# Noninvasive and Multicancer Biomarkers: The Promise of LINE-1 Retrotransposons

Aurelien J Doucet, Gael Cristofari

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## To cite this version:

Aurelien J Doucet, Gael Cristofari. Noninvasive and Multicancer Biomarkers: The Promise of LINE-1 Retrotransposons. Cancer Discovery, 2023, 13 (12), pp.2502-2504. 10.1158/2159-8290.CD-23-1063. inserm-04342008

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# Noninvasive and multicancer biomarkers: the promise of LINE-1 retrotransposons

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Aurelien J. Doucet* and Gael Cristofari*

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University Cote d'Azur, Inserm, CNRS, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, France

* Co-corresponding authors. Correspondence should be addressed to:

Aurelien.Doucet@univ-cotedazur.fr or Gael.Cristofari@univ-cotedazur.fr

## RUNNING TITLE

Circulating retrotransposon protein as cancer biomarker

## SUMMARY

LINE-1 retrotransposons are frequently active in epithelial tumors. In a new study, Taylor, Wu et al. now describe that one of the proteins encoded by LINE-1 elements, ORF1p, is detected in the bloodstream of cancer patients, and can be used as a noninvasive and multicancer biomarker for diagnosis or treatment monitoring.
Although almost half of our genome is made up of repeated DNA sequences, these are not routinely exploited as cancer markers, with the notable exception of microsatellite instability (MSI). Forming a distinct class of repeats, transposable elements are highly dispersed and abundant in the human genome. Among them, the Long Interspersed Element-1 (LINE-1 or L1) is the only element encoding a functional machinery and capable of autonomous mobilization in humans. L1 replication is based on a copy-and-paste mechanism known as retrotransposition, involving an RNA intermediate and a reverse transcription step (1) (Figure 1, top). Active L1 copies are 6kb-long expression units, containing an internal promoter as well as two open reading frames, ORF1 and ORF2. The first encodes an RNA-binding protein (ORF1p, also known as p40), while the second encodes an enzyme with endonuclease and reverse transcriptase activities (ORF2p), both essential for retrotransposition (1). Upon transcription and translation of the bicistronic L1 mRNA, ORF1p and ORF2p proteins assemble with the L1 mRNA to form L1 ribonucleoprotein particles (RNPs) that mediate reverse transcription in the nucleus at sites of integration through a process called target-primed reverse transcription (1).

In most tissues, L1 expression is repressed, preventing or limiting L1-mediated mutagenic activity. However, specific physiological or pathological situations are associated with a profound remodeling of the cellular epigenetic landscape, opening windows of opportunity for L1 expression and even mobilization (2). For instance, the germline and early embryo can harbor high levels of L1 activity. In these territories, new insertions create inheritable polymorphisms and thereby contribute to the genetic diversity of the human population. Occasionally they can also cause genetic diseases (1). L1 expression was also reported in a few somatic tissues, such as the brain or senescent cells thereby contributing to age-associated inflammation (2,3).

As early as the 90's, the L1 ORF1p was found as specifically expressed in some tumors but not in adjacent normal tissues, and L1-mediated somatic mutations could be identified in
colon cancer. More recently, the advent of high-throughput sequencing technologies and the development of monoclonal antibodies against ORF1p have extended these initial observations (4,5). A large survey suggested that nearly half of human epithelial tumors show L1 ORF1p expression (5). Moreover, L1 somatic retrotransposition, as well as other forms of L1-mediated genomic alterations, are detected in several epithelial cancer types such as lung, colorectal, liver, breast, uterine, ovarian, prostate, and head-and-neck cancers, but are virtually undetectable in leukemia or brain tumors (6). Even if examples of L1 insertions acting as drivers of tumorigenesis are limited, L1 promoter hypomethylation and ORF1p levels often correlate with poor prognosis in many cancer types. Moreover, the accumulation of ORF1p in tumors is associated with TP53 loss-of-function, consistent with the notion that high level of L1 activity is associated with genomic instability (5). Altogether, these observations have led to the notion that ORF1p expression in tumors could serve as a biomarker for cancer diagnosis or treatment outcome, but the translation of this idea into clinically relevant assays has been limited so far.

In this issue of *Cancer Discovery*, Taylor, Wu *et al.* (7) develop several ultrasensitive immunoassays to detect L1 ORF1p, and they identify circulating ORF1p as a highly specific cancer biomarker, that is thus amenable to liquid biopsy (Figure 1).

