2. Various ways to Detect HRD in Tumors

In the search for BRCAness, several tests have been designed to recognize patients with a HRD phenotype. Many tests are based on sequencing. The most obvious test is sequencing of known disease causing genes, i.e. BRCA1 or BRCA2. As an extension of this approach, panels of multiple genes can be chosen for example based on their involvement in the HR pathway. The PALB2 gene is an example of a HR gene that predicts PARPi sensitivity, but many other HR genes are also expected to have predictive value. It is currently not clear which additional genes should be included in such sequencing panels.

Alternative tests are based on loss and amplification of pieces of chromosomes or whole chromosomes. The basic idea behind these tests is that HRD is often accompanied by very specific chromosomal alterations, which give specific growth advantages to HRD tumors. For example, loss of function of the TP53 gene prevents apoptosis caused by inactivation of HR which results in replication stress. Such a pattern of chromosomal changes can therefore be used as a way to identify HRD tumors.

Finally, sequencing can also be used in the search for specific types of genomic changes that are a direct or indirect consequence of the DNA repair defect, so-called scars, which accumulate over the course of tumor evolution. BRCA-deficient tumors accumulate specific patterns of point mutations, as well as specific types of DSB repair events. A very characteristic type of repair event is rejoining of DSB with microhomologies at the junctions. Microhomologies are short stretches (generally 1-6 base pairs) of directly repeated nucleotides. Therefore, this type of repair causes loss of the intervening sequence. Detecting these sequences requires Whole Genome Sequencing (WGS), which is currently rather expensive. However, it is expected that new technology will make this approach affordable on a routine basis in the future.

A completely different approach directly investigates functional parameters in fresh tumor specimens. These so-called functional assays use material from the tumor to test a relevant treatment, followed by measuring the response of these living tumor specimens to assess whether the tumor harbors deficiencies in a relevant process. HRD can be identified by testing accumulation of the RAD51 protein on DSB in a small piece of tumor material. A major advantage of this approach is that it measures directly whether the tumor has a defect without assumptions about the genes involved or correlated consequences of the repair defect. However, the use of fresh tumor material requires specialized laboratory adaptations, which may be challenging in hospital settings.

These various ways of selecting patients have been described in more detail in a recent review, which also summarizes evidence for their analytical validity, clinical validity and clinical utility.