Targeting Homologous Recombination Repair in Cancer - DNA Repair in Cancer Therapy - page 146

g -H2AX Foci as Predictive Biomarker
There has been increasing interest in using the levels
of residual g -H2AX foci, i.e., typically ~24 hours after
genotoxic treatment, as a surrogate endpoint of DSB
repair efficiency in a tumor (reviewed in 388 e 391 ). Such
an assessment would not be specific for HRR as defects
in other repair pathways may also lead to increased
levels of foci after treatment with, for example, IR or
cisplatin. There is strong preclinical evidence to suggest
that the levels of residual g -H2AX foci indeed correlate
with loss of clonogenicity after genotoxic treatments. 394
However, measuring g -H2AX foci in post-treatment
biopsies in-vivo can be difficult due to tumor intra- and
inter-heterogeneity, similar to the technical challenges
encountered with HRR protein foci. 388,401,402 Another
limitation of using residual

HRR rescue is probably less of a problem if the HRR tar-
geted agent is given in conjunction with IR for two prin-
cipal reasons. First, even if drug resistance arises during
the course of treatment, the initial enhancement of the
efficacy of IR leading to increased tumor cell kill might
be enough to ensure elimination of the last tumor stem
cell on the final day of radiation. Second, because there
is no absolute radiation resistance and IR kills cells
largely indiscriminately, any emerging drug-resistant
cells may get promptly eliminated by IR during the
course of treatment.
Three instructive studies published in 2008 illustrate
the reversal of HRR gene mutations in response to treat-
ment. Two of these reports analyzed reversals of a BRCA2
frameshift mutation in the CAPAN-1 pancreatic cell line
in response to PARP inhibition or cisplatin treat-
ment. 406,407 All drug-resistant clones demonstrated at
least partial restoration of the wild-type allele through
deletions or insertions affecting the inactivating BRCA2
mutation. A deletion mutation in a cisplatin-resistant
breast cancer cell line also restored BRCA2 function by
expressing a truncated protein. 407 Interestingly, in 50%
of the cisplatin-resistant clones there was no restoration
of the BRCA2 gene, suggesting the existence of alterna-
tive mechanisms. This picture bears some resemblance
to the situation inNSCLCwithmutant EGFRwhere treat-
ment with an EGFR-targeted kinase inhibitor is associ-
ated with drug resistance via a secondary kinase
mutation in about 50% of cases, while in many of the
other cases tumors activated alternative signaling to
bypass the effects of the inhibitor. 408 Significantly, the
Taniguchi laboratory also showed that cisplatin resis-
tance in patients with ovarian cancer was due to the
reversal of BRCA1 or BRCA2 mutations, 407,409 demon-
strating the utility of preclinical cell-line-based resistance
models, again similar to the study of drug resistance
mechanisms in NSCLC. With regard to the reversal of
epigenetic mechanisms, the D'Andrea laboratory identi-
fied promoter hypermethylation of FANCF as the reason
behind the cisplatin sensitivity of ovarian tumor cell lines
and suggested that demethylationmay lead to drug resis-
tance. 162 However, whether this phenomenon also exists
in patients has not been convincingly demonstrated.
These studies stress the need not only for genotyping
and phenotyping of individual tumors prior to therapy
but also for a rebiopsy of any recurrent cancers in order
to identify resistance mechanisms and guide salvage
therapy. Acquired resistance to molecular targeted or
genotoxic anticancer drugs is an ubiquitous phenom-
enon and will ultimately require rational combination
therapies to overcome resistance and thereby effect
long-term survival or cures. Clinical trials with HRR tar-
geted agents should thus include correlative science
directed at biomarker discovery or validation and allow
for repeat biopsy procedures.

-H2AX foci as a surrogate
of cell survival is that cells must progress into S-phase.
Because the majority of cells in solid tumors are non-
cycling, cells must be given sufficient opportunity to
proliferate following damage induction. 394 Additional
problems include: (1) high baseline levels in some
untreated tumors; 403 (2) staining of apoptotic cells by
g -H2AX, 404 and (3) determining a common threshold
of foci numbers that signal abnormally high residual
DSB levels across individual tumors may be arbitrary.
Staining for g -H2AX in biopsies treated ex vivo , analo-
gous to the approach described above for HRR proteins,
may be useful for providing a better control of the inten-
sity of the damage signal and thus facilitate comparison
between tumors. Some of the challenges of heteroge-
neity and problems associated with multiple biopsies
could be potentially overcome by assaying g -H2AX
foci in circulating tumor cells. 405 With regard to the
study of HRR function in tumors, a parallel assessment
of g -H2AX foci or other DSB markers such as 53BP1
would also be useful for documenting that DNA
damage has been introduced in the first place, so that
a false negative lack of HRR protein foci induction can
be excluded.

g

Treatment Resistance Mechanisms
One of the biggest challenges of effective cancer treat-
ment is the development of tumor resistance to therapy
and progression as a consequence. In order to overcome
this problem, a thorough understanding of the mecha-
nisms underlying treatment resistance on a molecular
and cellular level is needed. There is a plethora of
ways how an initially therapy-sensitive tumor may
evade genotoxic treatment. With regard to the restora-
tion of HRR in an initially HRR-defective tumor, these
possibilities include reversal of the original genetic or
epigenetic defect or emergence of a compensatory repair
or tolerance mechanism. Resistance development due to