Studying How BRCA2 Reversion Mutations Arise in PARPi Resistant Prostate Cancer

By Michelle Bloom, PhD

The names of the BRCA1 and BRCA2 genes indicate their importance in the development of breast cancer. While women with mutations in either the BRCA1 or BRCA2 genes are at highest risk for breast cancer, other types of cancers also arise as a result of these mutations. BRCA1 and BRCA2 mutations are also just as likely to be found in men as in women; men’s risks for BRCA-mutated cancers, including breast and prostate cancer, however, are not as widely discussed as women’s risks. Scientists are making efforts, however, to understand how these other types of BRCA-mutated cancers develop and how to treat them. This article will highlight a research study that focuses on BRCA-mutated prostate cancer, which affects men, but may also give insight on how to treat all BRCA-mutated cancers.

In my previous article, The Power of PARP Inhibition, I discussed a promising treatment that targets many types of BRCA1- and BRCA2-mutated cancers called PARP inhibitors (PARPi). PARPi are currently only approved by the FDA for use in certain types of breast and ovarian cancers, but studies are showing that many other cancers may be successfully treated with PARPi, as well, including prostate cancer. While PARPi holds promise of successful treatment for BRCA-mutated cancers, patients can develop resistance to PARPi treatments over time. The development of resistance to these treatments means that while the PARPi may initially successfully kill cancer cells, over time, the cancer stops responding to the treatment and begins to grow again.

Resistance to PARPi has been associated with secondary reversion mutations, which restore the DNA repair functions inactivated by the original BRCA1 or BRCA2 mutation. In my recent post, BRCA: Bracing the Genome, I highlighted the important roles of the BRCA1 and BRCA2 proteins in repairing DNA damage through an error-free pathway called HR. When BRCA1 or BRCA2 are inactivated through mutation, an error-prone pathway called NHEJ kicks in to repair the damage. If, however, BRCA1 or BRCA2 are mutated again by a reversion mutation, the function of these proteins can sometimes be restored and DNA damage may properly be repaired again. PARPi, as I discussed in The Power of PARP Inhibition, takes advantage of the failure of the HR repair pathway and causes cells to die from the inability to repair DNA damage. When the HR pathway is, therefore, restarted by reversion mutations, PARPi is no longer effective.

Scientists are studying this phenomenon of PARPi resistance in order to figure out how to get around this problem in the future. One such study, published in Cancer Discovery in 2017, was led by Dr. David Quigley, a recipient of the BRCA Foundation’s Young Investigator Award. Dr. Quigley is a scientist in Dr. Alan Ashworth's laboratory at the Helen Diller Family Comprehensive Cancer Center at the University of California, San Francisco. His compelling study investigated how resistance to PARPi occurs in BRCA2-mutated metastatic prostate cancer.

Dr. Quigley, along with a group of other scientists, analyzed the DNA from the cancer cells of two patients with metastatic prostate cancer who were treated with PARPi and developed resistance to the drugs. They compared the DNA mutations in the BRCA2 genes both prior to and after treatment with PARPi. Using a few different methods of DNA sequencing, they uncovered different reversion mutations, showing the promise of a possible non-invasive method for testing for PARPi resistance.

In Patient #1, scientists sequenced the DNA of the BRCA2 gene from metastatic-tumor biopsies, both before and after treatment with the PARP inhibitor, talazoparib. When the scientists sequenced the tumor prior to treatment with talazoparib, they were able to identify the inherited mutation in BRCA2, although no normal (unmutated) copy of BRCA2 was present. This indicated that the cancer arose as a result of the loss of the second, protective, copy of the gene. After sequencing the DNA from the biopsy of the PARPi-resistant tumor, the scientists detected two additional mutations in the BRCA2 gene. Analysis of these two mutations showed they were deletions of DNA sequences within the gene that likely restored function to the BRCA2 protein.

DNA mutations, such as the ones detected in this study, can either restore or eliminate function of a protein due to the way DNA sequences code for proteins. DNA sequences in a gene comprise a series of 3-letter blocks called codons, each of which encodes a specific amino acid, which is the building block of a protein. Similar to Morse code, these 3-letter codons need to be maintained in register in order for the message to be properly translated. If one ‘dot’ or ‘dash’ is missing, the reading of the sentence is thrown off, as every letter after the deletion may now be different. Certain DNA codons do not encode an amino acid, but rather, tell the protein-building machinery to stop— similar to a period in a sentence. Omitting a DNA letter, therefore, can lead to a period being placed in the middle of the sentence and result in a truncation of the resulting protein.

