Patent ID: 12241124

The invention is now described by way of example which is intended to illustrate, rather than limit, embodiments of the invention as set out in the claims.

EXAMPLES

Example 1: Correlation of Joining and Constant Regions

TRBC transcripts were amplified using 5′RACE from 4 normal Human Tonsil samples. SeeFIG.2which shows NGS analysis of 4×106unique T cell transcripts.

PCR products of ˜530-620 bp in length were pooled and sequenced using Miseq illumina sequencing. Pair-end reads of 2×300 bp were acquired.

Results are shown inFIG.1.

Example 2: Application to DNA

We refer toFIG.3.

Suitably the DNA is genomic DNA.

Example 3: Read Out of Sequence

We refer toFIG.4.

IMGT output is shown.

Example 4

In this study various TRBC1/2 expressing cell lines were tested (Table 4).

Data from various T-cell lymphoma samples are also presented (Table 5).

We also provide an exemplary nucleic acid extraction protocol and exemplary NGS protocol.

In this example, data are obtained using the LymphoTrack® Dx TRB assay from Invivoscribe.

Methods

Formalin Fixed paraffin embedded (FFPE) cell lines (Jurkat, MJ, H9, HPB and Raji) and FFPE T-cell lymphoma samples were analyzed with the LymphoTrack Dx TRB Assay—MiSeq assay. DNA from each cell line and T-cell lymphoma sample was extracted from 10-15 5 μM FFPE sections using the Qiagen GeneRead DNA FFPE kit following the instructions from manufacturer unless otherwise mentioned herein.

DNA concentration was quantified using Qubit 3.0. DNA was tested in singles by the LymphoTrack Dx TRB Assay—MiSeq following the Assay IFU (280410). The FASTQ files from MiSeq runs were analyzed by the LymphoTrack Dx Software—MiSeq v2.4.3 following Software IFU (280344).

Sectioning of FFPE Tissue Blocks

1. Chill paraffin-embedded tissue blocks on ice before sectioning. Cold wax allows thinner sections to be obtained by providing support for harder elements within the tissue specimen. The small amount of moisture that penetrates the block from the melting ice will also make the tissue easier to cut.2. Fill a waterbath with ultrapure water and heat to 40-45° C.3. Place the blade in the holder, ensure it is secure and set the clearance angle. The clearance angle prevents contact between the knife facet and the face of the block. Follow the microtome manufacturer's instructions for guidance on setting the clearance angle. For Leica blades this is normally between 1° and 5° (FIG.1).4. Insert the paraffin block and orientate so the blade will cut straight across the block.5. Carefully, approach the block with the blade and cut a few thin sections to ensure the positioning is correct. Adjust if necessary.6. Trim the block to expose the tissue surface to a level where a representative section can be cut. Trimming is normally done at a thickness of 10-30 μm.7. Cut sections at a thickness of about 4-5 μm (you will probably need to discard the first few sections as they are likely to contain holes caused by trimming).8. Using tweezers, pick up the ribbons of sections and transfer to an autoclaved or DNAse free microtube (1.5 mL).9. Proceed with DNA isolation using the Qiagen GeneRead DNA FFPE kit following the instructions from the manufacturer.
Nucleic Acid Extraction Procedure

There are several DNA extraction kits that can be used. In this example, DNA is extracted using the Qiagen's GeneRead DNA FFPE Kit.

The GeneRead DNA FFPE procedure removes paraffin and reverses formalin cross-links from the DNA sample before it is bound to the QIAamp MinElute column. After heating to remove cross-links, the DNA is accessible for the specific removal of deaminated cytosine residues by the enzyme Uracil-N-Glycosilase (UNG). The optimized reaction mixture provides conditions in which the UNG can specifically remove artificially induced uracils from the DNA obtained from the FFPE sample. After the binding of DNA to the spin column, residual contaminants such as salts are washed away by Buffers AW1 and AW2, and ethanol. Any residual ethanol, which may interfere with subsequent enzymatic reactions, is removed by an additional centrifugation step. DNA is eluted and is now ready to use in next-generation sequencing workflows.

Sequence Determination Procedure

In this example sequence is determined using the NGS protocol—LymphoTrack Dx TRB Assay—MiSeq assay:1. Using gloved hands, remove the Master Mixes from the freezer. Allow the tubes to thaw; gently vortex to mix.2. In a containment hood or dead air box, pipette 45 ul of Master Mix into individual wells of a PCR plate. One well for each of the Master Mixes and one Master Mix per sample, positive, negative or no template controls.3. Add 0.2 ul EagleTaq DNA polymerase (EagleTaq @5 U/uL) to each of the Master Mixes.4. Add 5 ul of sample DNA (at a minimum concentration of 10 ng/uL) and 5 uL of control samples to wells containing the respective Master Mix reactions and pipette up and down 5-10 times to mix.5. Add 5 uL of molecular biology grade water to the well containing the respective Master Mix for no template control and pipette up and down 5-10 times to mix.6. Amplify target DNA using the following thermal cycler program:

StepTemperatureTimeCycle195° C.7minutes1295° C.45seconds29X360° C.45seconds472° C.90seconds572° C.10minutes1615° C.*forever*17. Remove the amplification plate from the thermal cycler8. Purify the PCR products using the Agencourt AMPure XP PCR Purification system. Add 35 ul of particles to each 50 ul reaction; elute DNA in 25 ul eluate.9. Quantify amplicons using the KAPA library quantification kit according to the kit instructions. Dilute amplicons 1:4,000 before proceeding to qPCR.10. Pool equal amounts of amplicons from samples (do not include the no template control), dilute 1:1,000 and quantify the library using the KAPA library quantification kit.11. Denature and dilute the library to 12 pM for MiSeq reagent kit v2 and 12-20 pM for MiSeq e=reagent kit v3 (MCS v2.6).12. Load 600 ul of denatured and diluted library to the MiSeq Reagent Cartridge.13. Set up a MiSeq sample sheet using the Illumina Experiment Manager (v1.4 through v1.13).14. Start the MiSeq run.15. Analyze and visualize the acquired data using the associated LymphoTrack Dx Software—MiSeq package.
LymphoTrack Dx Software—MiSeq Package Interpretation and Reporting

The Merged Read Summary report should be used to identify the top merged read sequences and their frequencies prior to clonality determination using the criteria listed in Table 3.

