Professor Liu Congrong: Clinical and Pathological Interpretation of "Ovarian Cancer Test Report"

[Editor's note] In recent years, with the clinical application of PARPi, it has effectively prolonged the survival of ovarian cancer patients and broke the treatment pattern for ovarian cancer patients
.

And biomarker testing can guide the clinical rational use of drugs and evaluate the prognosis of patients, especially HRD testing, which has many significances in guiding the clinical practice of precise treatment, precise prevention, and precise prediction

【Editor's Note】

1.
Clarify the concept-HRR vs.
HRD

1.
Clarify the concept-HRR vs.
HRD

Homologous Recombination Repair (HRR)

The substance in the cell nucleus that can be dyed dark by basic dyes is called a chromosome.
It is composed of DNA and protein.
DNA is the carrier of genetic information.
There are many genes on a DNA molecule, and genes are DNA fragments with specific genetic information.

.

In the living environment, the DNA in every cell of the body is damaged thousands of times every day, resulting in base-pairing errors in DNA, and missing DNA fragments on single or double strands of DNA

When DNA single-strand damage occurs, the side chain is usually used as a template for replication and repair
.

When DNA double-strand damage occurs, the opportunity to "resurrect" the other strand using one strand as a template is lost.

In DNA single-strand damage, the PARP enzyme is used as a template to repair the DNA single-strand damage to a normal state, that is, the high-fidelity repair method of base excision
.

In DNA double-strand damage, the method of "copying work" borrows another chromosomal DNA double-strand to repair the defective DNA double-strand with high fidelity.
This process is called HRR
.

In the HRR repair process, the main work team is called the HRR complex, and the core of the HRR complex is the BRCA1/2 protein

In addition, there is a low-fidelity repair method.
After the DNA double-strand breaks, since it cannot be repaired by "copy work", the defect and broken ends are joined together by non-homologous end joining.
, The repaired DNA double-strand is low-fidelity and can have various problems, either too short or too long, commonly known as "crippled legs"
.

Homologous recombination repair defect (HRD)

Homologous recombination repair defect (HRD)

The so-called HRD refers to the physiological functional defect that cannot be repaired by "copying" the DNA double-strand defect, such as the BRCA1/2 mutation
.

Second, the significance of HRD testing

Second, the significance of HRD testing

Since it is impossible to repair DNA double-strand defects through "copy work", only low-fidelity repair methods can be used to cause a large amount of "lame" DNA to accumulate, causing serious damage to DNA genetic material.
Lethal effects
.

PARP inhibitor

When there is HRD in tumor DNA, relatively speaking, there is only one high-fidelity repair method-single-strand high-fidelity repair.
For example, if PARP inhibitors are used to make DNA damage unable to perform single-strand repair, it will evolve into DNA double-strand breaks.
At this time, only low-fidelity repair is left as a DNA repair method, and the generated "lame" DNA is difficult to survive and has a lethal effect on cells, which is the so-called "joint lethality
.

"

Therefore, an important mechanism for the use of HRD detection is to use PARP inhibitors to block the high-fidelity repair of DNA single-stranded DNA once it is confirmed that there is a DNA damage "copy job" repair defect in the tumor, so that it will quickly accumulate into DNA double-stranded damage.
Because of its own defects, it is impossible to carry out high-fidelity repair of DNA double-strand homologous recombination, and only low-fidelity repair of non-homologous ends can produce a large amount of "lame" DNA, which will lead to the rapid death of tumor cells, while normal human cells Because there is no HRD, it can still survive after applying PARP inhibitors
.

This is also a precise treatment

Platinum sensitivity + PARP inhibitor sensitivity

At present, many doctors in the clinic use platinum sensitivity as one of the principles of PARP inhibitor application based on their clinical experience.
Then, does platinum sensitivity have HRD? Is platinum sensitivity the same as PARP inhibitor sensitivity? First, understand the impact of platinum drugs on DNA
.

