Lung cancer

Molecular analysis of lung cancer is currently limited to non-squamous non-small cell lung carcinomas, while there are virtually no advances in DNA-assisted guidance of the treatment of squamous and small-cell lung malignancies. Histological diagnosis of lung cancer is often complicated; therefore, it is recommended to test patients with high probability of actionable genetic findings, i.e., women and non-smokers, irrespective of tumor histology [4–6].

EGFR gene mutations are the most common druggable alterations in lung adenocarcinomas. They account for approximately 10–20% of patients of European race and 40–70% of subjects of Asian ancestry. Although these manifold differences in racial distribution of EGFR mutations were acknowledged immediately after their discovery, the causes of this phenomenon remain unknown. EGFR mutations are significantly more common in females and non-smokers [7]. EGFR exon 19 deletions (ex19del) are associated with higher tumor sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and demonstrate similar frequencies in patients of various ages. EGFR L858R mutations are particularly common in elderly subjects [8].

Approximately 20% of EGFR genetic events are defined in scientific literature as “rare” or “uncommon” mutations, i.e., they are represented by other than ex19del or L858R alterations [9]. The majority of these uncommon lesions are druggable by conventional inhibitors, with the exception of EGFR exon 20 insertions which require a distinct category of drugs [9–11]. Early EGFR PCR kits focused mainly on the detection of hotspot ex19del and L858R mutations, while current standards of EGFR testing call for comprehensive analysis of exons 18–21.

The cumulative frequency of tyrosine kinase gene rearrangements is around 10%, being 5% for ALK, 4% for RET, 1.5–2% for ROS1, and around 0.2% for the NTRK receptor family (NTRK1, NTRK2, and NTRK3). These fusions are strongly associated with young patient age, female gender, and non-smoking status. Reliable and time-efficient detection of gene fusions still presents a challenge, despite significant advances in techniques for molecular analysis. RNA hybridization-based NGS is the most proficient technology in this respect; however, its routine utilization remains limited due to extraordinary complexity, high costs, and significant turn-around time. Amplicon-based RNA and DNA NGS are more accessible, but these techniques have a risk of missing some translocations. Immunohistochemistry (IHC) analysis is widely used for ALK testing, and, to a lesser extent, for ROS1 and pan-NTRK screening, despite having significant drawbacks with regard to specificity and sensitivity. There are several elegant techniques for rapid and cost-efficient detection of ALK, ROS1, RET, and NTRK1-3 gene fusions that address the disadvantages described above, but their use is restricted to facilities that have managed to achieve scrupulous in-house adjustment and validation of these laboratory-developed protocols [6].

The medical significance of error-free ALK, ROS1, RET, and NTRK1-3 testing is perhaps the highest among all known predictive markers. For example, earlier studies suggested that patients with metastatic ALK-driven lung cancer may gain up to 7 years in their life expectancy upon targeted therapy, and these estimates are likely to exceed 10 years with more modern drugs [12, 13]. In addition to the above genes, NRG1 rearrangements appear to be more or less actionable; however, their testing has not been yet incorporated in minimal diagnostic standards due to the low occurrence of these fusions (around 0.2%) and moderate survival benefit from matched therapies [14, 15].

Activating MET mutations result in exon 14 skipping, thus significantly increasing the half-life of this tyrosine kinase. Their occurrence reaches approximately 2.5%. These events are confined to elderly patients, with almost all mutation-positive subjects being well above 70 years old [16, 17]. Surprisingly, IHC cannot be used for preliminary screening of these mutations. Exon 14 skipping MET alterations can be detected either by RNA-based NGS or specifically adjusted PCR allele-specific expression tests [18]. MET amplification coupled with gene overexpression is also associated with potential tumor sensitivity to MET inhibitors, although this variety of molecular testing and clinical attitudes towards MET-amplified tumors have not been standardized yet [19, 20].

BRAF activation occurs in approximately 4% of lung carcinomas. Only substitutions affecting codon 600 are druggable; they are observed in less than 2% of tumors. BRAF V600-mutated malignancies are treated with a combination of BRAF and MEK inhibitors [6, 21]. Other BRAF alterations are represented by exon 11 mutations, non-codon-600 substitutions located in exon 15, and gene rearrangements. None of the latter events are clearly actionable [22].

