was observed in one recurrent specimen (Figure 2). – The original mutation is a 1 bp insertion that puts the transcript out of frame, resulting in a premature truncation of the transcript. – The possible reversion mutation results in 3 amino acid changes (Gln→Pro, Asn→Gly, Arg→Gln), but puts the transcript back in frame. – Both the primary and recurrent specimens had high HRD scores. – All other primary-recurrent pairs had the same mutations. ● LOH at BRCA1 or BRCA2 was observed in all mutated tumors. All mutated pairs had high HRD scores. For additional information, please contact Jai Patel at [email protected] Figure 2. Possible BRCA1 Reversion Mutation Table 1. Tumor Characteristics Specimens Primary- Recurrent Pairs Total tumor specimens 104 52 Evaluable HRD scores 85% (88/104) 73% (38/52) High HRD score (≥ 42) 59% (52/88) 66% (25/38) a BRCA1 mutation 27% (28/104) 27% (14/52) a BRCA1 LOH – 100% (10/10) b BRCA2 mutation 6% (6/104) 6% (3/52) BRCA2 LOH – 100% (3/3) a Positive in either the primary and/or recurrent specimen b LOH could not be determined in both samples of 4 pairs 5385insC (Primary Tumor) Wild Type C A G G A C A G A A A G G T A C C A G G A C A G A A G G T T A c.5276_5277+1delinsGGT (Recurrent Tumor) In-Frame Splice Donor Junction C C A G G A C A G A A A G G T A OBJECTIVE ● Here we assess HRD in paired primary and recurrent HGSOC specimens to better understand possible changes in HRD during tumor recurrence. 0 20 40 60 80 100 0 20 40 60 80 100 HRD score in primary HRD score in recurrent BRCA1/2 wild type BRCA1 mutant BRCA1 reversion BRCA2 mutant Recurrent − primary score Median (IQR): BRCA1/2 wild type 0.0 (−5.0, 4.0) BRCA1/2 mutant 5.0 (2.0, 9.0) p−value = 0.064 HRD Low 2.0 (−3.0, 6.0) HRD High 2.0 (−1.5, 8.5) p−value = 0.65 HRD Low HRD High Confidential - Do Not Distribute
Transcript of Characteristics of homologous recombination …...HRD ANALYSIS All tumors were analyzed using the...
Figure 1. Correlation between HRD Scores in Primary and Recurrent Tumor Samples
RESULTS
Characteristics of homologous recombination deficiency (HRD) in paired primary and recurrent high-grade serous ovarian cancer (HGSOC)
J. Patel1, J. Sehouli2, K. Timms3, C. Solimeno3, J. Reid3, J. Lanchbury3, I. Braicu2, S. DarbEsfahani2, M. Ganapathi1, R. Ganapathi1
1. Department of Cancer Pharmacology, Levine Cancer Institute, Charlotte, NC, US 2. Department of Gynecology, Charité / University Hospital Berlin, Berlin, DE 3. Myriad Genetics Inc, Salt Lake City, UT, US
METHODSCOHORT● Paired primary and recurrent HGSOC samples were obtained for 54 subjects.
The majority of patients received platinum based therapy.
● A total of 116 samples were obtained. Samples from 2 individuals were excludeddue to uncertainty regarding the tumor histology.
HRD ANALYSIS● All tumors were analyzed using the HRD assay, a next generation sequencing
assay performed using DNA extracted from FFPE tumor tissue.8
● The HRD score is an algorithmic assessment of TAI (regions of allelic imbalancethat extend to the subtelomere but do not cross the centromere),1 LOH (numberof LOH regions >15 Mb but less than the length of a whole chromosome),2 andLST (breakpoints between regions of imbalance >10Mb after filtering out regions<3 Mb).3
● All tumor samples were also tested for BRCA1/2 mutations.
● An HRD threshold of 42 has been previously developed to detect 95% of tumorswith BRCA1/2 mutations.7
● Tumor samples are categorized as having a high (≥ 42) or low (< 42) HRD score.
CONCLUSIONS● All markers of HRD examined, including mutations in BRCA1
and BRCA2, LOH in BRCA1 and BRCA2, and HRD score, weremaintained between the primary and recurrent specimens.
● There was one possible reversion mutation in a recurrent sample;however, both the primary and recurrent tumors had high HRD scoresand would be classified as HR deficient.
● High HRD scores (≥42) were observed in all specimens with mutationsin BRCA1 or BRCA2; however, high HRD scores were more prevalentthan mutations in BRCA1 or BRCA2 (60% versus 34%, respectively).