The damage of platinum drugs is mainly DNA cross-linking, and the DNA repair methods include nucleotide excision repair (NER), trans-damage DNA synthesis (TLS) and homologous recombination repair (HRR).

So, whether platinum-sensitive patients are only treated with PARP inhibitors and do not need to be checked for HRD? The answer is no
.

In fact, although a considerable number of patients with platinum-sensitive tumors are sensitive to platinum due to the presence of HRD, a small number of patients have normal HRR pathways, and there are problems with nucleotide excision repair (NER) and trans-damage DNA synthesis (TLS) pathways.

Therefore, HRD and platinum sensitivity are two independent units.
Although they overlap, they have their own independent fields
.

In clinical practice, if there is no HRD detection method, patients with platinum-sensitive tumors can try PARP inhibitor treatment, but if they have the ability to detect HRD, they still need to be tested
.

3.
Clinicopathological significance of HRD detection for ovarian cancer

3.
Clinicopathological significance of HRD detection for ovarian cancer

1.
Guide precision treatment

HRD detection has guiding significance for the clinical application of postoperative platinum chemotherapy and PARP inhibitor targeted therapy, especially once HRD appears, it indicates that the tumor patient is highly sensitive to PARP inhibitors and can guide clinical precision treatment
.

2.
Genetic counseling, precise prevention

Common clinical examination results include gBRCA mutation and tBRCA mutation
.

gBRCA mutations refer to the germ cells with mutations, so the cells of the patient's body carry the mutations, and blood/normal tissues must be tested.
Half of the germ cells of this population carry mutations and are hereditary
.
Its significance is mainly reflected in the following three aspects: ① The gBRCA mutation in patients with ovarian cancer indicates that they are sensitive to platinum-based chemotherapy and have a good prognosis and can be treated with PARP inhibitors; at the same time, gBRCA mutation indicates a higher risk of breast cancer
.
②If you are a gBRCA mutation carrier, it is recommended to perform MRI screening every year from the age of 25, and resection of double appendages + breast cancer resection after the age of 40
.
③Prevent inheritance of immediate relatives and detect BRCA germline mutations
.

tBRCA mutation refers to the mutation of somatic cells.
Germ cells do not carry mutations and are not inherited.
They only affect part of the area.
Tumor tissues must be tested
.
The main significance is as follows: ① Patients with ovarian cancer are sensitive to platinum-based chemotherapy and have a better prognosis.
PARP inhibitors can be used for treatment, but the risk of breast cancer is the same as the general population
.
②Because the germ cells of patients with tBRCA mutation do not carry the mutation, the immediate family members do not need to be screened for BRCA
.

Therefore, HRD detection for ovarian cancer, especially gBRCA mutation, can guide clinical genetic counseling and precise prevention
.

3.
Assess the risk of recurrence and make accurate predictions

According to relevant clinical studies, in terms of overall ovarian cancer, tBRCA mutations indicate poor tumor prognosis
.
For example, patients with high-grade serous ovarian cancer often have tBRCA mutations, and in comparison, they often have a poor prognosis
.
Someone analyzed the proportion of patients who died of breast cancer/ovarian cancer and found that in the United States, the proportion of BRCA1/2 gene mutation carriers and patients under 70 years old accounted for 56% to 72%, while the general female population in the United States, under 70 years old, accounted for 56% to 72% of deaths.
The death rate of patients accounted for 11%
.
It can be seen that the death rate of breast/ovarian cancer patients with BRCA1/2 gene mutations is significantly higher
.
However, other studies have shown that BRCA1/2 gene mutation carriers have a good prognosis.
An important premise is that, as far as high-grade serous cancer is concerned, BRCAm patients are more sensitive to PARP inhibitors and platinum-based chemotherapy than BRCA wild-type patients.
So the prognosis is good
.

Therefore, for the prediction of the prognosis of ovarian cancer, it is necessary to know the BRCA mutation through HRD test before making judgments
.