HER2 amplification accompanied by gene overexpression plays a driving role in approximately 1% of lung carcinomas. These tumors can be managed by various anti-HER2 therapeutic agents [23–25]. Some lung malignancies produce excessive amounts of this receptor tyrosine kinase without being HER2-dependent. The analysis of other mutated genes in MAPK pathway, particularly RAS mutations, may help to discriminate between driver and passenger HER2 amplifications [25]. Activating HER2 mutations, mainly exon 20 insertions, occur in approximately 2–3% of lung carcinomas. These tumors can be targeted by novel low-weight TKIs or HER2 antibody-drug conjugates [26, 27].

KRAS mutations are seen in 30% of lung carcinomas. KRAS G12C substitution is amenable to therapy by sotorasib or adagrasib [28]. While actionable alterations affecting EGFR, BRAF, MET, HER2, ALK, RET, ROS1, and NTRK1-3 oncogenes are strongly associated with the lack of smoking history, KRAS G12C substitutions occur almost exclusively in smokers, with the frequency about 1 out of 6–7 tobacco-related cancers [29]. KRAS G12C also serves as a marker of lung cancer sensitivity to immune therapy, because smoking-induced lung malignancies have a high tumor mutation burden (TMB) [30]. Non-G12C KRAS mutations are not necessarily associated with smoking history and cannot be targeted by currently approved drugs.

EGFR, BRAF, MET, HER2, ALK, RET, ROS1, and NTRK1-3 targeted drugs render the best overall survival when applied in the first line [31]. Current laboratory facilities rarely provide comprehensive genetic profiling of lung carcinomas within an acceptable time frame, which is around two weeks or fewer. Consequently, many patients carrying druggable mutations start their therapy with non-specific interventions. Significant shortening of the turn-around time for the comprehensive analysis of actionable mutations is a primary need for lung cancer management [6].

Germline genetic testing is rarely applied to lung cancer patients, although the substantial incidence of these tumors in young non-smoking subjects remains an enigma. There are a few dozen subjects across the world, mainly of North American ancestry, who developed this disease due to inheritance of the EGFR T790M mutation [32]. In the practical sense, oncologists have to be aware of Li-Fraumeni syndrome, i.e., TP53 pathogenic variants, as a likely cause of young-onset lung cancer. Li-Fraumeni syndrome includes predisposition to early-onset breast, soft-tissue, brain, and hematological malignancies, so the combination of lung cancer with these tumors within a given patient or family calls for germline genetic testing. Of particular note, Li-Fraumeni related lung cancers almost always carry somatic EGFR mutations [33, 34].

Breast cancer

While molecular testing for lung cancer is focused mainly on the analysis of somatic mutations, germline DNA sequencing forms the backbone for genetic examination of breast cancer patients. Women with clinical features of hereditary cancer predisposition, e.g., younger than 50–55 years, or having family history of breast or ovarian cancer, or with triple-negative receptor phenotype, or with bilateral appearance of breast cancer disease, have to undergo testing for germline pathogenic variants in BRCA1 and BRCA2 genes. Historically, BRCA1/2 analysis was viewed as a purely cancer predisposition test aimed to estimate the risk of the development of the second tumor as well as to reveal mutation carriers among family members. The discovery of the vulnerability of BRCA1/2-driven tumors to DNA double-strand break-inducing agents, such as platinum compounds and PARP inhibitors, shifted the focus towards predictive value of BRCA1/2 testing [35–37].

Several issues regarding germline DNA analysis in breast cancer patients remain disputable. The increasing availability of NGS calls into question current attitudes towards the selection of breast cancer patients based on clinical criteria for hereditary syndromes. Some experts suggest universal BRCA1/2 testing for all consecutive patients with breast cancer [38–40]. It is beyond doubt that this practice will eventually take place, although some essential nuances, e.g., clinical decisions with regard to chance finding of germline BRCA1/2 pathogenic variants in family history-negative elderly women, have been neither discussed nor properly investigated.

