Patent Application
20220119893
The present invention concerns the classification of tumor brains and the choice of therapeutic options useful for treating patients with tumor brains, based on said classification.
A glioma is a type of tumor deriving from the glial cells of the brain or the spine. Gliomas represent about 30% of all brain and central nervous system tumors, and 80% of the malignant brain tumors.
Malignant gliomas are graded from grade II to grade IV, although benign gliomas are designated as gliomas of grade I.
According to the 2016 WHO classification (4th Edition) of tumors of the central nervous system (CNS), summarized in (Louis et al., 2016), malignant tumors of the CNS are classified according to immuno-histological and molecular criteria into different categories:
Gliomas are defined as tumors of the category “diffuse astrocytic and oligodendroglial tumors”. In this 4th edition of the WHO classification, gliomas are defined with molecular biomarkers, and then subclassed into the following subclasses: the oligodendroglioma (OD), the astrocytoma (A), the glioblastoma (GBM), the diffuse midline glioma and the oligoastrocytoma.
Molecular features define theses subclasses:
When no molecular marker is available, glioma are classified as being “NOS” (for Not Otherwise Specified). In this circumstance, glioma is designated with the term “oligoastrocytoma”.
Histological criteria such as vascularization, necrosis or proliferation further refine each subclass into different grades: diffuse (grade II) or anaplasic (grade III) glioma. Grade IV, the most advanced stage, defines glioblastoma.
Because of their diffusely infiltrating nature, grade II to IV gliomas cannot be completely resected and are not curable by surgical excision.
Each subclass of glioma is defined by a median time of overall survival (OS) of the patients affected by said glioma. A brief summary of features of each subclass is presented below.
Among glioblastoma, clinicians distinguish primary glioblastoma that have an IDHwt status, and secondary glioblastoma that present a mutation in IDH 1 and/or IDH 2 (IDHmt) and that are supposed to derive from low-grade, IDHmt astrocytoma.
Patients affected with a secondary glioblastoma present a better median overall survival than those affected with primary glioblastoma: the overall survival of patients with primary glioblastoma ranges from 7 to 15 months, whereas the overall survival of patients with secondary glioblastoma is about 2 years and a half.
Diffuse astrocytoma IDHmt (grade II), anaplasic astrocytoma IDHmt (grade III), glioblastoma IDHmt (grade IV), glioblastoma IDHwt (grade IV) are mainly reported in adults.
Histopathological classification is the basis of the World Health Organization (WHO) classification; however, it suffers from a high variability of interpretation from one practician to another. This is also due to the fact that tumors from the same category are highly heterogeneous. Consequently, therapeutic strategies may be wrongly chosen, if the glioma is incorrectly classified.
Recently, IMPACT-NOW, the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy, has produced additional guidelines for glioma sub-classification. These guidelines designate as «NEC» (Not Elsewhere Classified) tumors with histo-molecular characteristics that cannot be classified according to the WHO 2016 diagnosis (for example, because of a mismatch between clinical, (immuno)histological and/or genetic features)1-4.
Among NEC, the “astrocytoma IDHwt” subgroup consists in grade II and III glioma with astrocytoma features (based on morphological and immuno-histological parameters) but without any IDH mutation.
This “astrocytoma IDHwt” subclass is particularly heterogeneous in terms of response to treatment and overall survival, with a much more severe prognosis than the IDH mutated astrocytoma. The 3rd update of the IMPACT-NOW consortium advises to class these tumors as “Diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV” if any of the following markers are observed: EGFR amplification, whole chromosome 7 gain and whole chromosome 10 loss (+7/−10) or TERT promoter mutation. If none of these markers is retrieved, no guideline is available for the treatment of said “NEC” tumors.
Beside histological features and specific molecular biomarkers, an additional defining feature for gliomas is the characterization of the telomere maintenance mechanisms (TMMs).
Telomeres are DNA-protein complexes present at the end of chromosomes in eukaryotic cells, which play a crucial role in cellular survival. Indeed, in healthy cells, a gradual shortening of telomere happens at each replicative cycle. When telomeres reach a critical size, cells stop to proliferate and enter in senescence. Most cancer cells maintain the length of their telomeres by the reactivation of telomerase, or through the telomerase-independent alternative lengthening of telomeres (ALT) mechanism.
A retrospective study of 573 glioblastoma patients showed that ALT+ glioma patients had longer survival, which suggests that most ALT+ tumors may be less aggressive gliomas (McDonald et al., 2010). Another prognosis studies were realized on high-grade astrocytomas (Nguyen et al., 2012).
(Mangerel et al., 2014) and (Fogli et al., 2017) teach the use of a C-circle assay for determining the ALT status of human glioma samples. Both articles demonstrate that this specific C-circle assay is useful for the detection of ALT activation.
(Hakin-Smikth et al., 2003) reports the analysis of both telomerase activity (ALT status) and telomere lengths in tumors from 77 patients having glioblastoma multiforms, of primary or secondary types.
In view of the discordant findings from these studies, the prognosis value of the ALT status is still uncertain. In particular, a glioma classification method taking into account the ALT status of the tumor, among other parameters, is still to be defined.
The international application WO 2017/127803 proposes a glioma classification method based on multiple molecular features, comprising:
This method is based on the measure of at least six parameters, including molecular parameters. In view of (i) the quantity of biological material, (ii) the high cost and (iii) the time-consuming techniques that are necessary for measuring these parameters, this classification process would not be feasible in an usual clinical practice. Moreover, the timing for choosing a therapeutic strategy would be too long in regard to the duration of this diagnosis process.
A relevant classification of gliomas is the key for improving the therapeutic strategies and hopefully the clinical outcomes.
Currently, hospital services classify gliomas according to immuno-histological features and on the basis of available molecular biomarkers (such as mutation of TERT, mutation of histones, mutation of IDH, amplification of EGFR, deletion of CDKN2A). This classification process is fastidious, expensive, and variable from one hospital to another, since highly dependent on the pictures interpretation of the pathologist and on the molecular biology facilities available in the hospital.
Since the current classification process is not satisfying, active research is currently conducted in order to identify and classify as precisely as possible gliomas, in a clinical environment, for helping health practitioners to choose the best therapeutic approach for patients.
Ideally, an efficient classification process would answer to the following criteria:
The present invention discloses a process of classification of gliomas that present all the advantages listed above.
