HC7: Clinical relevance of genetic repair mechanisms

Introduction

BRCA and Lynch syndrome occur very frequently in the population. Both syndromes are caused by DNA repair genes.

Even though germline mutations are equally distributed through the genome, hereditary cancer cases associate with syndromes caused by mutations in DNA repair genes. On the other hand, cancers related to inherited APC-mutations are also relatively frequent.

Damage and clinical prognosis

There is an association between DNA repair defects and clinical prognosis. Lung cancers have much more mutations than breast cancers. However, if there are more mutations, a tumor doesn’t necessarily have a worse prognosis. There are several reasons for this:

• There can be too much DNA damage in cancer cells → essential cancer genes are hit → the cell dies due to excessive damage
• Point-mutations are not the only alterations that occur in a cancer cell → tumors with very few mutations may have very abundant chromosomal mutations

Colorectal cancer genetics and prognosis:

Colorectal cancer can develop in several different ways:

• A minority develops under very specific defects in the DNA repair machine
• A small proportion (less than 5%) develops under deficiencies in the proofreading domain of polymerase chains → accumulate to a lot of mutations in their coding gene
• 15-20% develops under the context of mismatch repair deficiency
• A large majority has a low number of mutations in their genome but a large chromosomal instability

Even though MMR-deficient (mismatch repair deficient) or POLE-mutated (polymerase mutated) tumors have more mutations than MMR-proficient tumors, they have a better prognosis up to stage 3. In stage 4, the prognosis is equally bad for all tumors. This means that more mutations actually result in a better prognosis. This can be explained by:

• There being too much damage → affects the tumor cells
• Chromosomal instability leading to a worse prognosis than mutations

MMR-deficient and POLE-mutated tumors have a lot of lymphatic infiltrates in their tumors.

Endometrial cancer and prognosis:

In endometrial cancer, MMR-deficient and POLE-mutated tumors are also present. Here, POLE-mutated patients do better than patients who are MSS (microsatellite stable) or MSI (microsatellite instable). MSI-patients are MMR-deficient and MSS-patients are MMR-proficient. POLE-mutated and MSI patients have more lymphocyte infiltrates, which mainly consist of CD8 T-cells, than MSS-patients.

BRCA status and breast cancer prognosis:

Approximately 10% of all breast cancers carry defects in the BRCA genes, of which half are due to germline mutations. There has been a long debate about whether there is a relation between prognosis in breast cancer and the status of BRCA mutations. Recently, a big study has shown that there is no difference between BRCA-positive and BRCA-negative patients.

Arguments supporting an association between BRCA1 mutations and worse prognosis are:

• 70-90% of BRCA1 related tumors are triple-negative
  • Don’t express estrogen-receptors or HER2-receptors

Arguments against an association between BRCA1 mutations and a worse prognosis are:

• BRCA-related cancers are detected at earlier stages
• If looking at triple-negative tumors, BRCA mutations are associated with a slightly better prognosis
  • More mutations
  • More inflamed profile → the immune system may be involved

In conclusion, more studies need to be conducted to confirm the relation between BRCA1 mutations and a worse or better prognosis.

Targeting homologous recombination defects in breast cancer

BRCA deficiency represents the failure of the homologous recombination DNA repair system in cells, but also in other responses to DNA damage. BRCA1 can be seen as a general DNA response protein.

Cisplatin:

Cisplatin is a chemotherapeutic which can be used for breast cancer treatment. It is both used in neoadjuvant and adjuvant situations:

• Neoadjuvant therapies are delivered before the primary treatment
• Adjuvant therapies after the primary treatment

Lab experiments show that loss of BRCA1 function increases the antitumor response of cisplatin against human breast cancer xenografts in vivo. When cisplatin is used in triple-negative breast cancers, the prognosis for patients with BRCA-mutations is slightly better than for patients without a BRCA mutation.

BRCA2-mutated ovarian cancer patients specifically benefit from platinum-based chemotherapies. However, there still isn’t a clear-cut association between the response of BRCA-patients to cisplatin.

There is a trend towards a relation between BRCA status and clinical responses, but this is non-significant. This can be explained by the fact that cisplatin mostly targets the resolution of intra-strand crosslinks, and not really the formation of double strand products.

Synthetic lethality:

If part of a system in a cell is down, another system can compensate for it → if homologous recombination is down it can be compensated. However, if both complementary and compensative systems to BRCA are taken down, the tumor cells are left without alternative.

The occurrence of single strand breaks of DNA can intervene with homologous recombination:

1. As single strand breaks occur, they are recognized by a DNA damaged response protein → PARP-1
2. PARP-1 recruits a BER
   • BER: base excision repair machine
3. BER repairs the single strand break

If the action of PARP-1 is inhibited, the replication fork may collapse when the DNA strand is being replicated. This requires homologous recombination to operate, otherwise the cell dies.

If BER can be inhibited in cells that are already homologous recombination deficient, the tumor cells are left without any alternative to repair these type of mistakes. Olaparib is a drug which specifically targets PARP-1 which stops BER of single strand breaks. This is useful to treat tumors which lack homologous recombination. This concept is called synthetic lethality.

Synthetic lethality arises when there is a combination of deficiencies in 2 or more genes which leads to cell death, while deficiency in 1 gene allows the cell to survive. PARP inhibition such as olaparib can be used in BRCA-mutant breast and ovarian cancer. However, tumors do develop resistances to these type of therapeutic approaches.

Testing for homologous recombination defects

To apply therapy targeting HR-defects, there needs to be a way to distinguish these cancers. Moreover, homologous deficiencies can also arise due to deficiencies in other components of the homologous recombination process. Different methodologies have been applied to assess whether cancer cells are homologous recombination deficient or not, without having to sequence actual genes.

BRCA1 or BRCA2 defects and mutational signatures:

BRCA1 or BRCA2 deficient cancers have specific mutational signatures which indicate the deficiency:

• Large chromosomal alterations
• Genomic scars that suggest that HR is not working properly

By reading these type of signatures, HR deficient tumors can be identified → are susceptible to PARP-1 treatment. HRDetect is superior to screening for the detection of HR-deficient cancers.

RAD51 foci for assaying HR-deficiency:

One of the final steps of homologous recombination includes the accumulation of RAD51 proteins → causes the DNA to break. If an antibody against RAD51 with immunofluorescence is used, dots become visible → demonstrates that RAD51 is accumulating in the nucleus and is binding naked chains of DNA.

Researches are trying to expose vivo patient material to ionizing radiation, making it possible for them to determine if RAD51 foci are formed:

• If RAD51 foci are visible, homologous recombination is functional
• If RAD51 foci aren’t present, homologous recombination isn’t working properly
  • These patients can receive drugs like olaparib

Mismatch repair deficiency and 5-FU chemotherapy:

Some tumors, such as Lynch syndrome related cancers, are mismatch repair deficient (MMR-deficient). 5-FU is a common chemotherapy used for colorectal cancer. It is a nucleoside analog which interferes in the metabolism of nucleotides that compose the DNA and RNA molecules. While it is metabolized, it binds enzymes that are involved in the normal processing of nucleotides → inhibits nucleotide metabolism and integrates itself in DNA and RNA molecules, disturbing their structure.

MMR-deficient tumors do not respond to 5-FU treatment. It is unknown what causes this, but a proposition is:

1. Cells can activate apoptosis by recognizing that something strange has been incorporated in their DNA → can be recognized through the activity of the MMR-system → apoptosis
2. If the MMR-system isn’t active, apoptosis pathways aren’t activated → MMR-deficient tumors can survive with 5-FU incorporated in their DNA