Patent Application
20180073083
This application claims the benefit of priority of Singapore provisional application No. 10201501928T, filed 12 Mar. 2015, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention relates to assays for tumours of the breast and in particular, assays for differentiating benign and malignant fibroepithelial tumours of the breast. More specifically, the present invention relates to multigene assays using samples obtained by biopsy or excision and methods for managing the treatment of a subject with a fibroepithelial tumour of the breast. Kits of reagents are also provided.
Fibroepithelial tumours are biphasic tumours that consist of epithelial and stromal tissue. In the breast, fibroepithelial tumours represent a heterogeneous group of tumours that range from benign to malignant. Of these, fibroadenomas account for a large percentage of benign fibroepithelial tumours, while phyllodes tumours (PT) range from benign to malignant and have unpredictable clinical outcomes.
The heterogeneous nature of fibroepithelial tumours equates to a wide range of clinical and pathological features. Accordingly, distinction between fibroadenomas and PTs is important in order to guide the surgical decision for margin width during resection of breast tumours and for appropriate clinical management.
However, it is not always possible to definitively diagnose fibroadenomas from PTs. Fibroadenomas and PTs share some overlapping morphology, and biopsy or excision material for diagnosis may be limited. Furthermore, morphologic predictors of malignancy such as mitotic activity, infiltrative borders, tumour necrosis, positive margins and tumour size are not definitive markers of malignancy.
There is therefore a need for a method to differentiate fibroadenomas and PTs that allows definitive diagnosis of a PT, which would in turn guide clinical management of the patient.
In one aspect, there is provided a method of determining a fibroepithelial tumour of the breast, in a biological sample in vitro, comprising: obtaining an expression profile of one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1, in said sample;
obtaining an expression profile of one or more normalized genes for use as a control;
determining the differential activity of the one or more genes relative to the control based on the expression profile of the one or more genes and one or more normalized genes;
correlating the differential activity of the one or more genes relative to the control to obtain a p-score; and
determining the type of fibroepithelial tumour based on the p-score, wherein a p-score of less than 0.5 is indicative of a fibroadenoma and a p-score of 0.5 and above is indicative of phyllodes tumour.
In one aspect, there is provided a method for managing the treatment of a subject with a fibroepithelial tumour of the breast, comprising:
obtaining an expression profile of one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1, in a biological sample obtained from the subject;
obtaining an expression profile of one or more normalized genes for use as a control;
determining the differential activity of the one or more genes relative to the control based on the expression profile of the one or more genes and one or more normalized genes;
correlating the differential activity of the one or more genes relative to the control to obtain a p-score;
determining the type of fibroepithelial tumour based on the p-score, wherein a p-score of less than 0.5 is indicative of fibroadenoma and a p-score of 0.5 and above is indicative of phyllodes tumour; and
selecting the treatment of the patient based upon the type of fibroepithelial tumour and p-score.
In one aspect, there is provided a kit when used in the method of any one of claims 1-15, comprising:
a primer pair for amplifying the one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1.
The terms “biological material” or “biological sample” as used herein refers to any material or sample, which includes an analyte as defined herein. Such samples may, for example, include samples derived from or comprising stool, whole blood, serum, plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, fluid harvested from a site of an immune response, fluid harvested from a pooled collection site, bronchial lavage, urine, biopsy material, for example, from all suitable organs, for example, the lung, the muscle, brain, breast, liver, skin, pancreas, stomach, etc., a nucleated cell sample, a fluid associated with a mucosal surface, hair, or skin.
The term “polymerase chain reaction (PCR)” refers to an enzyme-mediated reaction use to amplify a specific target DNA sequence. By amplifying the target DNA sequence in the DNA template, it is then able to produce millions more copies of the targeted DNA sequence. This is useful when a biological sample contains only small amounts of DNA. PCR is carried out in a mixture containing DNA polymerase, a pair of primers (forward and reverse) and four deoxynucleotide triphosphates (dNTPs) with the aid of thermal cycler. A PCR reaction cycle typically involves a denaturation step to yield single stranded DNA molecules, an annealing step to allow primers to anneal to the DNA molecules, an extension/elongation step to allow a new complementary strand of DNA to be synthesized. The PCR reaction cycle is repeated to allow amplification of the target DNA.