The present invention relates to an in vitro process for classifying a glioma, comprising the following steps:
Among said five classes, gliomas of low grades may be further refined as being of grade II or III, as a function of the histological grading, if available/known.
Furthermore, gliomas of the class “low-grade astrocytoma-like” may be subclassified as belonging to one of the two subclasses designated as t-low grade Astrocytoma (tLGA) and t-Astrocytoma grade IV (tA-IV).
In another specific implementation of this process, the ALT status in (a) is measured by performing a C-circle assay coupled to a telomere-specific PCR (TeloPCR), thereby obtaining a C-circle value and selecting the ALT status of the glioma after comparison of said C-circle value to threshold values, determining several classes, for example four classes: “ALT−”, “ALT intermediate”, “ALT+”, and “ALT++”.
In another specific implementation of this process, the ALT status in (a) is measured by performing a C-circle assay coupled to a telomere-specific PCR, thereby obtaining a C-circle value and selecting the ALT status of the glioma after comparison of said C-circle value to threshold values, determining three classes:
In another specific implementation of this process, step (a) further comprises the measure of the telomere length status of said glioma.
In this specific implementation of this process, the telomere length status is measured by quantifying the telomeric DNA with a telomere-specific PCR, thereby obtaining a T-length value and selecting the telomere length status of the glioma after comparison of said T-length value with threshold values determining the 3 following classes: “short”, “intermediate” and “long”.
The present invention also concerns the use of the in vitro process as described herein for reclassifying a glioma of the group “astrocytoma IDHwt” or of the group “NEC”, according to the WHO's classification, in one of the following classes: oligodendroglioma-like, glioblastoma IDHwt-like, glioblastoma IDHmt-like, t-low grade Astrocytoma (tLGA) and t-Astrocytoma grade IV (tA-IV), and other gliomas
Among said classes, gliomas of low grades may be further refined as being of grade II or III as a function of the histological grading, when available.
The present invention also concerns a process for choosing a therapeutic strategy for treating a glioma, comprising the steps of:
The present invention also concerns a process for adapting a therapeutic strategy for treating a glioma, comprising the steps of:
The present invention also concerns a computer program product comprising code instructions for implementing a process as defined above, for classifying a glioma.
The present invention also concerns a kit for the implementation of the processes as described above, comprising:
The present invention also concerns an inhibitor of the telomere maintenance mechanism for its use in the treatment of a glioma, wherein said glioma has been previously classified according to the process as described above.
The following abbreviations are used:
TL: T-length value; long, int. and short designate the three possible TL status;
C: C-circle value; int.: intermediate; ALT++, ALT+, ALT int. and ALT− designate the four possible ALT status;
IDH: IDH status is chosen among IDHwt (no mutation) or IDHmt (at least one mutation in IDH1 and/or IDH2)
Classification of gliomas “like” according to the algorithm of the invention: OD: oligodendroglioma; OD II: oligodendroglioma grade II; OD III: oligodendroglioma grade III; GbmOD: GBM or OD; GBM_IDHwt: primary glioblastoma; GBM_IDHmt: secondary glioblastoma; A_GBM_IDHmt: astrocytoma or secondary glioblastoma (mutated for IDH); A_IDHmt: astrocytomas; All_IDHmt: astrocytoma grade II; AIII_IDHmt: astrocytoma grade III.
The same abbreviations than in
qPCR were led using DNA from an ALT positive tumor (left: TeloPCR, right: qPCR targeting the reference housekeeping gene 36B4).
(A) representative amplification curves are shown with (+) or without (−) pre-incubation with the enzyme ϕ29, as shown by the corresponding arrows. Telomere length is determined in the ϕ29−, and the c-circle value is calculated by using +ϕ29 and −ϕ29 values. Note that the same amount of genomic DNA is used in the two conditions (same curves for 36B4).
(B) DNA from an ALT positive tumor was diluted and the CT value (y) is plotted against the log-transformed dilution factor (x) (TeloPCR on the left, and qPCR against 36B4 on the right). Note that the correlation coefficient (R2) is nearly 1 for both qPCR.
Gliomas (with the exception of anaplastic astrocytoma IDHwt) have been classified according to the standard algorithm according to the WHO 2016 classification (upper line), or with the algorithm of the invention (bottom line).
The algorithm of the invention allows a separation into five subclasses: “oligodendroglioma-like”, “glioblastoma IDHwt-like”, “glioblastoma IDHmt-like”, “low-grade astrocytoma-like” and “other” (the code is depicted in the legend).
(A) Only patients with molecular biomarkers in agreement with the immuno-histochemical classification are analyzed (N=180), the two classifications are concordant for 93.5% of gliomas, are different (noted misclassified) for 3.8%, and 2.7% of gliomas are classified as “others”.
(B) Considering only the gliomas with discordant molecular biomarkers and immuno-histochemical classification (N=29), the algorithm is in agreement with histological classification for 72% of gliomas.
Gliomas (with the exception of anaplastic astrocytoma IDHwt) have been classified according to the standard algorithm following the WHO 2016 classification (left curves), or with the algorithm of the invention (right curves).
The overall survival (OS, upper curves) and the disease free survival (DFS, bottom curves) have been followed for each patient. 210 patients were analyzed (Standard classification: 74 low-grade astrocytoma, 24 glioblastoma IDHwt, 57 glioblastoma IDHmt, 55 oligodendroglioma). Among these patients, 29 have discordant molecular parameters as regard with the immuno-histological classification.
The algorithm of the invention allows a separation into five subclasses: oligodendroglioma-like, glioblastoma IDHwt-like, glioblastoma IDHmt-like, low-grade astrocytoma-like (A_IDHmt like), and other gliomas.
The following abbreviations are used:
OD: oligodendroglioma; A_IDHmt: Astrocytoma_IDHmt; GBM_IDHmt: secondary glioblastoma (also called glioblastoma IDHmt); GBM_IDHwt: primary glioblastoma (also called glioblastoma IDHwt).
Note that the curves are highly similar regarding the overall survival, and that the algorithm is even more discriminant that the standard classification considering the low grade astrocytoma and the oligodendroglioma.
Results obtained by radio-activity determination or by multiplex teloPCR have been compared in 120 frozen tumors (A) and in 37 total blood sample (B). Characteristics of the tumors are depicted regarding the diagnosis and the molecular features.