The initial BRCA2 mutation in Patient #1 from Dr. Quigley’s study resulted in a premature stop that truncated the BRCA2 protein. The two reversion mutations they uncovered in the PARPi-resistant tumor were deletions that restored the register of the codons and allowed for most of the protein to be built again.

In addition to sequencing the tumor biopsy, which only contains cells from one part of the body, the scientists also tested DNA throughout the entire body by obtaining a blood sample. The researchers used a technique called cell-free DNA (cf-DNA) sequencing, which takes advantage of DNA fragments floating in the blood that come from cells that have died. The process of cellular death results in a breakdown and release of the cellular components. Generally, these fragments are cleaned up by the immune system. DNA fragments, however, are found at higher levels in the blood of cancer patients, possibly from inability of the immune system to keep up with the rapid rate of cellular growth in tumors. The use of cf-DNA to diagnose genetic conditions, including cancer, is a fairly new method that is gaining traction, particularly for prenatal screening due to its non-invasive nature.

Dr. Quigley and his colleagues utilized the cf-DNA sequencing method to analyze BRCA2 mutations from Patient #1 after PARPi resistance had been conferred. Through cf-DNA sequencing the scientists saw the normal copy of the BRCA2 gene that resides in non-cancerous cells, the inherited BRCA2 mutation that is present in every cell, and the two reversion mutations found in the biopsy. The scientists, surprisingly, also saw five unique mutations in the BRCA2 gene that were absent from the biopsy tissue. These five reversion mutations were all deletions of DNA sequences that eliminated the inherited BRCA2 mutation and restored the register of the codons, allowing the protein to be built again.

In the study, the scientists also used cf-DNA sequencing on Patient #2, who had BRCA2-mutated metastatic prostate cancer and was treated with olaparib. They sequenced the BRCA2 gene both before and after resistance to olaparib had been acquired and found 105 new mutations in the BRCA2 gene post-olaparib treatment. Most of these mutations were insertions or deletions of DNA sequences that were predicted to restore the register of the codons in the gene and result in the synthesis of a functional BRCA2 protein. Surprisingly, most of the new mutations clustered to one specific region of the BRCA2 gene sequence. The scientists predicted the likelihood that random mutations would occur in this region and found that mutations arose in this individual at a significantly higher frequency than would otherwise occur by chance. The elevated frequency of mutations was an indicator to the researchers that there was a selective pressure for these mutations to occur. The selective pressure seen among these tumor cells is similar to the black peppered-moths I discussed in my first article, The Double-Edged Sword of Mutation. The moths that acquired mutations that allowed them to blend into their sooty environment during the Industrial Revolution gained a survival advantage over those that could not blend in. The mutations that conferred resistance to the PARPi inhibitor, similarly, gave the tumor cells a survival advantage over cells that did not acquire mutations.

The major findings of this study are: 1) the first reported BRCA2 reversion mutations in prostate cancer; and 2) that multiple mutations can arise within both a tumor and body to confer resistance to the same drug. The study also showed that using cell-free DNA screening provides a non-invasive and highly sensitive method of detecting reversion mutations that confer resistance to PARPi, potentially allowing doctors to detect PARPi resistance earlier. The 105 mutations detected in Patient #2, in addition to the five mutations from Patient #1 that were not detected in the biopsy sample, indicate that using the cf-DNA sequencing method allows for detection of mutations from metastases throughout the body through a single test.

Testing for the presence of reversion mutations will help doctors tailor treatment before the cancer can return. Knowledge of mutations that predispose a person to cancer, similarly, allows detection of cancer at an early stage and for people to take measures to prevent cancer from arising in the first place.

Over the past four articles, I explored the genetics behind the BRCA genes by discussing the effects of genetic mutations, how they can lead to cancer, and how the unique genetic defects in BRCA-mutated cancers can be targeted for treatment. My next article will explore how testing for genetic mutations is affecting care of breast cancer patients. I will discuss a recent study by Dr. Allison Kurian, a BRCA Foundation-supported investigator at Stanford University, that examines how genetic testing is being integrated into care and affecting outcomes for patients with breast cancer. I hope you will join me as I explore this different aspect of genetic mutations.