This is a rather long blog post, and for that reason, I will first provide an outline of the whole blog post, so if you are interested in some specific segment of it, you can simply jump to the given section right away. So, here is the outline
Shortly About the ROS1 Gene
In this section, I will briefly introduce the ROS1 gene, explain what kind of protein encodes, and explain the function of the protein it encodes, which is actually a type of tyrosine kinase. You can learn more about tyrosine kinases in my short blog post here.
What Does the ROS1 Gene Stand For? Who Discovered the ROS1 Gene?
The ROS1 gene is an abbreviation for the ROS proto-oncogene 1, receptor tyrosine kinase gene, discovered by Matsushime et al. in 1986 [citation]. More, the abbreviation ROS stands for Reactive Oxygen Species.
Where Is ROS1 Located?
The ROS1 gene is located on the long arm of chromosome 6, roughly 138,713 bases MB long [citation].
What Does the ROS1 Gene Do?
The ROS1 gene encodes a protein, as mentioned above, the ROS proto-oncogene 1, a receptor tyrosine kinase. In this post, I will call it the ROS receptor tyrosine kinase. If you want to learn more about tyrosine kinases, check out this short blog post.
What Is the Function of the ROS1 Receptor Tyrosine Kinase?
The ROS receptor tyrosine kinase is a transmembrane receptor protein involved in cellular proliferation, differentiation, migration, and cell death processes [citation].
Why Is the ROS1 Receptor Tyrosine Kinase Important?
The ROS receptor tyrosine kinase is a surface receptor [citation] is involved in the signal transduction pathway. The signal transduction pathway is further essential for growth, cellular differentiation, proliferation, migration, and cell death.
Interestingly, the ROS receptor tyrosine kinase (RTK) protein has been characterized as an orphan RTK because its ligand has not yet been identified. You can check more about tyrosine kinases here.
As I mentioned, the ROS receptor tyrosine kinase is involved in signal transduction and cellular communication and it does this by regulating down-signaling pathways in the following ways:
It phosphorylates and activates and activates STAT3 (required for ROS1 anchorage-independent growth) [citation]
It phosphorylates VAV3
It activates the PI3K/AKT/mTOR signaling pathway.
It binds and activates PTPN6
It binds and activates the SH2 domain [citation].
To summarize, the ROS receptor tyrosine kinase is involved in regulating:
Cell proliferation
Cell differentiation
Cell migration, and
Cell death [citation].
Where Is the ROS1 Gene Normally Expressed?
The ROS1 gene is expressed during development, but little is expressed in adults [citation]. The ROS1 gene expression levels have been highest in the liver, platelets, T-cells, and monocytes but are found across nearly all cell types. Interestingly, and as mentioned above, the ROS receptor tyrosine kinase in wild type remains unclear with an undefined ligand [citation].
What Are Tyrosine Kinases?
I mentioned tyrosine kinases several times throughout this blog post. So, I would like to explain what tyrosine kinases are all about. Tyrosine kinase is a family of enzymes that can transfer a phosphate group from ATP to a protein in a cell, and this process is also called phosphorylation. Phosphorylation is crucial in many processes related to cell communication and cell proliferation, differentiation, migration, and cell death.
To learn more about tyrosine kinases and phosphorylation, check my blog posts here: phosphorylation and tyrosine kinases.
The ROS1 Gene and Mutations
In this section I will cover the ROS1 gene and its mutations. It is important to note that when I use the word mutations in this blog post it actually refers to any type of genetic alteration not only for example point mutations but also more complex genetic rearrangements such as gene fusions. From time to time, I will use specifically term gene fusion which is a special case of genetic rearrangement or just mutations to cover any type of genetic alterations in the ROS1 gene including gene fusions and any other type of genetic mutation or genetic altertions.
What Are the ROS1 Mutations? What Are the Most Important ROS1 Mutations
Just to remind you, the ROS receptor tyrosine kinase that is encoded by the ROS1 gene is involved in the processes of cell proliferation, differentiation, migration, and cell death; and mutations in the ROS1 gene have been associated with several human tumors including
Cholangiocarcinoma
Ovarian carcinoma
Gastric carcinoma and others [citation]
The specific type of genetic alterations in the ROS1 gene associated with various cancers are gene fusions. Gene fusion is a genetic phenomenon where a gene joins to a part of another gene, and as a consequence, in the case of the ROS1 gene, this leads to uncontrolled cell growth and cancer.
Genetic alterations (mainly gene fusions with other genes) lead to the oncogenic potential of the ROS1 gene, constitutively activating the ROS tyrosine kinase (constitutively activating means that the ROS tyrosine kinase can not be turned off), which likely drives malignant cell proliferation. Therefore, accurate identification of ROS1 fusions is critical to guiding targeted therapies [citation].
These gene fusions are not hereditary; they are acquired during life, meaning those are somatic alterations [citation]. Furthermore, there are other ROS1 mutations, point mutations, which also lead to the acquisition of resistance to the ROS1 inhibitors [citation].