C-circle level and telomere length have been determined by the dTeloPCR method, on 161 FFPE samples (A) and 152 frozen samples (B), as a function of glioma subtype (A: astrocytoma, GBM_IDHmt, GBM: glioblastoma, OD: oligodendroglioma). Control DNA from a TERT positive cell line HeLa or ALT positive cell line U2OS were used in each experiment (N=40 independent determination), results are shown (C).
(A) CC (C-circles) and TL (telomere length) have been determined in paired FFPE/frozen samples from 16 patients. The differences between CC reported in frozen minus FFPE is depicted (delta circle). The same has been calculated for TL (delta TL). 0 stand for no difference.
(B) DNA was re-extracted from 21 FFPE samples of ALT+ gliomas 3 (2016) to 8 years (2011) after their inclusion in FFPE. The c-circle content is compared with those obtained by analyzing DNA extracted at the time of initial diagnosis.
A) Classification of FFPE tumors. B) Classification of frozen tumors.
As a function of CC level and TL values, tumors are separated into two TMM subgroups: ALT+ or ALT−. The determination of IDH1/2 mutational status and grading allows to further classify the tumors in five subgroups (tOD, tGBM, tLGA, tGBM_IDHmt and tA-IV). Unclassified tumors are designated as “Other”.
Gliomas were assessed for C-circle by the dTeloPCR method, and classified in ALT+ an ALT− subgroups according to the first step (a) of the process/algorithm (TMM).
(A, C) 321 gliomas with molecular markers (IDH1/2, 1p19q deletion, TERTmt and ATRXloss) in agreement with the integrated WHO 2016 diagnosis (WHO diagnosis) are depicted. We observe a perfect concordance between the ALT+ subgroup and the ATRXloss, so as for the ALT− subgroup and TERT mutation.
(B, D) 96 tumors with discordant molecular features were next analyzed. Only 7 were not classified, all the other tumors were assigned to ALT+ or ALT− subgroups. nd:ND
(A,B) C-circle (dTeloPCR method) assays were performed on 321 tumors (frozen or FFPE) with coherent molecular and immune-histochemical markers. Classification of these tumors by the TeloDiag is compared to the WHO 2016 based classification. A diagram recapitulating the correspondence is shown. (C,D) The same analysis was done on 96 tumors with discordant molecular biomarkers.
C-circle assays were performed on 208 blood samples.
(A) results of C-circle detection and TL determination are shown as a function of glioma subtype, no difference regarding TL in blood DNA was noted (as expected) and a higher level of C-circle in blood from patients with ALT tumor (Astrocytoma, GBM-IDHmt) was observed.
(B) A cut-off value of 1.25 was used to classify the sample as C-circle positive (CC+) and C-Circle negative (CC not detected), whatever the TL result were.
(C-E) Detection of CC in total blood samples from patients with tumor showing concordant molecular and immune-histological markers (N=188) (C on the left, E), or bearing tumors with discordant molecular markers (NEC, N=20) (C on the right, D). (D-E) Summary of CC results as a function of gliomas subtype are shown.
Survival curves of N=307 gliomas (63 GBM IDHwt, 4 GBM ATRXloss, 65 GBM_IDHmt, 100 Astrocytoma IDHmt (A II-III), 2 oligoastrocytoma (OA), 64 oligodendroglioma (OD)), are shown following the WHO 2016 classification (with c-Impact-now updates). The same patients classified following the TeloDIAG were analyzed in the same way.
The TeloDIAG has the advantage to reduce the number of subgroups and to keep the same prognosis significance. Note that the crossing of the tLGA and tOD curves (not expected), is due to fewer disease events in these two subgroups (13 over 91 for the tLGA and 15 over 75 for the tOD).
“Glioma” is identified on the basis of histopathological analysis. Classification as a “glioma” is given by a health practician on the basis of:
The present invention relates to an in vitro process for classifying a glioma, comprising the following steps:
This classification process allows the distinction of subclasses of gliomas, different from the 2016 WHO standard classification of tumors of the central nervous system.
The subclasses proposed by the present invention are the following:
The phrase “low-grade astrocytoma” designates astrocytoma of grade II (diffuse astrocytoma) and of grade III (anaplastic astrocytoma). Nevertheless, it was found that this class “low-grade astrocytoma-like” actually includes two subclasses designated as t-low grade Astrocytoma (tLGA) and t-Astrocytoma grade IV (tA-IV).
Therefore, in a specific implementation of the invention, the in vitro process for classifying a glioma comprises the following steps:
The term “like” is used to mean that the patient in the group is expected to have similar response to treatment and overall survival as the group's name, but not necessarily have the corresponding immuno-histological or molecular parameters.
Each group of patients having glioma is defined by a median time of overall survival of said patients. For example, a patient whose glioma is classified in the class “glioblastoma IDHwt-like” can expect an overall survival time comprised between 7 and 15 months.
Advantageously, this classification process allows the re-classification of some gliomas that had been previously wrongly characterized, and consequently an incorrect expectation of median time of disease free survival and/or of overall survival had been announced to the patient. Furthermore, a non-adapted therapeutic strategy might be used for incorrectly classified patients.
In this case, the algorithm of the invention would allow to inform the patient that his/her median overall survival time would be 7 to 15 months (GBM IDHwt-like), instead of 15 years for an oligodendroglioma of grade II.
Also, the therapeutic strategies will not be the same, depending on the classification conclusions.
As presented in
Advantageously, this classification process is easy to implement in a clinical environment, since it necessitates only DNA extracted from said glioma sample, for the measure of parameter in step (a) and optionally (b).
Optionally, the grading of the tumor is measured on the basis of immuno-histochemical analyses of the glioma sample. Preferentially, the grading of the tumor is previously known from the anatopathologic analysis.
It is understood that the process according to the invention comprises at least two steps (a) and (c) but might also comprises other supplementary steps, well known by the man skilled in the art, such as the optional step (b), and also the detection of an EGFR amplification, the detection of a CDKN2A copy number loss, and/or the detection of chromosomal abnormalities like the gain in chromosome 7p correlated with loss of chromosome 10q (Inda et al., 2003).
In a specific embodiment of the invention, the process of the invention comprises both steps (a) and (c).
In a specific embodiment of the invention, the process of the invention comprises the three steps (a), (b) and (c).
In a specific embodiment of the invention, the process of the invention consists in both steps (a) and (c).
In a specific embodiment of the invention, the process of the invention consists in three steps (a), (b) and (c).
Surgery to remove as much of the tumor as possible is usually the first step in treating gliomas. After this procedure, a sample of the tumor is further analyzed.
