Structural variations are recognized as underlying hallmarks of cancer^1,2. At the same time, these genomic alterations can serve either as predictive or prognostic markers, to help physicians devise adequate plans and personalized treatment options for their patients. Unsurprisingly, there has been a growing interest for robust gene fusion screening methods. This increasing demand is also warranted by the fact that clinical trials have now transitioned - from site-of-origin and histological subtype-specific designs - towards basket trials, which focuses on testing targeted therapies in different diseases that share specific genetic alterations.³

In our newest Opinion & Review segment, we scrutinize 3 publications that evoke the topic of structural variations leading to oncogenic gene fusions. In our view, these single-handedly picked papers perfectly underscore the need and urgency for reliable gene fusion-calling testing, to further advance cancer diagnostics and improve patient outcome.

I. Oncogenic fusion partner: an emerging target for cancer diagnostics and treatment

In many rare cancer types (including sarcomas), NTRK gene fusions are known oncogenic drivers. Fortunately, this event can be counteracted with small molecule (TRK) inhibitors, which have shown extremely encouraging results in patients. Unnervingly, more common cancers have a low but significant frequency of NTRK fusion too.⁴ As such, a sizeable at-risk patient population should undergo NTRK fusions screening.⁵

Since most studies on mesenchymal tumors have primarily focused on soft tissues, data on bone tumors are rather scarce.^6,7 Therefore, Dutch LUMC researchers investigated the frequency of NTRK fusions in 3 of the most common bone sarcomas (i.e., osteogenic, chondrogenic tumors and Ewing sarcoma) – for potential treatment with targetable TRK-inhibitors. Of note, these scientists conducted their research in accordance with recommendations laid by the World Sarcoma Network (WSN), which advises immunohistochemical (IHC) pre-screening.^6,8

In this comprehensive study, the authors initially performed IHC evaluation and opted AMP-based (RNA) targeted NGS as a subsequent method to dissect fusions in selected cases.⁸ 19 cases were positive in IHC, albeit the majority with weak staining. Molecular analysis was successful in 6 of those, which were all found to be negative for NTRK fusions.

All in all, it was concluded that the likelihood of finding a NTRK fusion in bone tumors and in clinical practice are slim to none. This also appears to be true for tumors with complex genomes lacking driver alterations (e.g. osteosarcoma). Nevertheless, it is also important to acknowledge a limitation accompanying this study⁸. Indeed, false-negative results due to the effect of decalcification on pan-Trk expression cannot be ruled out. Therefore, the present and subsequent studies might benefit from an orthogonal fusion detection method that is (1) uninfluenced by decalcification and (2) able to detect any unexpected or complex fusions.

II. Inherent constraints that plague standard lab testing render their independent usage unreliable

Clinical researchers from the University of Colorado Anschutz Medical Campus have extensively studied the implications of ROS1 fusions in lung cancer. In short, patients who test positive for ROS1 are usually prescribed with crizotinib.^9,10 However, standard laboratory tests come with intrinsic limitations and can therefore, lead to confounding results. This hindrance poses great concern, since true ROS1-positive patients might go unnoticed. Consequently, this also means that the overlooked patients will be unable to benefit from appropriate treatments due to false negatives being returned. In a first-of-its-kind study, scientists decided to perform a comparative evaluation between break-apart FISH, RNA-based NGS and DNA-based NGS, to weigh in on each of their accuracies.¹¹

Challenges were found to stem from various factors, including:¹¹

• a fusion partner that occurs in close proximity to ROS1. In which case, the resulting split signals from break-apart probes might still be too close and could therefore, lead to false-negative calls with FISH assay

• large and/or hard to sequence regions (e.g. 2 large repetitive areas that sit in intron 31 of ROS1). If those were to harbor a ROS1 variant, it would prove challenging for conventional DNA-based NGS techniques to identify it (see Figure 1)
• RNA-based assay relies on high RNA quality. Therefore, this might not make it an ideal fit for clinical samples
  
  