The term “cycle threshold” or “Ct” as used herein refers to the number of cycles of a PCR reaction cycle that is required before a target DNA is positively detected. Ct levels are inversely proportional to the amount of target DNA in a sample.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
In a first aspect the present invention refers to a method of method of determining a fibroepithelial tumour of the breast, in a biological sample in vitro. The method may comprise: obtaining an expression profile of one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1, in said sample; obtaining an expression profile of one or more normalized genes for use as a control; determining the differential activity of the one or more genes relative to the control based on the expression profile of the one or more genes and one or more normalized genes; correlating the differential activity of the one or more genes relative to the control to obtain a p-score; and determining the type of fibroepithelial tumour based on the p-score, wherein a p-score of less than 0.5 is indicative of a fibroadenoma and a p-score of 0.5 and higher is indicative of phyllodes tumour.
The one or more genes may be selected from the group consisting of PRAME, TRIM29, FN1, CCL19, ABCA8, NPTX2 and APOD. In one embodiment, two, three, four or five or more genes may be selected from the group consisting of PRAME, TRIM29, FN1, CCL19, ABCA8, NPTX2 and APOD. In a preferred embodiment, the one or more genes may consist of PRAME, FN1, CCL19, ABCA8, and APOD.
The one or more genes may be compared against one or more reference genes. A reference gene is a gene that is used as a basis of comparison with a gene of interest. It will be understood to one of skill in the art that the expression level of a gene of interest may be normalized against one or more reference genes to obtain an expression level of the gene of interest relative to the reference gene. Normalization of the expression level of a gene of interest against one or more reference genes allows for the comparison of the expression levels of multiple genes of interest within a sample and/or between samples.
A reference gene may be a housekeeping gene or a normalized or normalization gene. One or more reference genes may be used alone or in combination. Housekeeping genes are well known in the art and refer to genes that are constitutively expressed in all cells of an organism under normal physiological conditions. Examples of housekeeping genes include but are not limited to β-actin, GAPDH and 18S. A normalized gene is one that has whose expression varies minimally across all samples. Normalized genes may be selected based on the smallest value of coefficient of variance (mean/standard deviation). A normalized gene may be a housekeeping gene or any other gene whose expression varies minimally across all samples.
In one embodiment, the one or more normalized genes may be selected from the group consisting of RMRP, RPL18, RPLP2, SNORA61 and SNORA45. In a preferred embodiment, the one or more normalized genes may consist of RMRP, RPL18, RPLP2, SNORA61 and SNORA45.
In one embodiment, the step of correlating the differential activity of the one or more genes relative to the control to obtain a p-score comprises calculating a ΔCt (Delta threshold cycle) value for the one or more genes. It will generally be understood that the ΔCt value is the Ct value for a gene normalized to one or more normalized genes. In one example, the ΔCt of a gene to be tested may be the Ct value of the gene, normalized to one or more normalized genes. In a preferred example, the ΔCt of a gene to be tested may be the Ct value of the gene, normalized to the geometric mean (Ct of the five normalized genes) as follows: ΔCt of test gene=Ct of test gene−geometric mean (Ct of five normalization genes)
It will generally be understood that one, two, three, four, five or more normalization genes may be used in the ΔCt calculation.
The ΔCt value may be used to calculate a p-score using a predictive algorithm. For example, a predictive algorithm may be:
In one embodiment, the biological sample may be selected from the group consisting of an organ, tissue, fraction, and a cell. A tissue sample may be obtained from tumour tissue selected from the group consisting of frozen tissue, tissue biopsies, circulating tumour cells, bodily fluids or other biological sample.
The biological sample may be a fresh, frozen, or fixed sample. In one embodiment, the biological sample may be formalin-fixed and paraffin embedded (FFPE).
In one embodiment, the bodily fluids may be selected from the group consisting of ascites, effusions, cerebrospinal and urine.
In a one embodiment, the biological sample may be a malignant tumour sample. In another embodiment, the biological sample may be a benign tumour sample. In a further preferred embodiment, the tumour sample may be a sample from the breast.
In one embodiment, RNA may be extracted from the biological sample in order to obtain the expression profile of the one or more genes and one or more normalized genes. In another embodiment, the expression profile of the one or more genes and one or more normalized genes is obtained from the sample biological sample. In yet another embodiment, the expression profile of the one or more genes and one or more normalized genes may be obtained by a quantitative PCR method.
In another aspect, the present invention refers to a method for managing the treatment of a subject with a fibroepithelial tumour of the breast. The method may comprise: obtaining an expression profile of one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1, in a biological sample obtained from the subject; obtaining an expression profile of one or more normalized genes for use as a control; determining the differential activity of the one or more genes relative to the control based on the expression profile of the one or more genes and one or more normalized genes; correlating the differential activity of the one or more genes relative to the control to obtain a p-score; determining the type of fibroepithelial tumour based on the p-score, wherein a p-score of less than 0.5 is indicative of fibroadenoma and a p-score of 0.5 and above is indicative of phyllodes tumour; and selecting the treatment of the patient based upon the type of fibroepithelial tumour and p-score.