If no surgery is performed, a stereotaxic biopsy may be realized to obtain a tumor sample. From this tumor sample, DNA is extracted by any technique well known by the man skilled in the art.
In an embodiment of the invention, the parameters ALT status and IDH status are measured on the basis of DNA extracted from a tissue sample of said glioma, that has been recovered by any technique known by the man skilled in the art.
In most cases, glioma samples are not analyzed directly but are conserved before extraction of DNA. Typically such collected tissue samples are frozen or are processed as paraffin blocks. Advantageously, these tumor samples are annotated with clinical information on the patient.
In an embodiment of the invention, the parameters ALT status and IDH status of tumor samples are measured on the basis of DNA extracted from a glioma sample, in particular a tissue sample that has been conserved in paraffin or frozen.
Advantageously, the techniques for measuring parameters ALT status and IDH status of tumor samples can be realised on the basis of DNA extracted from a glioma sample that has been conserved in paraffin or frozen, even if said DNA is of poor quality and/or in a small amount.
Frozen tissues show a higher level of C-circles but a lower level of telomeric sequence amplification, thereby producing a bias toward a lower TL evaluation (
The FFPE samples (conserved in paraffin) are thus preferred, but should be extracted during the first year following the biopsy/surgery, to avoid the degradation of C-circle due to prolonged FFPE conservation and the risk of false negative (
According to a preferred embodiment of the invention, the step (a) of the process further comprises the measure of the telomere length status of said glioma. Technique for this measure is described in a later chapter of the specification.
Implementation of the Process on DNA Extracted from Blood of Cerebrospinal Fluid of a Patient with Glioma
In an aspect of the invention, the parameters ALT status and optionally IDH status of tumor samples are measured on the basis of DNA of glioma cells, extracted from a blood sample or a cerebrospinal fluid sample from a patient having said glioma.
Glioma cells have a tendency to necrose and cell debris and/or free DNA circulate in blood and cerebrospinal fluid of patients affected by glioma. Moreover, cells designated as Circulating tumor cells (CTCs) issued from a primary tumor are able to circulate around the body in the blood circulation. From circulating DNA (circDNA) and/or CTCs, tumor-specific DNA can be purified and analyzed in order to assess the parameters ALT status and IDH status.
In this aspect of the invention, the term “glioma sample” designates the DNA from the glioma cells, that is obtained from the blood or from the cerebrospinal fluid of the patient.
In the sense of the invention, “blood sample” designates a sample comprising all types of blood cells (white cells, red cells and platelets). Peripheral white cells consist of lymphocytes (T cells, B cells, NK cells) monocytes and polynuclear cells. After total centrifugation of blood, all blood cells are collected to obtain the “blood sample” of the invention. This blood sample is expected to comprise circulating tumoral cells (CTC).
This implementation of the process on a blood sample is advantageous since, in the preliminary steps not included in the process, the invasive step of biopsy for obtaining a tumor sample is avoided.
A disadvantage of the use of a blood sample is that mutated DNA from CTC is drowned in a huge amount of “normal” DNA from other blood cells (for IDH1/2), which would prevent from establishing the IDH status of the glioma cells.
Another interesting biological sample is the cerebrospinal fluid, wherein a sufficient amount of tumoral DNA is available for determining the IDH status of the glioma.
Furthermore, when the process is implemented on the basis of blood or cerebrospinal fluid of a patient with glioma, the parameter “telomere length” cannot be used: indeed, these samples contain different types of cells, and the measure of the telomere length status concern all cells of the sample. Therefore the telomere length status of the glioma only cannot be determined. It is to be noted, however, that this measure of telomere length status can be performed anyway on the sample, even if results will not be used in the classification process of the invention.
In this embodiment, the in vitro process for classifying a glioma comprises the following steps:
If the ALT status and IDH status are known (a and b) the four following classes are “available”: oligodendroglioma-like, glioblastoma IDHwt-like, Astrocytoma_IDHmt (grade II to IV), and other gliomas.
If only step (a) is performed, glioma are classified either:
In this embodiment of the invention, usually no solid glioma sample is available. Therefore, the histological grading of the tumor is not available. The process is therefore limited to the classification in two classes, without any further sub-classification of the gliomas according to their histological grade II or III.
In a specific embodiment of the invention, the process of the invention comprises both steps (a) and (c).
In a specific embodiment of the invention, the process of the invention comprises the three steps (a), (b) and (c).
In a specific embodiment of the invention, the process of the invention comprises a further step of determination of the TERT status (mutated or wild-type) of the glioma.
In a specific embodiment of the invention, the process of the invention consists in both steps (a) and (c).
In a specific embodiment of the invention, the process of the invention consists in three steps (a), (b) and (c).
In a specific embodiment of the invention, at least two glioma patient biological samples obtained at different time points are submitted to said process of classification of the glioma, for a follow-up of the patient over time. Indeed, the implementation of the process on a blood sample is particularly advantageous for the following up over time of a patient having a glioma, in particular for detecting the evolution or recurrence after treatment of said glioma.
In vertebrates, chromosome extremities, designed as telomeres, consist of:
Telomeres play vital roles in eukaryotic cells, in particular by limiting DNA replication and therefore avoiding unlimited cell proliferation, via their gradual shortening at each replicative cycle.
Telomerase is a ribonucleic reverse transcriptase enzyme, able to add the telomere repeat sequence to the 3′ end of telomeres. It consists of a catalytic subunit called TERT (for Telomerase Reverse Transcriptase), an essential RNA component TERC that functions as the RNA template for the addition of the telomeric repeats, and a series of auxiliary components.
In most tumour cells, the TMM is linked to the reactivation of telomerase. Nevertheless, about 10% of tumour cells acquire immortality through the telomerase-independent alternative lengthening of telomeres (ALT) mechanism.
Glioma can therefore be classified as “ALT-dependent tumors”, when the telomerase-independent mechanism is in place, and “ALT-independent tumors” when a telomerase-dependent mechanism is activated in the cell.
Within the ALT-dependent group, different subgroup can be identified: ALT intermediate, ALT+ and ALT++.
The ALT-independent tumors define the ALT− subgroup, wherein no C circle is detected.
Any suitable assay for determining the ALT status of cells can be used for the implementation of the process.
Henson et al. (2009) have disclosed an assay based on partially single-stranded telomeric (CCCTAA)n DNA circles that allows a specific measurement of the ALT status. This assay is known as “the C-circle assay” or “CC assay”.