4.
How to perform HRD test?

4.
How to perform HRD test?

The HRD test for ovarian cancer can guide the clinical practice of precise treatment, precise prevention, and precise prediction.
So, how should HRD be tested?

1.
Causes of HRD

After the DNA double-strand break on one side of the chromosome, the repair team (HRR complex) led by BRCA1/2 uses the contralateral DNA double-strand as a template, and through complicated operations, the DNA is finally copied The double strand is repaired
.
It can be seen that BRCA mutation can cause HRD, but it is not the only cause.
An abnormality in any member of the repair team (HRR complex) will cause HRD
.
At present, the main causes of HRD include BRCA1/2 mutations, other genetic mutations in the HR pathway, promoter methylation, and unknown causes
.
So, from the perspective of reasons, is it feasible to conduct HRD testing?

Only use NGS to detect BRCA1/2 mutations

Taking high-grade serous cancer as an example, studies have shown that 42% of the cases are related to HRD, and 14% of the cases can be detected in blood samples, which are gBRCA1 mutation 8% and gBRCA2 mutation 6%; if it is detected in a tumor sample , Can expand the case to 19%, in addition to gBRCA mutation, it also includes tBRCA1 mutation 4% and tBRCA2 mutation 3%
.
Therefore, for treatment, tumor samples detected by BRCA are better than blood samples
.
But even so, using only NGS to detect BRCA1/2 mutations, the detection rate of ovarian cancer HRD was less than 50%, and 11% of methylated BRCA1 and 11% of other HRR genes could not be detected
.
Therefore, for HRD detection, it is far from enough to only detect BRCA1/2 mutations
.

Use NGS to detect HRR gene panel

Also take high-grade serous cancer as an example.
There are a total of 42% of HRD-related cases.
The g/tBRCA1/2 mutation test can cover 19% of the relevant cases.
The HRR gene panel can detect other HRR genes (11%).
Part of the HRR gene mutation
.
However, other HRR gene abnormalities include deletion of large gene fragments, abnormal expression regulation such as microRNA and transcription factors, protein degradation and other factors that affect mRNA and protein, which cannot be detected.
In addition, methylated BRCA1 (11%) is also undetectable.
Undetectable, therefore, there are still 30% of HRD undetected
.

Therefore, even if NGS is used to detect the HRR gene panel, the detection rate of ovarian cancer HRD is less than 70%, and the coverage rate is still incomplete
.

2.
Ending due to HRD

Since the detection is conducted from the perspective of the cause of HRD, it cannot be fully covered, can you change your mind? From the perspective of the results caused by HRD, think backward, and use the detection results to infer whether there is HRD
.
HRD directly leads to two states-HRD functional defect and genome instability.
Regarding these two HRD functional states, there is currently no direct test commercially available kit in the world
.
HRD functional defects and genome instability further develop, and the outcome will lead to scarring of the HRD genome
.
The "genomic scars" that have been proven to be closely related to HRD include loss of heterozygosity (LOH), telomere allelic imbalance (TAI), large segment migration (LST), and there are already commercially available genomic scar detections.
HRD detection kit
.
One is FOUNDATIONNE's kit, covering tBRCA and 28 HRR genes, can detect LOH, and set a threshold (LOH/full genome ratio Cutoff ≥ 16%), its HRR gene detection is comprehensive, but the genomic scar coverage is incomplete
.
The other is MYRIAD's kit.
Compared with FOUNDATIONNE, its HRR gene detection is incomplete, only g/tBRCA is detected, but its genomic scar coverage is more comprehensive, covering all three genomic scars, LOH+TAI+LST Cutoff Set at 42 points
.
These two kits detect both genetic mutations and genomic scars.
Although HRD testing is more comprehensive, both are FDA certified and there is no SFDA certification
.