The technical drawbacks of existing technologies for NGS analysis are rarely acknowledged in medical literature. For example, many NGS services cannot reliably detect so-called large gene rearrangements (LGRs), i.e., gross deletions or duplications involving one or several exons of BRCA1/2 genes [41]. Importantly, patients with BRCA1/2 LGRs are the best responders to PARP inhibitors, so this is a significant deficiency in that current DNA testing practices are likely to miss women with the most prolonged benefit from targeted therapy [42, 43].

The extension of NGS panels beyond BRCA1/2 is a particularly difficult topic. CHEK2 and ATM genes are the most common and established contributors to breast cancer predisposition after BRCA1/2; however, the clinical significance of the testing of these genes is unclear because they appear to be associated with only a two-fold increase of the risk of malignant disease [44–46]. Furthermore, ATM- and CHEK2-associated tumors apparently do not have a therapeutic window for PARP inhibitors [47, 48]. Similar concerns are applicable towards BLM and NBS1 gene testing [49, 50].

A recent study provided novel insights into the testing of moderately penetrant breast cancer predisposing genes. Many genes associated with hereditary cancer syndromes render a relatively insignificant increase in cancer risk in heterozygous mutation carriers but are associated with fatal medical conditions in subjects with biallelic gene abnormalities. The inclusion of BLM in the breast cancer NGS diagnostic panel led to the identification of a woman with Bloom syndrome, who remained surprisingly healthy until she developed a malignancy. These chance findings may have profound impact both for the well-being of particular patients and for medical research. Indeed, compensatory mechanisms in subjects with otherwise fatal mutations deserve detailed investigations, as they may suggest novel treatments for life-threatening conditions [51].

The analysis of TP53 germline mutations may reveal subjects with Li-Fraumeni syndrome, thus calling for genetic analysis of family members; it is particularly relevant to women with young-onset breast cancer [52]. Tumors arising in these patients have a high frequency of HER2 gene amplification and overexpression [53]. Li-Fraumeni syndrome is a severe condition, so many carriers of the TP53 mutation cannot transmit this defect to children due to limited life span or decreased chances to find a spouse. At the same time, a significant portion of subjects with this disease have de novo alteration in the TP53 gene, so they do not have a family history of cancer disease [54].

PTEN mutations are associated with Cowden syndrome [55]. It is of interest that clinical activity of the AKT inhibitor, capivasertib, has already been demonstrated for breast patients with somatic PTEN mutations, therefore, this drug deserves studies in subjects with hereditary PTEN-related tumors [56].

Several “BRCA1/2-like” genes, i.e., members of DNA repair by homologous recombination pathway, have been incorporated in breast cancer genetic testing relatively recently. Perhaps only PALB2 may be regarded as a true equivalent of BRCA genes [40, 57]. Some RAD и FANC family members, e.g., RAD51C and RAD51D, demonstrated more or less consistent association with breast cancer, although neither the degree of the increase in cancer risk nor the somatic status of the remaining allele of the involved gene, and consequently, tumor sensitivity to PARP inhibitors, have been investigated in sufficient detail [58, 59]. Similar limitations apply to BRIP1 and BARD1 genes [40, 45, 59]. The impact of other genes from RAD и FANC families is even less studied. Overall, it is important to continue the analysis of rapidly growing data sets in order to clean diagnostic NGS gene lists from irrelevant genes while encouraging sequencing of novel breast cancer predisposing loci.

Somatic analysis of breast tumors is limited to the testing of only a few genes in metastatic hormone receptor-positive HER2-negative carcinomas. Some of these tumors are intrinsically resistant to endocrine therapy due to activation of PI3K/PTEN/AKT pathway. Patients with PIK3CA mutations may benefit from the addition of PI3K inhibitors to fulvestrant [60]. Capivasertib has somewhat larger indications, as it also demonstrated activity in women with somatic mutations affecting PTEN or AKT [61]. Importantly, a breast cancer study involving comprehensive genetic profiling, i.e., exhaustive analysis of all potential drug targets, demonstrated no benefit from extended testing for somatic mutations [62].