To learn more about somatic mutations and or gene fusions check my short blog posts about these gene fusions and on somatic and germline mutations.
As already mentioned, oncologists have focused attention on the ROS1 gene fusions because ROS1-activated kinase is a crucial feature for cellular proliferation and survival [citation].
The ROS1 fusions are, as already briefly explained, translocations due to chromosomal rearrangements and are the most common type of clinical mutations in the case of the ROS1 gene. To date, more than 20 ROS1 fusion partner genes have been identified. Among these gene fusion partners, the CD74-ROS1 is the most frequently detected ROS1 fusion [citation]. Again, to get the full picture of what gene fusion is check my blog post about gene fusions here.
What Causes ROS1 Mutation?
Changes in the ROS1 gene are caused by the above-described genetic alterations, specifically by the above-mentioned gene fusions. However, the mechanism by which the ROS1 fusion proteins become constitutively active is not precisely known [citation].
The ROS1 Gene Fusions and Cancer
In this section, I will shortly present the link between ROS1 gene fusions and specific types of cancers for which ROS1 gene fusions act as driver mutations.
What Is the Role of ROS1 in Lung Cancer?
Several studies have shown aberrant/overexpression of wild-type ROS1 in several cancers, including non-small cell lung cancer (NSCLC) [citation].
The gene fusion SLC34A2-ROS1 fusion [citation], and CD74-ROS1 fusion [citation] are marked as likely driver mutations in NSCLC. You can read more about gene fusions here.
ROS1 gene fusions has been identified with an incidence of 0.5–2% in NSCLC [citation]. Detection of ROS1 gene fusions is critical for the optimal treatment of ROS1-positive NSCLC patients [citation], but more about in my other blog post that is focused on the ROS1 tyrosine kinase and its inhibitors that you can check here [INSERT].
What Is the ROS1 Gene Rearrangement?
ROS1 rearrangement (particularly gene fusions) is first detected in a glioblastoma cell line. The entire ROS1 kinase domain is retained in the rearrangement of ROS1 at the 3′ ends in human glioblastoma cells. The genetic alteration occurs in particular gene fusions that make a constitutively active ROS1 tyrosine kinase, which means that after that mutation, it can not be turned off, leading to uncontrolled cell proliferation [citation]. ROS1 gene fusions were independently associated with: (i) female sex, (ii) younger age at diagnosis, and (iii) absence of smoking history [citation].
That information is based on a meta-analysis (of 9898 patients [citation]). ROS1 rearrangement is found in many malignancies, including cholangiocarcinoma, ovarian carcinoma, gastric carcinoma, and non-small cell lung cancer (NSCLC) [citation].
How Are Common ROS1 Mutations in Non-Small Cell Lung Cancer (NSCLC)?
ROS1 rearrangements characterize about 0.5%–2% of unselected non-small cell lung cancer (NSCLC) patients [citation]. Data about studies related to ROS1 presence vary. ROS1 gene fusion was found in 141 of 5547 patients (2.54%) with adenocarcinoma. The prevalence is higher among patients in Asia than in western countries (Europe and USA) [citation].
What Is Gene Fusion?
Gene fusions are products of joining the parts from two different genes. This causes the uncontrolled function of fused genes. You can check my short blog post about gene fusion here.
What Are the Most Common ROS1 Gene Fusions?
At present, 26 genes were found to fuse with ROS1 [citation]. FIG was first identified gene partner for fusion with ROS1. Fusion with FIG was found in glioblastoma, cholangiocarcinoma, and lung adenocarcinoma [citation]. The most common gene fusions of ROS1 in lung adenocarcinoma are with the next genes:
CD74 (41%)
SLC34A2 (18%)
EZR (10%)
SDC4 (9%)
TPM3 (4%)
GOPC (2%) and
with other genes (16%) [citation].
All rearrangements involve the fusion of the 3′ regions of the kinase domain of ROS1 to the 5′ regions of the partner gene.
What Is the Effect of ROS1 Gene Fusions On Other Genes?
Gene fusions are detected in solid tumors. The distribution of fusion genes is very important for scientists in discovering combinations and connections between ROS1 and other genes. The function of the ROS1 gene is crucial for, for example, signaling pathways, and any disruption of the normal flow of these processes affects the development and form of the disease.
ROS1 fusion appears frequently in inflammatory myofibroblastic tumors [citation], adenocarcinoma of the stomach [citation] and the colon [citation]. SLC34A2, fusion has been identified in gastric, colon, and lung adenocarcinoma [citation].
What Are the Consequences of ROS1 Gene Fusions?
From what I have written so far, it is evident that ROS1 gene fusions can be considered driver mutations in cancer development. There are treatments with inhibitors on ROS1-positive patients (more about that in a separate blog post I wrote here [INSERT]). Still, over time, mutations can occur that further create a problem related to resistance to inhibitors. There are no approved second-line targeted therapies for crizotinib, ceritinib, or entrectinib-resistant ROS1+ NSCLC, but there are promising data from next-generation ROS1 inhibitor trials [citation].