● These results suggest that characterization of HRD in primary orrecurrent HGSOC specimens may be beneficial in selecting treatmentstrategies.
BACKGROUND● Defects in the homologous recombination (HR) pathway occur in up to 50% of
epithelial ovarian tumors1 and have a major impact on treatment response toDNA damaging agents.
● A homologous recombination deficiency (HRD) assay has been developed toassess 3 metrics of genomic instability: TAI (telomeric allelic imbalances)2, LOH(loss of heterozygosity)3, and LST (large-scale state transitions).4
● Previous studies have shown that the HRD score is significantly correlated withprogression-free and overall survival in ovarian cancer1,5 and predicts responseto DNA-damaging agents in breast cancer.6,7
● Previous studies have been based on the HRD score in the primary, diagnostictumor specimen.
REFERENCES1. Cancer Genome Atlas Network. Nature.
2011;474, 609.2. Birkbak NJ, et al. Cancer Discov. 2012;2:366.3. Abkevich V, et al. Br J Cancer. 2012;107:1776.4. Popova T, et al. Cancer Res. 2012;72:5454.
5. Hennessy BT, et al. J Clin Oncol. 2010;28, 3570.6. Isakoff SJ, et al. J Clin Oncol. 2015;33:1902.7. Telli ML, et al. Clin Cancer Res. 2016;22:3764.8. Timms KM, et al. Breast Cancer Res.
2014;16:475.
Figure 3. Genomic Profiles of Primary and Recurrent Tumor Samples.
Examples are shown for tumors with similar (Left) and very divergent (Right) primary and recurrent pairs. The yellow line indicates regions of LOH (allele dosage of 0) and non-LOH (allele dosage of 1). Areas of divergence (red arrows) and similarity (green arrows) are indicated. HRD scores are at the bottom right of each profile.
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Chromosome Location1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23Chromosome Location
KEY FINDINGS● 17/52 (33%) primary-recurrent pairs had a mutation in BRCA1 (n=14)
or BRCA2 (n=3) (Table 1).
● 10/38 (26%) primary-recurrent pairs had high HRD scores with intactBRCA1/2 (Figure 1).
● HRD scores between primary and recurrent specimens were highlycorrelated (ρ = 0.88) (Figure 1), despite evidence of additionalacquired genomic rearrangements (Figure 3).
● HRD scores in specimens with BRCA1/2 mutations were higher in therecurrent than in the primary sample (p=0.064).
● A possible reversion mutation in BRCA1 was observed in onerecurrent specimen (Figure 2).
– The original mutation is a 1 bp insertion that puts the transcript out offrame, resulting in a premature truncation of the transcript.
– The possible reversion mutation results in 3 amino acid changes (Gln→Pro, Asn→Gly, Arg→Gln), but puts the transcript back in frame.
– Both the primary and recurrent specimens had high HRD scores.
– All other primary-recurrent pairs had the same mutations.
● LOH at BRCA1 or BRCA2 was observed in all mutated tumors. Allmutated pairs had high HRD scores.
For additional information, please contact Jai Patel at [email protected]
Figure 2. Possible BRCA1 Reversion Mutation
Table 1. Tumor Characteristics
Specimens Primary-Recurrent Pairs
Total tumor specimens 104 52Evaluable HRD scores 85% (88/104) 73% (38/52)High HRD score (≥ 42) 59% (52/88) 66% (25/38)a
BRCA1 mutation 27% (28/104) 27% (14/52)a
BRCA1 LOH – 100% (10/10)b
BRCA2 mutation 6% (6/104) 6% (3/52)BRCA2 LOH – 100% (3/3)
a Positive in either the primary and/or recurrent specimenb LOH could not be determined in both samples of 4 pairs
5385insC(Primary Tumor)
Wild Type C A G G A C A G A A A G G T A
C C A G G A C A G A A G G T T Ac.5276_5277+1delinsGGT(Recurrent Tumor)
In-Frame Splice Donor Junction
C C A G G A C A G A A A G G T A
OBJECTIVE● Here we assess HRD in paired primary and recurrent HGSOC specimens to
better understand possible changes in HRD during tumor recurrence.
0 20 40 60 80 1000
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D s
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BRCA1/2 wild typeBRCA1 mutantBRCA1 reversionBRCA2 mutant
Recurrent − primary scoreMedian (IQR):BRCA1/2 wild type 0.0 (−5.0, 4.0)BRCA1/2 mutant 5.0 (2.0, 9.0) p−value = 0.064HRD Low 2.0 (−3.0, 6.0)HRD High 2.0 (−1.5, 8.5) p−value = 0.65
HRD Low HRD High
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