Note: Identifying patients eligible for PARP inhibitor treatment: from NGS-based tests to 3D functional assays

Note: Identifying patients eligible for PARP inhibitor treatment: from NGS-based tests to 3D functional assays

doi: 10.1038/s41416-021-01295-z

• ไม่ใช่ทุกคนจะได้ PARP เพราะการจะให้ PARP ต้องมีการตรวจ genetic ก่อนว่าสามารถรับ PARP ได้ไหม 

• โดยปกติจะใช้การตรวจ NGS แบบ specific gene panels

• แต่ว่ามันก็มีข้อจำกัด คือ มันจะมีกลุ่มคนป่วยบางกลุ่มที่ sensitive ต่อ PARP แต่ negative NGS results

• ข้อจำกัดอีกอย่างก็คือ เวลาทำ NGS เราทำกับ tissue จากคนป่วยเลย แต่เราไม่สามารถที่จะตรวจหา reversion mutation/secondary mutations -- ซึ่งมีผลทำให้ HR กลับมาทำงานเป็นปกติได้

In this paper;

• tumour models such as patient-derived xenografts/tumour-derived organoids

• Tailoring the clinicians to make better decision on treatment

- giving the overview of currently available NGS-based tests

- Expand to 3D functional assays

-     NGS+functional assays

PARP inhibitor

• Accumulation of DSB in BRCA1/2 defective

• Causing PARP-DNA complex trap

Other HRD besides BRCA1/2 defective (at coding level)

• BRCA1/RAD51C promoter methylation

Four PARPi (olaparib, niraparib, rucaparib, talazoparib) -- FDA approved to treat breast, ovarian, pancreatic and prostate cancers with recurrent, locally advanced, metastatic 

How to select patients receiving PARPi

• NGS -- CDx

• On targeted gene panels/genomic signatures (genomic scares)

• Having limitation -- fail to identify HRD status up to 18% of cases in OVCA

Perspective of this paper

• Companion diagnostic test (CDx) available -- diagnostic test which helps clinician to select specific drugs according to their genetic profiles

• Sample processing

• Interpretation

• Clinical success

• Challenges

• Outline potential functional assays to determine PARPi sensitivity

NGS is gold standard for patient selection

• Detecting somatic as well as germline mutations

• 5 CDx approved by FDA

1. BRACAnalysis

2. Mychoice HRD

3. FoundationOne

4. FoundationFocus CDxBRCA Assay

5. FoundationOne Liquid CDx

Sample processing

• Detecting defective in HR

• Germ line mutations

• Whole blood

• Somatic mutations

• Fixed frozen tissue

• FFPE

• Determining cancer cell proportion in tumour samples

• Sequencing must be in high-coverage to detect low allelic fraction for somatic alterations

• 200 ng DNA isrequired

• ACGM classification -- 5 classes

• Pathogenic (5)

• Likely pathogenic (4)

• Variant of uncertain significance (3)

• Likely benign (2)

• Benign (1)

Interpretation

• All CDx can detect class 4-5

• CDx can detect genomic instability profiles

• Given as a score -- genomic instability score (GIS)

• GIS -- defined by assessing the unweighted sum of three individual,  validated markers of genomic instability -

• Loss of heterogeneity (LOH)

• Telomeric allelic imbalance (TAI)

• Large scale state transitions (LST)

Clinical assessment of CDx testing

• Comparing with placebo/conventional chemotherapy -- PARPi gives significant benefits in a range of cancer, esp in patients both w BRCA mutations and HRD positive (defined by NGS)

• Clinical trial

• SOLO-1

Challenges associated with CDx testing

• difficult to identify strong and robust predictors of HR defects beyond alterations in BRCA and/or other mutational signatures or genomic instability signatures.

• Lack of consensus guidelines from the international task force to use NGS and selecting the patients to receive PARPi

• promoter methylation (BRCA1, RAD51C) or a new mutational signature of HRD, called “Sig3” (mutational signature established from whole genome sequencing, independent of the effect of these mutations on the functionality of the encoded proteins) -- could be candidate biomarkers for PARPi sensitivity

TRITON-2 trials (metastatic castration-resistant prostate cancer)

• Using DNA damage response beyond BRCA mutations as biomarkers for PARPi

• ATM

• CDK12

Current limitation of current HRD analysis

• It requires to detect before treatment but some cancer like OVCA -- biopsy is hard to do

• Perhaps, we might need to get the biomarkers from liquid biopsy (ctDNA or CTC)

• NGS is low in sensitivity -- hard to detect variants with low allelic fraction (tumour is very heterogeneity) and can not detect the epigenetic changes

• Thus, NGS could give

• False-negative and False-positive (10.1007/s10689-016-9955-8) with regard to HR-status

• Thus, requires complementary functional assays

Functional assays to assess PARPi sensitivity

Indirect assessment -- require clinical validation

• Not detecting sensitivity toward PARPi directly but observing repair capacity test (RECAP) instead

• Formation of RAD51 foci formation in proliferation cells in S/G2 phase following irradiation of breast cancer samples or fresch primary ovarian cancer cells

• Required prospective study to distinguish clinical responders from non-responders to PARPi

• Combination of RAD51 activity + other factors involved in DNA repair pw.

• Using multi fluorescence microscopy analysis of PARPi toxicity -- simultaneously measure

• DNA-danage-associated gH2Ax

• HR competency (RAD51 foci)

• DNA replication

• Cell cycle blockade

• Level of PARP inhibition

• Mitotic disorders

Direct assessment

• 3D tumour model -- freshly resected samples from solid tumours

• Spheroids -- fluid paracentesis

• CTC --  from blood

• Using zebrafish saves time for engraftment (mouse 3-12 mo, 1-3 mo in zebrafish) thus it might be suitable for the clinical decision-making time frame.

Challenges and opportunities

• PARPi mostly used in solid tumour as indicated in table but now many studies have extended to haematological malignancies

• Major challenge -- way to say which group of patients will be benefits

• Genetic testing might not be enough thus it is suggested to consider with functional assay to improve the therapeutic management of PARPi

• Patient-derived tumour organoids seems to be the most promising, still some limitations need to be issued -- 

• Success rate to establish is wide 10-80% depending on cancer type and culture conditions

• 3D based-assay on PARPi sensitivity must be done in a short time, thus the clinician can make the decision, esp. in a front-line/first-line setting

• By scaling down at the level of microfluidic-based culture system -- would offer a good opportunity to do so

• Microenvironment which impacts tumour response to treatment ~ esp. immunological response within the tumour area

• We might combine immune checkpoint inhibitor+PARPi but the biomarker to select this group of patients is unknown.

Conclusion remarks

• It is possible that we might need functional assay + genetic testing to prevent unnecessary treatment/consideration of new treatment options

• However, at this stage, functional assay requires clinical validation in the prospective cohort