Briefly, this C-circle assay can be summarized as follow:
Telomeric extrachromosomal DNA (ECT) is particularly enriched in ALT-dependent cells versus telomerase-positive or mortal cells. Among different species of ECT, C-circles is a specific and sensitive marker of the ALT process. A C-circle is composed of a circular DNA of C3TA2 sequence, partially double stranded with a short T2AG3 sequence. The technique of Rolling Circle Amplification (RCA) has been used to detect telomeric circles in ALT+ cells. The “CC-assay” involves the use of ϕ29, a highly processive DNA polymerase that is auto-primed by the partial G-strand (TTAGGG)n.
The amplified telomeric DNA is then quantified together with the genomic telomeric DNA after and before incubation with ϕ29. A ratio between these two quantities of DNA is calculated, and compared to the same ratio obtained in telomerase positive cells and in ALT-dependent cells. (Henson et al., 2017).
The amplified telomeric DNA can be quantified by all techniques well known by the man skilled in the art, such as hybridization and flow cytometry methods.
In a preferred embodiment of the invention, the quantification of the amplified telomeric DNA circles is performed as described in (Gil & Coetzer, 2004) with a real-time polymerase chain reaction (PCR)-based method, and particularly with a telomere-specific PCR.
A “C-circle” value, expressed in arbitrary units, equal to the following ratio:
Quantity of total telomeric DNA (including RCA-amplifiedC-circles)/Quantity of total telomeric DNA (without amplification of C-circle content)
is measured by the means described above for each glioma sample.
This value is then compared to at least three thresholds values, previously defined according to the general knowledge of the man skilled in the art.
In particular, the inventors have defined four thresholds as follow:
In a specific embodiment of the invention, in the in vitro process, the ALT status in (a) is determined by performing a C-circle assay coupled to a telomere-specific PCR, thereby obtaining a C-circle value and selecting the ALT status of the glioma from:
In a specific embodiment of the invention, the in vitro process for classifying a glioma comprises the following steps:
wherein the ALT status is measured by performing a C-circle assay coupled to a telomere-specific PCR, thereby obtaining a C-circle value and selecting the ALT status of the glioma.
Measurement of telomere length is another important parameter of the process, measured in step (a) in certain implementation of the process of the invention.
The original method for determining telomere length employed Southern hybridization to determine the mean terminal restriction fragment length. However this technique was labor-intensive, time-consuming and necessitates high amount of DNA (over 1 μg), not available in standard clinical use. Various alternative techniques have since been proposed, including slot blots, next generation sequencing, hybridization protection assays and flow cytometry.
Although any technique for determining the telomere length status might be employed in the implementation of the process of the invention, in a preferred embodiment, telomere length status in step (a) is determined by quantifying the telomeric DNA with a telomere-specific PCR.
In particular, the real-time quantitative PCR named TeloPCR described in (Gil & Coetzer, 2004) can be used for this quantification.
This TeloPCR has been adapted into a duplex TeloPCR, both amplifying the housekeeping gene (using a fluorescent hydrolysis probe) and the telomeric sequences (SybrGreen).
According to this embodiment, the quantification technique comprises the following steps:
The telomere content value representative of the mean quantity of telomeric sequences in cells for each sample is obtained. This value is next normalized by internal controls obtained from DNA extracted from ALT+ and ALT− cell lines (such as U2OS and HeLa, respectively), as it is well known by the man skilled in the art. The resulting value is designated as “T-length” value.
Four threshold have been defined as a function of increasing TL: short, middle-long, long and very long. With these thresholds, the telomere length status of the glioma can be determined as follow:
In a specific embodiment of the invention, the in vitro process for classifying a glioma comprises the following steps:
In a specific embodiment of the invention, the ALT status and the T-length status are measured concomitantly with the same experimentation, comprising:
Specifically, both substeps of step (a) are concomitantly performed by one duplex PCR as presented above.
The present invention also relates to a process for determining the Telomere Maintenance Mechanisms status (TMM status) of any tumor, comprising the measuring, from a patient biological sample, of both the Alternative Lengthening of Telomeres (ALT) status and the telomere length status of said tumor,
wherein the ALT status is measured by performing a C-circle assay coupled to a telomere-specific PCR, thereby obtaining a C-circle value and selecting the ALT status of the tumor from:
wherein the telomere length status is measured by quantifying the telomeric DNA with a telomere-specific PCR, thereby obtaining a T-length value and selecting the telomere length status of the tumor from:
and wherein both measures are concomitantly performed by one duplex PCR.
In a specific embodiment of the invention, the values of the parameters “ALT status” and, where appropriate, the telomere length status, are measured and then compared each one to at least one threshold value.
For the purposes of carrying out the above process, the term “threshold value” is intended to mean a value determined with a group of glioma samples that are well characterized, and whose ALT status and telomere length status have been previously determined.
Based on the results obtained for these groups of specific gliomas, inventors have defined threshold values, also designed as cut-off values, for ALT status and telomere length that are defined below.
Other threshold values can be easily determined by those skilled in the art by means of their general knowledge.
Therefore, the value obtained for the ALT status is compared to at least one ALT threshold value, in order to determine the ALT status of the glioma; and the value obtained for the TL status is compared to at least one T-length threshold value, in order to determine the telomere length (TL) status of the glioma.
Thresholds and classification may evolve during the implementation of the process, and in particular may be function of the nature of the tested biological sample.
Recently, a novel classification, more precise, has been defined with the implementation of the process according to the invention on a higher number of biological samples.
The second algorithm designated as “Telo-DIAG” is illustrated in
This specific implementation of the process “Telo-DIAG” is described in details below:
The thresholds for the C-circle (CC) level are the following for FFPE samples:
The thresholds for the C-circle (CC) level are the following for frozen samples:
The thresholds for the telomere length (TL) status are the following for FFPE samples:
The thresholds for the telomere length (TL) status are the following for frozen samples:
The TMM categories are defined as follow for FFPE samples:
The TMM categories are defined as follow for frozen samples:
On the basis of the TMM status, the TeloDIAG is defined as follow:
This specific classification process takes into account high grade glioma without mutation in IDH such as pontine gliomas (with or without histone mutation) and includes the classification of IDHwt astrocytoma that are now considered as grade IV in respect with the Impact-now update 3.
The parameter of IDH status has previously been described for the classification of gliomas.