It will be generally understood that the treatment of a subject with a fibroepithelial tumour of the breast may be tailored based on the indication of a malignant phyllodes tumour or a benign fibroadenoma.
In a third aspect, the present invention relates to a kit when used in the method of as described herein. The kit may comprise: a primer pair for amplifying the one or more genes selected from the group consisting of PRAME, ADH1B, CTHRC1, NPTX2, NEFL, ABCA8, DAPL1, TP63_v2, COL17A1, GCNT2, CCL19, MMP3, FN1, TRERF1, TRIM29, TESC, KIF20A, UHRF1, HEPACAM2, APOD, SERHL2, KIF15, HOXD13, GAGE2B, CALML5, C2orf40, ADH1C, CYP1B1, SPAG11B, GRB7, UBE2C, SYNGAP1, TP63_v1, LAMB1, OR5P3, SPC25, SHISA2, SCARA5, LHX2, RORC, DPYSL4, CH25H, and CHST1.
In one embodiment, the one or more genes may be selected from the group consisting of PRAME, TRIM29, FN1, CCL19, ABCA8, NPTX2 and APOD. In another embodiment, the one or more genes may consist of PRAME, FN1, CCL19, ABCA8, and APOD.
In one embodiment, the kit further comprises a primer pair for one or more normalized genes selected from the group consisting of RMRP, RPL18, RPLP2, SNORA61 and SNORA45. In a preferred embodiment, the one or more normalized genes may consist of RMRP, RPL18, RPLP2, SNORA61 and SNORA45.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Study Population
The study received approval from the Institutional Review Board. Forty eight formalin-fixed, paraffin embedded (FFPE) samples of fibroadenomas (FA) and phyllodes tumours (PT) were selected from the database of Department of Pathology, Singapore General Hospital (Table 1). Haematoxylin and eosin (H&E) stained slides were retrieved and reviewed. Diagnoses for the core biopsy samples were confirmed on corresponding excisions.
a5 paired core biopsies and surgical excisions;
b4 paired core biopsies and surgical excisions;
c1 paired core biopsy and surgical excision
Gene Expression Profiling
Sample Preparation and RNA Extraction
Representative tumour area was identified and marked. 3-7 sections of 10 μm sections from the same FFPE tumour block were obtained. Sections were deparaffinised in two changes of xylene for 2 minutes each then in three changes of absolute ethanol for 1 minute each. Macrodissection was performed immediately to retrieve the tumour area. RNA was extracted from the macrodissected tissue using the RNeasy FFPE kit (Qiagen, Germany) according to manufacturer's protocol with slight modifications. Briefly, buffer PKD and proteinase K was added after all ethanol was removed from the tissue. The tissue mixture was incubated overnight at 56° C. instead of 15 minutes as stated in the protocol, then it was incubated at 80° C. for 15 minutes followed by incubation on ice for 3 minutes. DNase I and DNase Booster Buffer was added and incubated in room temperature for 15 minutes after which buffer RBC was added to adjust for binding condition. Absolute ethanol was added and the entire lysate was then passed through a filter cartridge and washed with buffer RPE according to the manufacturer's instructions. Finally, RNA was eluted in 30 μl nuclease-free water and stored at −80° C. immediately.
Quality Assessment for RNA Extracted
RNA extracted was quantified by Nanodrop Spectrophotometer (Thermo Scientific, USA). 100 ng was used for quality assessment by real-time amplification of the RPL13A gene (forward primer, 5′-CACTTGGGGACAGCATGAG-3′ (SEQ ID NO: 1), and reverse primer, 5′-TAACCCCTTGGTTGTGCAT-3′ (SEQ ID NO: 2) using the PowerSYBR® Green RNA-to-CT™ 1-Step Kit (Life Technologies, USA) on the CFX96™ Real-Time PCR machine (Bio-Rad Laboratories, USA), Samples with threshold cycle (Ct) below 29 passed for subsequent experiments.
Expression Profiling by Whole-Genome DASL HT Assay
1 μg of RNA were submitted for expression profiling at the Biopolis Shared Facilities, Agency of Science, Technology and Research (A*Star), Singapore. Samples were further subjected to quality assessment on a bioanalyzer before subjected to expression profiling on the Whole-Genome DASL (WG-DASL) HT Assay (Illumina, Inc., USA), The assay interrogates 29,377 features using the HumanHT-12 v4 BeadChip. Quantile-normalized gene expression data pre-analyzed using GenomeStudio® was delivered.