Ovarian cancer

Ovarian cancer, being a common disease, has an unusually high proportion of patients who developed this disease due to germline genetic defects. As many as 25–40% of women with high-grade serous carcinoma of the ovary carry pathogenic alleles in BRCA1 or BRCA2 genes [63, 64]. Unlike for breast cancer, the selection of patients based on clinical criteria, such as young onset or family history, is discouraged; hence, all consecutive patients with high-grade ovarian cancer disease need to be tested for germline BRCA1/2 mutations [65]. The addition of new genes to this list is even more questionable than for breast cancer. Given that current NGS panels tend to pool together all cancer-predisposing genes in the same test, not a mere DNA analysis but the medical interpretation of the obtained data is likely to possess some problem in the future.

Approximately 30% of high-grade serous ovarian carcinomas do not have apparent alterations in BRCA1/2 or similar genes but resemble BRCA1/2-driven tumors by genomic architecture as well as by sensitivity to PARP inhibitors and platinum compounds. This feature was historically defined as BRCAness, although in the current literature the term “homologous recombination deficiency” (HRD) is more common. HRD testing is utilized for the selection of patients for the treatment by PARP inhibitors. HRD analysis is a highly complex, expensive, and time-consuming test that involves NGS-based genome scanning followed by the bioinformatics analysis of the integrity of chromosomal arms [66].

Colorectal cancer

Molecular testing for colorectal cancer is no less complex than that for lung cancer, although it is often mistakenly perceived by oncologists as a relatively straightforward pipeline. The utmost challenge lies in the comprehensive detection of mutations in KRAS and NRAS genes. These substitutions occur at frequencies of approximately 50% and 5%, respectively, with surprisingly little impact of ethnic or geographic variations [67]. RAS testing guides the choice between anti-EGFR antibodies and bevacizumab when considering the addition to chemotherapy backbone. While patients with wild-type KRAS and NRAS derive significant benefit from cetuximab or panitumumab, erroneous administration of these drugs to subjects with mutations, which were missed by laboratory tests, is associated with the risk of stimulation of tumor growth [68].

Initial clinical trials on anti-EGFR antibodies relied on PCR testing for a limited repertoire of RAS hotspot mutations [69]. Subsequent studies utilized Sanger sequencing, which may be prone to false-negative results when the proportion of the tumor cells in the sample is low [68]. This disadvantage can be overcome by NGS testing or by the use of relatively sophisticated laboratory procedures combining allele-specific PCR, high-resolution melting (HRM) analysis, pyrosequencing, etc. [67]. Importantly, unlike for lung cancer, molecular analysis of colorectal tumors is not a particularly time-sensitive procedure. Clinical studies demonstrate that the choice between anti-EGFR antibodies and bevacizumab can be safely postponed until the second cycle of chemotherapy; therefore, one month is an acceptable turn-around time for RAS testing [70].

The analysis of BRAF codon 600 mutations is not complicated. BRAF V600E substitutions occur in 4–8% of colorectal carcinomas and are associated with significantly worsened disease outcomes [67, 71]. BRAF inhibition alone or in combination with MEK downregulation is not effective due to collateral activation of EGFR-driven signaling cascade. The efficacy of combined administration of BRAF-targeted drugs and anti-EGFR therapeutic antibodies has been demonstrated in several clinical trials involving different agents; however, formal approval for colorectal cancer treatment has been granted only to encorafenib and cetuximab [72].

Microsatellite instability (MSI), i.e., accumulation of multiple alterations in tandem nucleotide repeats, reflects the deficient mismatch repair (dMMR). This mechanism of tumor development is relevant to approximately 5–10% of colorectal carcinomas. Two distinct routes underlie the emergence of MSI. Some colorectal cancer patients have developed their disease due to heterozygous germline defects in one of the dMMR genes (MLH1, MSH2, MSH6, PMS2, or EPCAM). This condition is called Lynch syndrome (hereditary non-polyposis colorectal cancer); the majority of patients belonging to this category are aged below 50 years and/or have family history of colorectal or endometrial cancer. In addition to young-onset patients, MSI is characteristic of elderly subjects. Indeed, colorectal carcinomas arising in patients aged above 70–80 years frequently have somatic inactivation of the MLH1 gene due to methylation of its promoter.