In a specific embodiment of the invention, the IDH status in optional step (b) is determined by sequencing both genes encoding proteins IDH1 and IDH2.
Sequences of human isocitrate dehydrogenase proteins 1 and 2 are well known of the man skilled in the art, and can be found in proteins database such as GeneBank and OMIM.
Wild-type sequence of human IDH1 is represented in SEQ ID NO:1, and wild type sequence of human IDH2 is represented in SEQ ID NO: 2, as presented in table 1 below:
The IDH status of the glioma cells is determined as follows:
Step (b) of the process according to the invention is optional. Indeed, and as illustrated in
could be an oligodendroglioma or a glioblastoma.
In this case, a step (b) of determination of the IDH status is necessary for the final classification, where:
Advantageously, the tumor grading has been previously determined on the basis of immuno-histochemical analyses of pictures of said tumor, thereby selecting the tumor grade of the glioma from stage II, III or IV. The histological grade of the glioma is said to be available.
These grades are defined in the actual WHO classification of gliomas, and are based on the presence of the following criteria of the tumor cells: mitosis, vascular proliferation and necrosis.
The grade of the glioma will advantageously be determined by a man skilled in the art such as a neuro-specialized physician.
When no solid glioma sample is available, for example in the case of the implementation of the process on glioma DNA extracted from blood or cerebrospinal fluid of the patient, the histological grading of the tumor is not available and therefore not known, since it has not been determined.
As previously presented, the subclass of “astrocytoma IDHwt” as determined by WHO's classification is particularly heterogeneous and difficult to classify.
Often, these gliomas are not actually conventional “astrocytomas” as defined by the WHO classification.
Therefore, the median overall survival is highly variable with a median of 2.5 years, which is much more severe than for classical astrocytoma. Moreover, therapeutic options are not defined by guidelines and might be wrongly chosen.
Therefore, there is a need for reclassifying these specific gliomas, as well as unclassified gliomas called “others” or “NEC”, with a novel combination of parameters representative of the nature of said glioma.
In a particular aspect, the invention concerns the present in vitro process of classification, wherein a glioma of the class “astrocytoma” with a IDH status “IDHwt”, or a glioma classified “other” or “NEC”, is re-classified in one of the following classes: oligodendroglioma-like, glioblastoma IDHwt-like, glioblastoma IDHmt-like, t-low grade Astrocytoma (tLGA) and t-Astrocytoma grade IV (tA-IV), and other gliomas.
In this process, the histological grade of the “astrocytoma IDHwt” is usually known before the implementation of the process, and therefore is available. However, optionally, the histological grade of the “astrocytoma IDHwt” might be determined during the process.
Although the IDH status of this kind of glioma is IDHwt, the glioma might be re-classified in a class such as “glioblastoma_IDHmt-like” or “low-grade astrocytoma-like”, although tumors in this class present an IDHmt status. Nevertheless, it is understood that in this case, the glioma previously classified as “IDHwt astrocytoma” keeps its status of IDHwt, but is categorized in this astrocytoma class because the patient affected with said glioma would present the same median overall survival than those affected with such astrocytoma.
In this implementation of the process, further steps could be included such as determination of the status of TERT (mutated or wild-type) (Vinagre et al., 2013) and determination of the status of ATRX (present or absent) (Nandakumar et al., 2017).
The present invention also relates to a process for choosing a therapeutic strategy for treating a glioma, comprising the steps of:
These therapies can be the actual “gold standard” therapies: radiotherapy with a concomitant or adjuvant chemotherapy, with chemotherapeutic agents such as Temozolomide or PCV, a combination treatment for brain tumors including procarbazine, lomustine, and vincristine.
The present invention also relates to a process for adapting a therapeutic strategy for treating a glioma, comprising the steps of:
A promising therapeutic approach for the treatment of gliomas is the administration of inhibitors of any telomere maintenance mechanism. In particular, it has been shown that telomerase inhibitors increase the response to radiotherapy in a murine orthotopic model of human glioblastoma (Ferrandon et al., 2015).
Thus, the present invention relates to an inhibitor of at least one telomere maintenance mechanism (TMM) for its use in the treatment of a glioma, wherein said glioma has been previously classified according to the process of the invention.
Advantageously, the determination of the ALT status of the glioma allows the practician to choose between TMM inhibitors specific of the ALT mechanism, or specific of the telomerase reactivation mechanism.
Implementation of the Process with a Computer
In a specific implementation of these processes, data obtained in steps (a) (b) and (c) are recorded on a computer device, into a software program that is configured to memorize said data and to execute steps to classify the glioma in function of said parameters data.
Accordingly, the present invention also concerns a computer program product comprising code instructions for implementing a process as described above, for classifying a glioma.
The present invention also concerns a kit for the implementation of the processes as described, comprising:
wherein both substeps of measure of C-circle and telomere length are concomitantly performed by one duplex PCR.
In particular, internal controls are chosen among cell lines expressing the telomerase (ALT− cells), such as breast cancer-derived cells (HeLa), and among cell lines using the process of “Alternative lengthening of telomere” (ALT+ cells), such as osteosarcoma cell lines.
Other useful internal controls can be also present in said kit for the implementation of the processes of the invention.
Although the present invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
Rolling circle amplification of C-circle is performed as described in (Henson et al., 2009) and (Henson et al., 2014). Briefly, 3.2 μl of total genomic DNA (5 ng/μL) were incubated for 18 h at 30° C. with 3.75 units of φ29 DNA polymerase (New England Biolabs) (0.375 μL of 10 U/μL), in 0.2 μg/μL of BSA, 0.1% Tween, 4 μM DTT (Dithiothreitol), 1 mM dNTP, 1 μL of 10×NEB buffer. Enzyme is heat-inactivated at 65° C. for 20 min. The same reaction is performed without the enzyme φ29 (φ−).
For each experiment, two internal controls are added, namely TA and ALT. TA and ALT correspond to total genomic DNA extracted from HeLa (ALT−) and U2OS (ALT+) cell lines respectively.
The 10 μL of φ− and φ+ reactions are then diluted by adding 30 μL of water (molecular biology grade), 5 μL are used to performed each qPCR reaction.