Testing for ROS1 Gene Rearrangements
In this section, I will provide a short overview of the testing methodologies and actual genetic tests used to detect ROS1 gene rearrangements.
What Is the Preferred Test for Confirming Potential ROS1 Rearrangements?
Detection of the ROS1 gene rearrangements is mandatory in order to treat patients with positive-ROS1 non-small cell lung cancer (NSCLC).
Testing for ROS1 rearrangements involves a biopsy of lung cancer from patients and using FISH (dual color “break-apart” probes) methodology which is considered as the gold standard in light of its use in determining ROS1 positivity in clinical trials [citation].
ROS1 biomarker testing determines ROS1 status in patients, whether mutation (gene rearrangements) of the gene is present or absent. The identification of ROS1 rearrangement is of crucial interest in NSCLC patients due to the therapeutic consequences it generates about which I wrote in a separate blog post about the ROS1 gene and tyrosine kinase inhibitors used for treatment of the ROS1-positive cancers [citation].
When I mention ROS1 gene rearrangements I am actually referring to ROS1 gene fusions. So, from now on in the post I will use the term ROS1 gene fusions as the main type of ROS1 gene rearrangements.
How Do You Test for ROS1 Gene Fusions?
FISH testing does represent routine practice in laboratories. FISH testing for ROS1 is applicable either on biopsy or in cytologic specimens. Important to note that some break-apart FISH assays for ROS1 fusion cannot detect intrachromosomal deletion [citation].
The first step in the detection of ROS1 gene fusion is histological examination of the tissues collected through biopsy.
Then, screening for rearrangement of the Activated anaplastic lymphoma kinase (ALK) gene (positive or negative result).
In case of a positive result for the ALK1 gene, the sample is used for testing on ROS1 Immunohistochemistry (IHC).
Positive ROS1 IHC leads to FISH analysis [citation]. The FISH probes label the 3′ (centromeric) part of the fusion breakpoint with green fluorochrome and the 5′ (telomeric) part with an orange fluorochrome (dual color break apart probe) [citation].
IHC is an effective screening tool to detect ROS1-positive NSCLC since either sensitivity or specificity may reach over 90% compared to FISH results. IHC is cost-effective [citation].
What Are Gene Technologies Used to Test for ROS1 Alterations, Including Mutations and Gene Fusions?
The following gene technologies are used to build tests for the detection of the ROS1 gene alterations [citation]:
Immunohistochemistry
Fluorescence in situ hybridization (FISH)
Real-time reverse transcription-polymerase chain reaction (RT-PCR), and
Next-generation sequencing (NGS)
With the development of genetic sequencing methodologies that new type of assays for detection of ROS1 gene fusions is becoming a new promising tools. These assay are called extractive assays and are based on RT-PCR and massive parallel NGS and these methods use single and very small amount of tumor RNA. RT-PCR exhibits quite good sensitivity and specificity.
The European Board of Pathologists suggested that standard clinical practice algorithm based on IHC screening with further confirmation by ROS1 break-apart FISH assay in IHC-positive and doubtful cases is sufficiently good.
However, a single-tube essay (above-mentioned RT-PCR and NGS based methodologies) with a high extractive method are probably the future in detecting ROS1 gene fusions [citation] and actually there are already two FDA-approved NGS panels for detection of ROS1 gene fusions: FoundationOne CDx Test and Oncomine Dx Target Test.
What Are the Commercial Tests for Testing ROS1 Gene Fusions Available?
As I already explained above,there are fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) based tests, and non-in situ approaches such as NGS and RT-PCR are commercially available assays [citation].
Currently, there are two cleared or approved companion diagnostic tests by FDA for the detection of ROS1 gene fusions:
Test FoundationOne CDx developed by Foundation Medicine, Inc. for which Non-Small Cell Lung Cancer (NSCLC) tissue is used and ROS1 gene fusion-positive patients may benefit from treatment with Rozlytrek® (entrectinib).
The other one is Oncomine Dx Target Test developed by Life Technologies Corporation, where Non-Small Cell Lung Cancer (NSCLC) tissue is used, and ROS1 gene fusion-positive patients may benefit from treatment with Xalkori (crizotinib).
What Does ROS1-Negative Mean?
The ROS1-negative means that no ROS1-gene fusions are detected. The ROS1-negative serves as a control in testing for ROS1-positive detection [citation]. If there is a suspicion that ROS1-negative patients are still positive, although they are not positive for other oncological driver genes, it is recommended to use another test for detection (a verification test) [citation].
What Does ROS1-Positive Mean?
In ROS1-positive patients, adenocarcinoma is presented as a predominant histological subtype. The ROS1 chromosomal rearrangements were described in non-small cell lung cancer (NSCLC) in 2007. Patients are usually younger females with no smoking history. ROS1-fusion-positive (ROS1-positive) NSCLC accounts for 1–2% of 80% of NSCLC-positive patients [citation].