The authors first produced and characterized a new set of monoclonal antibodies and nanobodies targeting ORF1p with high specificity and affinity, which they use to implement digital immunoassays (Figure 1, bottom). These single-molecule approaches are based on a “sandwich” immunocomplex similar to Enzyme Linked Immunosorbent Assays (ELISAs). Beads coated with a first antibody capture the target protein, while a second antibody allows the detection of the first immunocomplex through an enzymatic reaction. However, unlike ELISA, quantification is digital, achieved by recording the number of negative and positive beads (like the 0 and 1 in binary coding) rather than measuring signal intensity, enabling highly sensitive detection of low-concentration targets. These technical developments have
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led to a remarkable sensitivity, reaching the attomolar range in the most sensitive versions of their assays, and to the discovery that free circulating ORF1p can be detected in the bloodstream.

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By comparing healthy donors with patients diagnosed with various forms of cancers of epithelial origin, Taylor, Wu *et al*. revealed that circulating ORF1p is a highly specific multicancer biomarker (7). Patients affected by breast, ovarian, uterine, colorectal, gastroesophageal, pancreatic, liver, lung, head and neck, or prostate cancers display detectable levels of circulating ORF1p, in the range of 10 to 75% of patients depending on the cancer type, while it was only detectable in around 1% of the healthy donors. With a second-generation assay, circulating L1 ORF1p could be detected in as much as 75% of colorectal cancer patients and 90% of ovarian cancer patients. Combining the detection of circulating ORF1p with other currently available biomarkers, such as CA125 and HE4 in ovarian cancer, improved the performance of the diagnosis. Anecdotally, one of the healthy donors tested had a higher level of circulating ORF1p than any of the cancer patients in the study. Although the patient was considered healthy at the time of blood donation, he was in fact subsequently diagnosed with cancer. While most of the ORF1p-positive patients were at a late stage, the sensitivity of the assay also allowed identification of patients with early stages of ovarian and lung cancers. Circulating ORF1p could thus become a powerful multicancer and noninvasive biomarker for early diagnosis and early treatment at a stage when patients are often asymptomatic.

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Finally, Taylor, Wu *et al*. (7) investigated the levels of circulating ORF1p in the plasma of patients that underwent therapy. In a group of gastroesophageal cancer patients that received systemic therapy, circulating ORF1p levels are correlated with the clinical response to treatment. Patients with low ORF1p levels prior to treatment showed a better clinical response than patients with high levels. In this cohort, high levels of circulating ORF1p

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before therapy were also associated with reduced survival. Thus, circulating ORF1p levels could also serve as an early predictor of response to therapy.

Understanding why some tumors express ORF1p and other not is a key question as ORF1p-negative tumors represent a potential source of false negatives. Some cancer types, not studied in the present work, such as neurologic or hematological cancers, appear to reactivate L1 elements to a very limited extent (6). However, even within cancer types of epithelial origin, some patients do not show ORF1p-positive tumors and have undetectable circulating ORF1p (5,7). ORF1p positivity was found to be strongly associated with TP53 loss of function (5,7). By identifying other factors influencing L1 expression in tumors, it may be possible to rationally design panels of complementary circulating biomarkers. In addition, individual human genomes contain a unique combination of inherited L1 loci, some being fixed in the human population, but others being polymorphic with respect to their presence or absence, or to their internal sequence (4,8,9). Among these loci, only a small subset has the potential to express a functional ORF1p and an even more restricted subset is capable of somatic retrotransposition. Thus, it is conceivable that the ability of tumors to express ORF1p or not depends on the ancestry of the patient. Being able to identify which loci contribute the most to circulating ORF1p could help improve and personalize the accuracy of the diagnosis.

Defining the baseline level of circulating ORF1p in a large cohort representing the general population will be essential in the future. What are the tissues of origin of the low ORF1p levels detected in healthy individuals? As mentioned above, L1 expression can occur in cancer cells but also in germ cells and senescent cells. The work by Taylor, Wu et al. (7) suggests that circulating ORF1p is not correlated with age or sex. It is possible that, in some cases, circulating ORF1p in healthy individuals originate from microtumors that would never grow and lead to disease, or would be naturally eliminated by the immune system, raising a risk of over-diagnosis. The question of how ORF1p is specifically released into the
bloodstream by cancer cells and not by other cell types expressing L1 remains open. The
level of expression could simply be higher in cancer cells, but cancer cells may also exhibit
unique release pathways. Size exclusion chromatography experiments suggest at least that
the process does not involve extracellular vesicles (7).

