Clustering genomic alteration in chromosome 7 in EGFR-activating lung cancer

1. JOURNAL CLUB
Shivali Jasrotia
23 Jan 2011

3. EGFR
Chromosome 7
Exons 1–16
Exon 17
Exons 18–24
Exons 25–28
Extracellular domain
Transmembrane domain
TK domain
Regulatory domain
EGFR transcript EGF protein
Introduction
EGFR

4. Mutations in the EGFR gene found in 10-15% of NSCLC patients
Exon 19 deletions & L858R (Exon 21) make up 85-90% of all mutation
positive NSCLC patients.
Exon 20 mutations (e.g. T790M) commonly resistance mutations.
EGFR mutations
45 %
~5 %
42 %
~5 %

5. Copy number variation (germline, inherited)
• inherited: also present in parents’ genome
• de novo: absent in parents’ genome
Example: 500kb region is deleted from a maternally derived
chromosome, but is present for the paternally derived chromosome
(cn=1)
Copy number alteration (somatic, e.g. in cancer cells)
The variation is from cell-to-cell (somatic changes) within the same
person.
Example: deletion in cancer cells and normal in other tissues of the
same person.
5
Basic Terminology

6. Alteration in DNA Copy Number: possible
mechanism
Molecular Biology of the Cell, Alberts et. al. (4th edition, figure 23-33)

8. Array CGH: Interpretation

9. Real Time qPCR
I. Real-time monitoring of the
amplification reaction.
II. Based on the detection and
quantification of a fluorescent
reporter.
III. Focus is on the first significant
increase in the amount of PCR
product.
IV. The time of the increase
correlates inversely to the initial
amount of DNA template

10. • To characterise the molecular difference between tumours with
EGFR- activating mutations and wild- type tumor at whole
genome level.
• To compare the survival prediction ability of CNA profiles of
genes between the EGFR-activating mutation group and the
wild type group.
• To establish the association of the CNA score with treatment
response.
Purpose of the study
10

11. 1st part: 138 frozen tumor tissues collected b/w 2000 and 2008.
2nd part: 114 consecutive patients with completely resected stage I
to IIIA lung ADC from April 2000 to February 2006, excluding
those receiving preoperative or postoperative chemotherapy and
those with a history of additional malignancy.
3rd part: DNA samples 25 patients; age younger than 70 yrs; with
EGFR mutation and undergoing EGFR-TKI.
Patients and Methods
11
Patients

12. Only lung adenocarcinomas and patients with activating EGFR
mutations (exon-19 deletion and L858R) or wild type were
included for study.
Disease-free survival (DFS) was measured from date of surgery
to date of recurrence or death.
Overall survival (OS) time was measured from date of surgery to
date of death.
Patients and Methods
12

13. Statistical Analysis
1. T test is used to determine the DNA gain or loss status of each probe-
block for each sample separately and then collectively.
2. A block is considered as a gain block (or loss block) if at least 30% of the
138 samples showed gains (or losses).
3. For comparative CNA analysis with respect to EGFR mutation status, the t-
test was applied to compare two group means.
4. Both univariate and multivariate Cox regression models were applied for
prediction of patient survival.
5. Statistical significance and confidence interval for Cox model and log-rank
test for comparing Kaplan-Meier curves were carried out by using R
software.
13

14. Conti…

15. Conti…

17. Results
17
Chromosome 7p Has Highest Rate of DNA Gain in Gene-Harbouring Regions
A total of 3,187 probe blocks of DNA gain and 6,029 probe blocks of DNA loss
were obtained.
Among gene-harbouring regions, chromosome 7p had the highest DNA-gain
rate.
EGFR was on the DNA-gain list, along with other notable genes like HDAC9,
DGKB, MEOX2 and POU6F2.
With regard to the ERBB family, although EGFR and ERBB4 (2q34) had DNA
gains, ERBB2 (17q12) and ERBB3 (12q13.2) had DNA losses.
To confirm the genomic array CGH results, RT- qPCR was performed and
validated the DNA gain/loss pattern for several genes.