Figure 1. DNA-based NGS coverage. IGV screenshots whereby sequencing coverage are represented by grey and colored bars. Blank areas reflect a drop of coverage, which is accounted (in this case)
by large (hard-to-sequence) repetitive sequences found within intron 31 of ROS1. The black bars denote the approximate positions of L1PA8A and L1PA10 (2 large long interspersed nuclear elements).
Figure retrieved from the paper “Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples” by Davies et al.¹¹

In a nutshell, this paper calls for careful attention on the deficiencies that go along with these classical assays, to correctly interpret results. In fact, the authors caution to not hastily rely on one result derived from a single test. Instead, they recommend validating any preliminary finding with a secondary orthogonal assay.¹¹

III. Hunting for optimal gene fusion detection methods

In a similar vein, the European Society for Medical Oncology (ESMO) has joined forces with Precision Medicine Working Group (TR and PM WG) to perform a comprehensive evaluation of currently available methods (see Table 1). Through this joint effort, they aimed to detect RET (rearranged during transfection) rearrangements, which are frequently found in non-small-cell lung cancer (NSCLC) and are key drivers of tumorigenesis.¹²

Table 1. Comparison of currently available technologies to detect RET rearrangements.
Figure retrieved from paper “ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research” by Belli et al.¹²

Nevertheless, ESMO takes it a step further by mapping out a clear step-by-step guideline for routine clinical detection of RET fusion genes (rearrangements) and mutations, in order to identify patients eligible for anti-RET therapy. As such, their recommendations are also featured in this same review (see Figure 2A).¹²

Figure 2. (A) Recommended RET testing algorithm for non-small-lung cancer (NSCLC), thyroid tumors other than medullary thyroid cancer (non-MTC) and other solid tumors.
Figure retrieved from paper “ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research” by Belli et al.¹²

There is an unquestionable need to discern optimal assays for the detection of RET alterations, as they promote cancer in various solid tumors. New selective RET inhibitors, such as pralsetinib and selpercatinib, have already shown marked and durable antitumor activity in patients. As part of ESMO’s recommendations, it is interesting to see that RET IHC testing has been excluded from their guideline. In fact, the sensitivity and specificity of IHC was considered to be suboptimal and therefore, deemed as an unreliable testing modality in this setting.¹²

IV. Filling an important gap in the cancer diagnostics market

As soon as gene fusions were found to drive oncogenesis, researchers raced to identify assays that could reliably characterize those oncogenic fusion partners.^11,12 To date, the search is ongoing as current NGS-based solutions appear to still be suboptimal. In the meantime, many have been relying on the concurrent usage of 2 or more methods to confirm their results and to circumvent potential false-negative results.¹¹ Indeed, standard techniques are often challenged by several limitations, most of which are inherent.¹¹ Therefore, the robust detection of all genetic mutations, including structural variants in and around genes of interest still remains a challenge (especially for FFPE specimens).¹³ In response to this unmet medical need, we have committed ourselves to developing a new revolutionary tool for cancer diagnostics.

In an unprecedented nationwide effort, we introduced our novel method (called FFPE-TLC) for accurate and automatic detection of clinically relevant translocations in FFPE tumor biopsies. In close collaboration with experts from the Hubrecht Institute and 5 Dutch hospitals, we underscored the advantages of our FFPE-TLC approach over conventional NGS-based technologies (in terms of sensitivity and specificity) by analyzing 149 (anonymized) lymphoma and control FFPE samples. A work that eventually culminated in a recent Nature Communications paper.¹⁴

Unlike other standard approaches, our FFPE-TLC can:

• identify rearrangements in areas difficult to target or sequence
• pinpoint fusions between genes in close proximity within the same chromosome
• distinguish between relevant and non-relevant rearrangements
• uncover both known and previously identified complex rearrangements
• avoid the need for manual curation
  
  

“After discussing with several key opinion leaders in the oncology diagnostics field, we are now convinced that we have a unique method to offer. That is why we are determined to find the fastest route to bring this to patients” highlights Harma Feitsma, our Head of Business Development of Oncology.