MSI tumors have an excessive number of mutations and, consequently, are highly immunogenic, have relatively low relapse rates after surgery, and can be efficiently managed by therapeutic inhibitors of immune checkpoints. The incidence of MSI demonstrates pronounced interstudy variations, depending on the proportion of localized and metastatic tumors, population-specific contribution of Lynch syndrome in cancer incidence, prevalence of elderly people among analyzed patients, and, possibly, technical nuances of MSI detection [73]. MSI is often combined with activating events in MAPK pathway genes, particularly KRAS, NRAS, and BRAF mutations. Up to a third of KRAS/NRAS/BRAF mutation-negative tumors carry druggable rearrangements in receptor tyrosine kinases [74].

POLE mutation testing has been introduced into clinical practice relatively recently. POLE encodes for DNA polymerase, so the tumors with altered POLE accumulate an excessive number of mutations. Clinical significance of POLE mutations is essentially similar to that for MSI-H, as they are associated with high tumor responsiveness to immune therapy and may indicate the presence of hereditary cancer syndrome [75].

HER2 amplification followed by gene overexpression is detected in approximately 1–2% of colorectal cancers. It is essential for treatment decisions to ensure that HER2 activation plays a driver but not a passenger role in a given tumor, i.e., at least to exclude the presence of KRAS mutations [25]. HER2-positive KRAS-negative tumors can be efficiently managed by a number of HER2-targeted drugs [76, 77].

Pancreatic cancer

KRAS mutations are detected in approximately 80–90% of pancreatic carcinomas [78]. The majority of them are currently not druggable, although KRAS G12C substitution, which is rare in pancreatic malignancies, can be managed by sotarasib or adagrasib [79, 80]. KRAS mutation-negative tumors often carry genetic alterations associated with sensitivity to available drugs, particularly BRAF V600E substitutions and rearrangements involving receptor tyrosine kinases (NTRK1-3, ALK, etc.). Approximately 1–2% of pancreatic tumors are microsatellite-unstable [81].

The contribution of germline pathogenic variants is a controversial topic. It is beyond doubt that heterozygous inactivating variants in BRCA2, and possibly PALB2 genes, are associated with the development of pancreatic tumors in some individuals, and these tumors arise via inactivation of the remaining allele of the involved gene, being, therefore, highly sensitive to platinum compounds and PARP inhibitors. The impact of BRCA1, which is similar to BRCA2 with regard to the spectrum of associated cancer types and the biological role, is less proven, both for the increase of the disease risk and for the tumor sensitivity to DNA double-strand inducing agents [82, 83]. Other genetic causes of pancreatic cancer are exceptionally rare.

Biliary tract tumors

Activating mutations and rearrangements affecting the FGFR2 gene are the most common events in this variety of tumors: they are detected in approximately 1 out of 5 biliary tract carcinomas. HER2 activation in this cancer type occurs via gene amplification and overexpression as well as via point mutations; overall, HER2 upregulation is observed in approximately 4% of biliary tumors. In addition, 2% of biliary carcinomas carry V600E substitution in the BRAF oncogene, and 2% of cases are microsatellite unstable. Intrahepatic cholangiocarcinomas, but not other tumors from this category, have frequent involvement of FGFR2 receptor tyrosine kinase and IDH1/2 genes. In total, more than a third of biliary malignancies are amenable to targeted therapy [84].

Melanoma

Approximately 60% of cutaneous melanomas carry BRAF V600E substitution. These tumors are often associated with excessive ultraviolet exposure and, therefore, a high mutation burden and increased antigenic load. The optimal sequence of BRAF inhibitors and immune oncology drugs depends on the particular clinical situation [85, 86]. The incidence of NRAS mutations in melanomas of the skin approaches 15% [87]. These alterations are not currently druggable; however, it is advisable to perform NRAS testing: the presence of NRAS mutations confirms BRAF-negative status of the tumor, while the absence of this event may call, at least in some circumstances, for the search of other alterations in MAPK signaling pathway [88]. Approximately 15% of mucous and acral melanomas carry activating events in KIT receptor tyrosine kinase; unfortunately, only a portion of KIT mutations are druggable by available KIT inhibitors [89].