Retrotransposon-derived antigens have recently emerged as potential targets for antitumoral
immunotherapy (10). In the present study, Taylor, Wu *et al*. (7) extend the use of
retrotransposon proteins to cancer diagnostics. They provide a proof-of-concept that the
early detection of circulating ORF1p by digital immunoassays is a promising strategy to
diagnose cancer and monitor the early-response to treatment in a noninvasive manner,
through a simple and cost-effective blood test. Such a noninvasive follow-up could help to
dynamically adjust treatment of cancer patients. Its adoption in the clinical practice will
depend on automation for implementation in clinical laboratories, and on future clinical trials
on larger cohorts to define the practical use of this new biomarker.

## ACKNOWLEDGMENTS

We thank Dr. Dominic van Essen for critical review of the manuscript. This work was
supported by the Agence Nationale de la Recherche (Labex SIGNALIFE, ANR-11-LABX-
0028; Idex UCAJEDI, ANR-15-IDEX-01; ImpacTE, ANR-19-CE12-0032; ActiveLINE, ANR-
21-CE12-0001), the Institut National du Cancer (INCa PLBIO 2020-095), Inserm (GOLD
cross-cutting program on genomic variability), CNRS (GDR 3546). This work was funded, in
whole or in part, by the Agence Nationale de la Recherche, Grant ANR-21-CE12-0001. A
CC-BY public copyright license has been applied by the authors to the present document
and will be applied to all subsequent versions up to the Author Accepted Manuscript arising
from this submission, in accordance with the grant's open access conditions.
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VERSION OF RECORD

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Noninvasive and Multicancer Biomarkers: The Promise of LINE-1 Retrotransposons. Doucet

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AJ, Cristofari G. *Cancer Discov*. 2023 Dec 12;13(12):2502-2504. doi: 10.1158/2159-

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8290.CD-23-1063

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The pdf of the Version of Record is available at the following address:

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<https://aacrjournals.org/cancerdiscovery/article-abstract/13/12/2502/731617/Noninvasive-

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and-Multicancer-Biomarkers-The-Promise

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6. Rodríguez-Martín B, Alvarez EG, Baez-Ortega A, Zamora J, Supek F, Demeulemeester 166 J, et al. Pan-cancer analysis of whole genomes identifies driver rearrangements promoted 167 by LINE-1 retrotransposition. Nat Genet. 2020;52:306-19. 168

7. Taylor MS, Wu C, Fridy PC, Zhang SJ, Senussi Y, Wolters JC, et al. Ultrasensitive 169 detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker. Cancer Discov 170 [Internet]. 2023 [cited 2023 Sep 14]; Available from: https://doi.org/10.1158/2159-8290.CD- 171 23-0313 172

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**Figure 1 – A circulating L1 retrotransposon protein, ORF1p, as a noninvasive, broad usage, multicancer biomarker.**

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**Top:** Schematic representation of an L1-expressing cancer cell within a vascularized tumor. An active L1 locus is transcribed (blue box, in black chromosome) to synthesize bicistronic L1 mRNAs (wavy lines). Translation occurs in the cytoplasm to produce ORF1p (blue circle) and ORF2p (purple oval). An L1 ribonucleoprotein particle (RNP) is formed by the association between the L1 mRNA, ORF1p and ORF2p. By entering in the nucleus, the L1 RNP can insert a new L1 copy into another locus (black box in grey chromosome) through DNA cleavage and a process called Target-Primed Reverse Transcription (TPRT) (1).
Taylor, Wu *et al*. describe the presence of ORF1p in the bloodstream (blue dots in red vessels) of patients diagnosed with a subset of epithelial cancers (7). The mechanism by which ORF1p is released from cancer cells and reaches the bloodstream is currently unknown (dashed arrows with question mark).

**Bottom:** Ultrasensitive detection of circulating ORF1p from blood samples. The procedure is based on a simple blood test. Taylor, Wu *et al*. measured plasmatic levels of ORF1p by digital immunoassays relying on a bead-based “sandwich” immunocomplex, which combines capture and detection antibodies or nanobodies with high affinity for ORF1p (7). Two strategies were employed: Single Molecule Arrays (Simoa), and Molecular On-bead Signal Amplification for Individual Counting (MOSAIC). Simoa is performed in a plate format with each well containing a single bead through an enzymatic detection, whereas MOSAIC relies on the evaluation of positive and negative beads by flow-cytometry through a secondary amplification process and fluorescence. Ultimately, the presence of circulating ORF1p can be used for the diagnosis and monitoring of cancer patients in a noninvasive manner, and can apply to multiple cancer types, such as colorectal or ovarian cancers.

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