Therefore, for HRD inspection, there is a missed diagnosis of cause detection.
The direct result-HRD functional defect and genomic instability detection.
There is currently no kit.
The final result-HRD genomic scar is used as the test "content", and the Cutoff value is set to exceed The “conclusion” given by the author of the functional status of HRD is a mechanism that is currently widely used
.
The PAOLA-1 study found that through HRR genetic testing + HRD testing, tBRCAm can rise from 29% to HRD+ 48%
.
In the PRIMA study, it was found that 51% of patients had HRD+ through HRR gene test + HRD test
.
The results of these large-scale clinical studies indicate that HRR gene testing + HRD testing can benefit more patients and is currently the most comprehensive detection of PARP inhibitor markers for ovarian cancer
.

From this, we can better understand the first recommendation in the 2020 NCCN Guidelines for Ovarian Cancer Diagnosis and Treatment, that is, all ovarian cancer, fallopian tube cancer and primary peritoneal cancer are recommended for genetic risk assessment and BRCA1/2 germline and somatic mutation detection, and Patients who have not found BRCA1/2 germline and/or somatic mutations should undergo corresponding HRD testing to guide PARP inhibitor treatment
.

Five, common confusion in HRD testing practice

Five, common confusion in HRD testing practice

1.
The BRCA1/2 mutation rate of Chinese ovarian cancer patients is lower than that of Westerners?

This inherent perception is actually wrong
.
The latest data show that the BRCA1/2 mutation rate of ovarian cancer patients in China is actually higher than the average level in countries such as the United States, Australia, and Japan
.
Therefore, HRD testing in China is more clinically meaningful
.

2.
Do all ovarian, fallopian tube, and peritoneal cancers need to be tested for HRD?

Yes
.
In fact, the total frequency of HRD in all ovarian cancer, fallopian tube cancer and peritoneal cancer is 30%~50%, of which 20% are HRD caused by BRCA1/2 mutations, and 20% are caused by BRCA1 methylation and other HRR gene mutations HRD
.
In recent years, there have also been more studies on the HRD status of ovarian cancer in different histological subtypes
.
According to databases in China and Japan, multi-center data research on the HRD status of different histological subtypes of ovarian cancer shows that the BRCA1/2 mutation rate and the HR-related gene mutation rate in different histological types of ovarian cancer both account for a certain proportion, and the detection significance is clear
.
Therefore, HRD testing is required for all histological subtypes
.

3.
Interpretation of BRCA Variation Classification

BRCA gene variants are divided into the following five categories according to the degree of risk: pathogenic, category 5, with a pathogenic probability of> 0.
99, likely pathogenic, category 4, with a pathogenic probability of 0.
95～ 0.
99; Unclear significance (uncertainsignificance), category 3, the probability of disease is 0.
05 to 0.
949; likely benign, category 2, the probability of disease is 0.
001 to 0.
049; benign (benign), category 1, the probability of disease is ＜0.
001
.

There are two solutions to the "3 types of BRCA gene variants with unclear significance".
One is that qualified genetic testing laboratories can verify the VUS function through animal models; Multiple members in the test
.

4.
Obtaining test samples

The sample tissue can be fresh, fixed with formaldehyde or paraffin, blood or saliva, as shown in the figure below
.

5.
BRCA gene clinical testing pathway

In 2019, the Chinese Journal of Pathology published the "Guidelines for BRCA1/2 Gene Detection Based on Next-Generation Sequencing Technology (2019 Edition)".
The clinical detection path of BRCA genes in the guideline is as follows
.
It is worth noting that when BRCA1/2 mutations are found to be negative in the clinic, whether it is germline or system, do not give up, and continue HRR-related gene and HRD testing
.

In summary, HRR is an important physiological mechanism for repairing DNA double-strand damage and maintaining gene stability.
The mechanisms leading to the occurrence of HRD are diverse and partly unknown
.
BRCA1/2 mutation detection is of great significance for the "precision prevention" of ovarian cancer, but only meets about 1/3 of the "precision treatment"
.
The precise treatment of PARP inhibitors for ovarian cancer relies on comprehensive detection of HRD status.
Current detection methods include detection for the cause (HRR gene mutations such as BRCA) and detection for the outcome (gene scar)
.

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