20. Results
20
Chromosome 7p Contains Highest Proportion of Most Notable Amplification
for EGFR-Activating Mutation Group
The top 1% of probe blocks with the largest and smallest mean values of CNAs
were obtained.
79 of 363 (21.7%) probe blocks with the highest CNAs fell on chromosome 7p.
The same pattern still occurred for the two EGFR mutation types studied
separately.
However, for the EGFR wild-type group, the pattern was completely different,
and chromosome 7p became insignificant.

22. Results
22
Chromosome 7p Is Most Enriched in Containing Sites of Differential CNAs
Between EGFR-Activating Mutation and Wild-Type Groups
We found that 46.62% of the probes located in chromosome 7p had CNA
differences.
We also examined the size of the difference and found the largest differences to
occur on a segment (869K bps in length) at 7p11.2 harbouring EGFR, LANCL2,
VOPP1, and others. The CNA values in this region were higher for the mutation
group.
We further compared the difference between L858R mutation and wild type and
found that chromosome 7p was still the richest arm in containing sites of
differences in CNA values. We also compared the deletion group with the wild-
type group. Last, we compared the differences between the deletion group and
L858R mutation group.

24. Results
24
Clustered Genomic Alterations in Chromosome 7p Predict Clinical
Outcomes in Lung Adenocarcinoma With EGFR-Activating Mutation
I. The CNA difference between the EGFR-activating mutation and WT
groups was confirmed by t test for each of the six genes.
II. The average of the copy numbers of the six genes was used to predict
patient survival in the EGFR-activating mutation group.
III. KM analysis on overall and disease-free survival indicated the stage effect
only, with no mutation status effect.
IV. Copy number–based risk score (CNA risk score) was able to identify the
high-risk and low-risk patients for both OS and DFS prediction.
Multivariate Cox regression was performed.

26. Conti….

29. Results
29
CNAs of Six Genes Are Associated With Response to EGFR-TKIs
Genomic qPCR to measure the CNAs of the six representative genes in tumor
DNA from the 23 patients who received EGFR-TKI treatment.
I. less favorable response group = SD and PD were compared with
II. favorable response group = PR
The simultaneous presence of four or more genes with CNAs higher than
average was associated with less favorable drug response.

31. Discussion
31
Chromosome 7p as the main chromosome arm enriched in notable sites of DNA
copy number alterations for lung ADC.
6 qPCR-validated genes from chromosome 7p yield a copy number–based risk
score, which is an effective predictor for both overall and disease-free survival in
patients with EGFR-activating mutation, independent of cancer stage.
Yet for the EGFR wild-type patients, we also showed that the same signature is
uncorrelated with both overall and disease-free survival.
It is insufficient to use only the EGFR-activating mutation as the marker in
predicting EGFR-TKI targeted treatment response. The genetic alterations
clustered in the chromosome 7p region that we identified may also play a crucial
role, and the risk score derived from these genetic alterations may determine
whether a patient will have a favorable response to EGFR-TKI therapy.

32. Discussion
32
The six representative genes from chromosome 7p CNA aberration sites have
important biologic functions.
SDK1 (sidekick homolog1, cell adhesion molecule)
GLI3 (glioma-associated oncogene family zinc finger 3) are best known for
their roles in development.
NFE2L3 (nuclear factor [erythroid-derived 2] -like 3) is a member of the cap-
and-collar family of basic-leucine zipper transcription factors that can bind the
antioxidant response element.
LANCL2 has reduced P-glycoprotein expression and increased sensitivity
to doxorubicin.
VOPP1 is co amplified with EGFR and over expressed in cancer.
EGFR (Epidermal Growth Factor Receptor)

33. Conclusion
33
Chromosome 7p contains rich genomic information that can shed more light on
the complex disease of lung ADC.
These findings provide clues to why patients with EGFR-activating mutation
may still have heterogeneous response to EGFR-TKI targeted therapy.
They may be useful for clinicians to make better predictions about treatment
response.
The detailed characterization of this chromosome arm using techniques like
next-generation sequencing would be rewarding.

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