TeloPCR
Oligonucleotides for the qPCR reaction have been previously described in (Gil et al., 2004) and (Lau et al., 2013) and are listed below
The sequence for oligonucleotides used in the qPCR are presented in table 2:
Telo-PCR and qPCR against 36B4 are run in duplicate for each condition φ− and φ+, on a 480 Light Cycler Thermocycler (Roche, Houwald, Luxembourg), in 1× final LightCycler® DNA Master SYBR Green I (10 μL), 200 nM final of TeloPCR-specific primers or 300 nMm final of 36B4-specific primers. Details of thermocycling conditions are detailed below
For each qPCR, the exact conditions are summarized in tables 3 and 4 below:
dTeloPCR
Oligonucleotides for the amplification of telomeric sequences have been previously described in (Gil et al., 2004) and (Lau et al., 2013). The oligonucleotides and the probe targeting RPLP0 have been customed-designed and are described below.
The sequences of oligonucleotides used in the qPCR are presented in table 5:
Duplex Telo-PCR is run in duplicate for each condition φ− and φ+, on a 480 Light Cycler Thermocycler (Roche, Houwald, Luxembourg), in 1× final LightCycler® DNA Master SYBR Green I (10 μL), 0.4 μM final of TeloPCR-specific primers or 1 μM final of 36B4-specific primers and 0.22 μM of 36B4 probes. Details of thermocycling conditions are detailed in table 6 below.
The fluorescence in logarithmic scale is analyzed as a function of PCR cycle, the threshold is determined by the second derivative method (all experiments). Intersection between the threshold of amplification curve gives the CT for each reaction. The fluorescence channel corresponding to the TeloPCR is the following: SYBR Green (465-510). For the dTeloPCR, two channels are analyzed: SYBR Green (465-510) for the telomeric sequence and CY5 (618-660) for RPLP0/36B4.
Efficiency of TeloPCR (Etelo) and 36B4 (E36B4) PCR are respectively 1.70111 and 1.9672.
For each reaction, the following value is calculated: Etelo−CT/ E36B4−CT, and annotated as φ+ and φ− as a function of the initial circle reaction.
φ− correspond to the telomere length (T-Length)
Cr correspond to the Circle score and is calculated as follow: φ+/φ−
The difference of T-Length between the two internal controls (U2OS and HeLa) A is calculated and correspond to x units (determined for each batch of controls) with Δ=φ−Hela−φ−U2OS
TLnorm is then calculated for each sample as follow: φ−/(Δ/x)
A standard curve for calculation of qPCR efficiency is presented in
As shown in
GbmOD category is then subclassified in step (b) according to the IDH status:
All glioma are then classified according to their grade in step (c). In particular, tumors of the category “A_GBM_IDHmt” will be subclassified, according to their grade, into All-IDHmt like (AD), AIII_IDHmt like (AA) or GBM_IDHmt like (GBMII).
An example of equations is given below; however, the indicated numbers have been defined for this experiment only, and are not limitating the scope of the claimed invention.
An example of equations is given below; however, the indicated numbers have been defined for this experiment only, and are not limitating the scope of the claimed invention.
A=(SI(TLnorm>4;“A_GBMII”;SI(ET(Cr>1.5;TLnorm>0.8);“A_GBMII”;SI(ET(TLnorm<0.66; Cr<1.9);“OD”;SI(ET(Cr<1.28; ET(TLnorm>0.66; TLnorm<3));“GbmOD”;SI(Cr>2;“A_GBMII”;“other))))))
2nd Step (b) with IDH Status (MT or WT)
B=SI(ET(A=∂GbmOD”;IDH=“MT”);“OD”;(SI(ET(A=“GbmOD”;IDH=“WT”);“GBM”;A)))
3rd Step with Grading G (II, III, IV)
C=SI(ET(B=“A_GBMII”;G=“III”);“AA”;SI(ET(B=“A_GBMII”;G=“II”);“AD”;SI(ET(B=“A_GBMII”;G=“IV”);“GBMII”;B)
Alternatively, if using the dTeloPCR method, as shown in
TMM Categories are then subclassified in the second step (Telostep1) according to the IDH status:
All glioma are then classified according to their grade in the third step. In particular, tumors of the category “IDHmt_AA_GBMII” will be subclassified, according to their grade, into tLGA for grade II and III, or into tGBM_IDHmt for grade IV.
An example of equations is given below; the indicated thresholds are function of the nature of the sample, and have been defined for this experiment only.
dTeloPCR Associated Algorithm
For FFPE Sample:
A=(SI(OU(TLnorm>4; Cr>2);“AA_GBMII”;SI(ET(Cr>=1.24; TLnorm>1.1);“AA_GBMII”;SI(ET(TLnorm<1.5; Cr<1.24);“GBM_OD”;SI(ET(Cr<1.24; ET(TLnorm>=1.5; TLnorm<2.6));“GBM”;“other”)))))
B=SI(ET(OU(A=“GBM_OD”;A=“GBM”);IDH=“MT”);“tOD”;SI(ET(OU(A=“GBM_OD”;A=“GB M”);IDH=“WT”);“tGBM”;SI(ET(A=“AA_GBMII”;IDH=“MT”);“IDHmt_AA_GBMII”;A)))
C=SI(B=“IDHmt_AA_GBMII”;SI(OU(grading=“II”;grading=“III”);“tLGA”;“tGBM_IDHmt”);SI(B=“AA_GBMII”;“tAIV”;B))
For Frozen Sample:
A=(SI(ET(TLnorm<1.2; Cr<=1.3); “GBM_OD”; SI(Cr>1.3;“AA_GBMII”; SI(TLnorm>1.8;“AA_GBMII”;“autre”))))
The following steps: TElostep1 and TeloDIAG are the same as for FFPE samples
For Circulating DNA
A=SI(Cr>1.25;“AA_GBMII”;“other”)
First, only 180 patients with molecular biomarkers in agreement with the immuno-histochemical classification (i.e. with the exception of anaplastic astrocytoma IDHwt) were analyzed.
These gliomas have been classified according to the standard algorithm according to the WHO 2016 classification (upper line), or with the algorithm of the invention (bottom line) and are presented in
The algorithm of the invention allows a separation into five subclasses: “oligodendroglioma-like”, “glioblastoma IDHwt-like”, “glioblastoma IDHmt-like”, “low-grade astrocytoma-like” and “other” (the code is depicted in the legend).
Both processes of classification are concordant for 93.5% of gliomas. However for 3.8% of gliomas, the classifications are different (noted misclassified). With the algorithm of the invention, 2.7% of gliomas are classified as “others”.