Thyroid cancer

Medullary thyroid carcinomas are characterized by the high occurrence of activating mutations in RET receptor tyrosine kinase. Importantly, this histological diagnosis calls for germline RET testing irrespective of other clinical features, such as age or family history, as 10–25% of consecutive medullary thyroid carcinomas are hereditary in nature. In addition to germline mutations, a significant portion of these tumors arise due to somatic events affecting the same oncogene. Papillary thyroid cancers have a totally distinct spectrum of druggable genetic events. Approximately half of these carcinomas carry BRAF V600E substitutions. A significant portion of papillary tumors contain druggable rearrangements: RET fusions are relatively common, while NTRK and ALK translocations occur at moderate frequency [90, 91].

Perhaps the most striking molecular feature of thyroid cancer is a commonality of scenarios involving RET oncogene activation, especially given recent invention of RET inhibitors. While medullary cancers develop via mutation-driven activation of RET kinase, papillary tumors often arise due to the emergence of rearrangements affecting this oncogene [90, 91].

Endometrial carcinomas

Endometrial carcinomas have the highest rate of MSI among common tumor types: this event is detected in approximately 20% of malignancies belonging to this category. Although the majority of MSI-positive uterine cancers are sporadic, the possible presence of Lynch syndrome and, consequently, germline testing should be considered in patients with young-onset disease and/or a family history of endometrial or colorectal cancers. POLE mutations are also a mandatory component of DNA analysis in endometrial cancer patients. A subset of endometrial tumors carry amplified and overexpressed HER2 oncogene and, therefore, are amenable to therapeutic HER2 inhibition [92, 93].

Prostate cancer

Similar to pancreatic cancer, the involvement of BRCA2 germline mutations in the pathogenesis of prostate cancer is beyond any reasonable doubt, while the role of the BRCA1 gene is far less clear [82, 94, 95]. Castrate-resistant prostate cancer patients are recommended to undergo testing for somatic mutations in homologous recombination repair (HRR) genes. The HRR test is sometimes confused with the HRD assay, given that both tests are intended to reveal tumors with sensitivity to PARP inhibitors and extend their spectrum beyond BRCA-driven carcinomas. As already explained above, the HRD test relies on the genomic scanning of the tumor karyotype and identifies instances of “BRCA-like” chromosomal instability. In contrast, the HRR assay is not capable of revealing the consequences of deficient homologous recombination, but instead simply supports the analysis for somatic mutations in genes presumably involved in this pathway.

The most well-known HRR panels contains up to 15 genes, including BRCA1 and BRCA2 [94, 96]. The reliance on HRR gene analysis has significant limitations. First of all, even the detection of a somatic mutation in a well-known gene, like BRCA2, does not guarantee the inactivation of the remaining gene allele, i.e., functional BRCA2 inactivation [43]. Furthermore, several genes, which have been included in HRR panels, do not play a role in rendering tumor responsiveness to DNA double-strand break inducing agents [97]. ATM and CHEK2 are relevant examples in this respect, because they usually do not undergo somatic biallelic inactivation even in tumors with germline pathogenic variants. Furthermore, there are several lines of evidence suggesting that ATM and CHEK2 deficiency is not associated with tumor sensitivity to PARP inhibitors or platinum compounds [47, 48]. Molecular genetic testing for prostate cancer deserves to undergo substantial revision in the next few years.

Urothelial carcinomas

FGFR3 up-regulation is the most common druggable event in urothelial cancers. Somewhat surprisingly, activation of this receptor tyrosine kinase is observed in more than a quarter of localized cancers, while this estimate falls to about 15–20% in metastatic forms of this disease. The majority of alterations are point mutations affecting “hot codons”. However, some carcinomas arise due to FGFR3 gene rearrangements. Alterations in other genes belonging to the FGFR family are significantly less common. A significant portion of urothelial malignancies have evidence of activation of the HER2 oncogene via point mutation or gene amplification followed by overexpression. MSI is detected in up to 1% of tumors of this type [98].