Therefore, our revolutionary FFPE-TLC approach paves the way for personalized therapy and holds great promise for improved cancer diagnostics. Noteworthily, our FFPE-TLC method is compatible for integration with standard targeted NGS workflows.

To find out more about Cergentis’ future plans in the oncology space and the commercialization avenues that we seek to explore, we invite you to read this recent interview conducted by GenomeWeb, which features our CEO Joris Schuurmans.¹⁵ 

If you would like to get in touch, to discuss about a potential collaboration, please reach out to our Harma Feitsma (Head of Business Development, Oncology) at: harma.feitsma@cergentis.com

References

[1] Li, Y. et al. (2020). Patterns of somatic structural variation in human cancer genomes. Nature 578, 112–121.

[2] Macintyre, G., Ylstra, B. & Brenton, J. D. (2016). Sequencing structural variants in cancer for precision therapeutics. Trends Genet. 32, 530–542.

[3] Solomon JP, Benayed R, Hechtman JF, Ladanyi M. Identifying patients with NTRK fusion cancer. Ann Oncol 2019; 30; viii16-viii22.

[4] Cocco E, Scaltriti M, Drilon A. (2018). NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol; 15; 731-747.

[5] Solomon JP, Benayed R, Hechtman JF, Ladanyi M. (2019). Identifying patients with NTRK fusion cancer. Ann Oncol; 30; viii16-viii22.

[6] Demetri GD, Antonescu CR, Bjerkehagen B et al. (2020). Diagnosis and management of tropomyosin receptor kinase (TRK) fusion sarcomas: expert recommendations from the World Sarcoma Network. Ann Oncol; 31; 1506-1517.

[7] Siozopoulou V, Smits E, De Winne K, Marcq E, Pauwels P. (2021). NTRK Fusions in Sarcomas: Diagnostic Challenges and Clinical Aspects. Diagnostics (Basel); 11.

[8] Lam SW, Briaire-de Bruijn IH, van Wezel T, Cleven AHG, Hogendoorn PCW, Cleton-Jansen AM, Bovée JVMG. (2021, June 20) NTRK fusions are extremely rare in bone tumours. Histopathology. DOI: 10.1111/his.14432. Epub ahead of print. PMID: 34148257.

[9] Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012; 30: 863–870.

[10] Davies KD, Le AT, Theodoro MF, et al. (2012). Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res.; 18:4570–4579.

[11] Davies KD, Le AT, Sheren J, Nijmeh H, Gowan K, Jones KL, Varella-Garcia M, Aisner DL, Doebele RC. (2018, October). Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples. J Thorac Oncol.; 13(10):1474-1482. DOI: 10.1016/j.jtho.2018.05.041. Epub 2018 Jun 20. PMID: 29935306; PMCID: PMC6286810.

[12] Belli C, Penault-Llorca F, Ladanyi M, Normanno N, Scoazec JY, Lacroix L, Reis-Filho JS, Subbiah V, Gainor JF, Endris V, Repetto M, Drilon A, Scarpa A, André F, Douillard JY, Curigliano G. (2021, March) ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research. Ann Oncol.; 32(3):337-350. DOI: 10.1016/j.annonc.2020.11.021. Epub 2021 Jan 14. PMID: 33455880.

[13] de Vree, P., de Wit, E., Yilmaz, M. et al. (2014). Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025. https://doi.org/10.1038/nbt.2959

[14]Allahyar, A., Pieterse, M., Swennenhuis, J. et al. (2021). Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing. Nat Commun 12, 3361. https://doi.org/10.1038/s41467-021-23695-8

[15] Petrone, J. (2021, July 01). Cergentis Highlights TLA Tech in Lymphoma Study, Seeks Commercialization Partner. GenomeWeb. https://www.genomeweb.com/sequencing/cergentis-highlights-tla-tech-lymphoma-study-seeks-commercialization-partner#.YOhtJugzZPY