In conclusion, with the classification process according to the invention:
In this case, the classification obtained with the algorithm of the invention is in agreement with the standard process of classification for 72% of gliomas.
The
The overall survival (OS, upper curves) and the disease free survival (DFS, bottom curves) have been followed for each patient.
210 patients were analyzed, classified as follow according to the standard classification: 74 low-grade astrocytoma, 24 glioblastoma IDHwt, 57 glioblastoma IDHmt, and 55 oligodendroglioma.
Among these patients, 29 have discordant molecular parameters as regard with the immuno-histological classification.
The algorithm of the invention allows a separation into five subclasses: oligodendroglioma-like, glioblastoma IDHwt-like, glioblastoma IDHmt-like, low-grade astrocytoma-like (A_IDHmt like), and other gliomas.
Note that the curves are highly similar regarding the overall survival, and that the algorithm is even more discriminant that the standard classification considering the low grade astrocytoma and the oligodendroglioma.
The C-circle assay combine a first step of rolling circle amplification of circular DNA (partially double stranded), and a second step wherein the telomeric DNA is quantified. The technic of reference proceeds through an hybridization of amplified telomeric DNA by radioactive probe. The quantification of the radio-active signal is proportional to the CC content in a linear manner.
To validate the duplex TeloPCR (dTeloPCR) as a detection mode, independent RCA assays have been run, either coupled with dTelo-PCR or with radio-activity detection on 120 DNA extracted from frozen tumors (
Considering the blood sample (
False negative/positives results are probably in relation with samples of poor quality and/or deleterious storage conditions.
These results show that the dTeloPCR detection method is concordant with the radio-active detection method.
152 DNA from frozen gliomas were analyzed (
(i) if C-circle can be detected in DNA extracted from FFPE sample, and
(ii) if the telomeric parameters determined by dTeloPCR can be combined to improve the detection of ALT+ sample in both types of samples.
Control DNA from a TERT positive, ALT− cell line HeLa or ALT positive cell line U2OS were used as internal controls in each experiment (N=40 independent determination), results are shown in
The C-circle (CC) rate is effectively positive (over 0 in logarithmic values,
By comparing frozen and FFPE samples, a higher TL but a lower CC level were noticed in FFPE sample in ALT+ samples.
To confirm this observation, DNA from paired frozen and FFPE samples was extracted from 16 different tumors; a higher rate of CC and a lower TL in frozen sample was confirmed (
Formaldehyde fixative is known to induce DNA single and double strand breaks due to oxidative lesions. Break in C-circle impedes the RCA reaction, which would explain a lower CC rate. Moreover, fragmentation of long double-stranded-telomeric sequences (over 5 kb) would favor TeloPCR reaction by increasing the content of DNA matrix and by producing shorter but more numerous amplicons, thereby biasing the TeloPCR reaction toward an increase in the TL.
A long conservation of FFPE specimen before extraction of DNA is expected to decrease the CC level. All DNA samples analyzed herein have been extracted during the months following the inclusion in FFPE.
To test this hypothesis, additional DNA extractions from 21 FFPE samples from ALT+tumors, 3 to 8 years after inclusion, have been realized. Results are shown in
Among the 21 samples, two (from 4 and 3 years) turned to be below the C-circle threshold of 1.23 in the newly made extraction and would have been false negative.
Using the results of
The concordance of this TMM classification was validated in regard to the presence of TERT mutation for ALT− subgroup, and in regard to the loss of ATRX for the ALT+ subgroup. This is in particular shown in
When taking into account the 321 gliomas, even if molecular markers are missing, all of the A and GBM-IDHmt tumors were detected as ALT+, and all of the OD and GBM (classically TERT positive) were classified as ALT− (
The TMM status of 96 tumors with discordant molecular and immuno-histochemical parameters was determined and are shown on
GBM, 1p19q not deleted OD, IDHwt astrocytomas or tumors with incoherent telomeric parameters (TERTmt and ATRXloss or TERTwt and ATRXwt).
Regarding the 60 tumors with no telomeric markers (TERTwt and ATRXwt), 19 ALT+ tumors (9 GBM, 8 GBM_IDHmt and 2 OD) were identified. Half of the double positive (TERTmt and ATRXloss) tumors were classified as ALT+ (6 Astrocytoma, 1 GBM and 1 GBM-IDHmt).
It is thus possible to assign a “TMM status” even in discordant tumors, and this is of great help for the classification and therefore for diagnosis.
The C-circle assay results (CC and TL) have been combined with the IDH1/2 status and the histological grading, to create a simple algorithm “TELODiag” to assign 5 glioma subtypes: this algorithm is schematically represented on
The five subtypes of glioma are the following:
The classification of glioma obtained on the basis of the algorithm TeloDiag was compared to the WHO 2016 classification (taking into account the impact now updates), first in concordant tumors (
311 tumors over the 321 tested were classified as expected with the WHO classification, five are attributed to a different groups (5 were unclassified, “other”).
Regarding the discordant tumors, 51 were classified on the same corresponding WHO section, the other 45 were classified in a different subgroup.
Total blood cells from 208 patients were collected by centrifugation and subjected to CC assay.
As expected, the TL reflecting the length of telomere in the blood nucleated cells were unchanged among the different glioma subgroups (
Modalities of blood collection, CTC preparation and conservation are under investigation to maximize the detection of C-circle.
We have analyzed 103 DNA extracted from buffy coat and obtained only 12% of positive result regarding patients with ALT+ tumors. Thus a total blood centrifugation of 1000 g 10 min should be operate to collect CTC. The delay between surgery and blood sampling has to be the lowest as possible, however we detected CC in blood drawed months after surgery (maybe due to relapse).
We have established a new easy, fast and low-cost assay, based on qPCR detection, able to determine the TMM on frozen and FFPE glioma samples. We established a new diagnosis tool, the “TeloDiag”, an algorithm combining the TMM, the IDH mutation and the histological grading.
This classification gives concordant results with the reference WHO 2016 classification with a refined number of prognosis groups, and could be helpful in tumors with discordant molecular and histological feature, as is presented in
We also detected ALT+ circulating cells in blood from 57% of patients with an ALT+ tumors. This is of importance: (i) to secure the initial diagnosis in some critical situations (radio-necrosis, differential diagnosis . . . ) and to avoid deleterious biopsy, (ii) to follow the treatment response of ALT+ tumors, without invasive analysis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305215.6 | Feb 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/054346 | 2/19/2020 | WO | 00 |