Selection of Normalization Genes
Normalization genes were selected based on the smallest value of coefficient of variance (mean/standard deviation) among all samples. Five genes with the least variable expression were selected. The genes are RMRP, RPL18, RPLP2, SNORA61, and SNORA45 (
Selection of Potential Differentiating Genes
Significance Analysis of Microarrays (SAM, R version 3.1.1) was employed to select significant genes that were differentially expressed between phyllodes tumours (PT) and fibroadenomas (FA). Then, genes were filtered based on the following criteria: 1) q-value less than 0.05; 2) mean difference of expression above 500; 3) R-fold above 1.5 (for genes highly expressed in PT) or less than 0.67 (for genes highly expressed in FA). List of genes after criteria applied is shown in Table 2. 43 genes were selected from the list for downstream applications.
phyllodes tumours.
Validation of Selected Genes by Quantitative Polymerase Chain Reaction (qPCR) Assay
Primers Design
Primers were designed using Primer-BLAST (NCBI) with accession number listed in Table 3. Criteria for qPCR primers designed were as follows: 1) amplicon size of 50 to 80 basepair (bp); 2) at least one primer spanning across an exon-exon boundary; 3) At least 7 bases must anneal to the 5′ and 3′ side of the junction. List of primers designed is as shown in Table 3.
†normalization genes
Synthesis of cDNA and qPCR Assay
cDNA was synthesized from 1 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems®, Life Technologies, USA) on a thermal cycler according to manufacturer's protocol. cDNA was then diluted 10-fold for subsequent qPCR assays.
qPCR assays were performed on the CFX96 machine. Each qPCR reaction consisted of 1× Power SYBR® Green PCR Master Mix (Life Technologies, USA), 0.5 μM of forward and reverse primer each and 1 μl of the diluted cDNA as template in a final total volume of 10 μl. Non-template control acted as a negative control. Specificity of the amplicons was checked with meltcurve analysis.
To validate the expression observed on the WG-DASL Array, six representative samples were used as a pilot run for each potential gene tested.
Data Analysis
Expression data from qPCR assays was obtained in terms of threshold cycle (Ct). Ct above 40 was deemed as below limit of detection and is converted to 40. Delta Ct (ΔCt) was quantified as follows:
ΔCt of test gene=Ct−geometric mean (Ct of five normalization genes)
ΔCt data was transformed to 2−ΔCt as a positive linear scale for comparison with expression value of DASL. Significance of correlation was analyzed with Pearson's correlation test. Table 4 shows the results of the pilot run. Genes with good correlation value (r≧0.6) were subjected to testing on remaining 40 samples. Two samples had no sufficient RNA for the qPCR assays after characterization on WG-DASL and hence were not included in the analysis.
Development of an Algorithm Model Based on qPCR Assay Results on 46 Samples
Variable Selection Using Random Forest
ΔCt values of all 23 genes on 46 samples were analyzed with Random Forest (RF) classifier algorithm (R version) to rank importance of genes differentiating fibroadenomas and phyllodes tumours. Results are as shown in
Development of an Algorithm Model Using Logistic Regression
Top seven genes (TRIM29, FN1, CCL19, ABCA8, NPTX2, APOD, PRAME) were used for logistic regression modeling. All possible models were screened to find the best model using the glmulti package (R version) including the interactions terms. The best model was selected based on the lowest AIC (Akaike information criteria) value. The coefficient of the best model generated is as shown in Table 5. The AIC for the model was 14.2.
Validation of the Predicting Algorithm on an Independent Set
An independent set of 230 core biopsy samples (189 fibroadenomas and 41 phyllodes tumours) was employed to evaluate the performance of the algorithm. RNA was extracted and cDNA synthesized as per the protocol above. ΔCt values were obtained as per the protocols above. Probability (p) of each sample whether it is a fibroadenoma or phyllodes tumours are calculated in the following manner:
A sample is predicted as a phyllodes tumour when p is 0.5 and above. Otherwise, the sample is predicted as a fibroadenoma. The outcome of the multigene assay was compared against the final diagnosis on the corresponding surgical excisions. Cases without subsequent surgical excisions were free from progression for at least two years and diagnosis made based on the initial core biopsy was used as the reference instead. The five-gene assay has an overall accuracy of 92.6% (
Protocol for a Multigene Assay Classifying Breast Fibroepithelial Lesion
Equipment
Materials
Consumables
Protocol
ΔCt of each gene=Ct−geometric mean (Ct of RMRP, RPL18, RPLP2, SNORA45 and SNORA61)
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201501928T | Mar 2015 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2016/050117 | 3/14/2016 | WO | 00 |