TABLE 3Criterion 1Criterion 2Criterion 3Criterion 4CallThe totalThe topThere is at leastThe % readsEVIDENCE OFnumber ofmergedone D-Jfor aCLONALITYreads for eachsequencerearrangementsuspectedDETECTEDsamplehas ≥2.5%detected in theclonal mergedis ≥20,000.of thefour mostsequencetotal reads.frequent mergedis >2X the %sequencesreads for the5th mostfrequentmergedsequence.1The % readsNo evidence offor aclonalitysuspecteddetectedclonal mergedsequenceis ≤2X the %reads for the5th mostfrequentmergedsequence.1There areThe % readsEVIDENCE OFno D-Jfor aCLONALITYrearrangementsuspectedDETECTEDdetected in theclonal mergedfour mostsequencefrequent mergedis >2X the %sequencesreads for the3rd mostfrequentmergedsequence.1The % readsNo evidence offor aclonalitysuspecteddetectedclonal mergedsequenceis ≤2X the %reads for the3rd mostfrequentmergedsequence.1The totalThe topThere is at leastThe % readsEVIDENCE OFnumber ofmergedone D-Jfor aCLONALITYreads for eachsequencerearrangementsuspectedDETECTEDsamplehas ≥5.0%detected in theclonal mergedis ≥10,000of thefour mostsequenceand <20,000.total reads.frequent mergedis >2X the %sequencesreads for the5th mostfrequentmergedsequence.1The % readsNo evidence offor aclonalitysuspecteddetectedclonal mergedsequenceis ≤2X the %reads for the5th mostfrequentmergedsequence.1There areThe % readsEVIDENCE OFno D-Jfor aCLONALITYrearrangementsuspectedDETECTEDdetected in theclonal mergedfour mostsequencefrequent mergedis >2X the %sequencesreads for the3rd mostfrequentmergedsequence.1The % readsNo evidence offor aclonalitysuspecteddetectedclonal mergedsequenceis ≤2X the %reads for the3rd mostfrequentmergedsequence.1The totalN/AN/AN/ANot evaluablenumber ofreads for eachsampleis <10,000.1Software calculations are rounded to the nearest tenth for comparison.
Results

Diagnostic and histology data for the T cell lymphoma samples can be found in Table 6.

Referring to the tables below, “total count” means the total number of reads obtained from the NGS instrument. “Length” means the length of read obtained. “V-gene” refers to the V gene type detected. “J-gene” refers to the J gene type detected. “Percentage total reads” is the percentage out of the total number of reads which matched this specific gene sequence as determined.

It will be observed that certain rows in the table(s) show more than one V gene or J gene type from a single sample—this can happen when a D-J join is detected as well as a V-J join. As explained elsewhere in this document, any D-J joins detected are discarded since they represent incomplete recombination events. Suitably the J gene type determined is from a V-J joined J gene (J region).

Cell Lines

DNA concentrations, amplicon concentrations and Top 1 and/or 2 clonal rearrangements for the cell lines are summarized in Table 4:

TABLE 4Top 1 and/or 2 clonal rearrangements for the cell linesDNA inputCellinto PCRTotal% totalTRBCType(ng)CountLengthV-geneJ-genereads(C1 or C2)Jurkat30658,707198Vb12-4Jb1-275.92C1HPB-ALL2533,238219Vb5-5Jb2-51.43C2H950588,668205Vb13Jb1-289.18C1MJ6.592,995219Db1Jb1-119.69C1189Vb28Jb1-117.55Raji2546,883201Db2Jb2-20.08Non-clonal
Shading Represents Incomplete D-J Rearrangements

One cell line sample (MJ) was detected with 2 clonal rearrangements. The D/J rearrangements are an incomplete rearrangement and will spliced out in V-J-C combination. Data from incomplete rearrangement(s) such as D/J rearrangements is suitably discarded. Focus is on the complete V/J rearrangements. Thus for ‘MJ’, the ‘Db1’ row of data is discarded due to being incomplete rearrangement. The Vb28 row of data is retained.

One cell line sample (Raji) was detected with the top % total reads less than 1.0% and is non-clonal. Data which is non-clonal is suitably discarded. Focus is on data which is clonal.

Following discarded data as explained above, Table 4A is produced.

TABLE 4ATop 1 and/or 2 clonal rearrangements for the cell linesDNA inputCellinto PCRTotal% totalTRBCType(ng)CountLengthV-geneJ-genereads(C1 or C2)Jurkat30658,707198Vb12-4Jb1-275.92C1HPB-ALL2533,238219Vb5-5Jb2-51.43C2H950588,668205Vb13Jb1-289.18C1MJ6.592,995189Vb28Jb1-117.55C1

Based on J1-C1 and J2-C2 correlations, MJ is C1.

Three cell line samples (Jurkat, HPB-ALL and H9) were detected with one V-J rearrangement. Based on J1-C1 and J2-C2 correlation, Jurkat and H9 cells are C1 and HPB-ALL cells are C2.

All cell line data is consistent with the reported literature.

Samples

DNA concentrations, amplicon concentrations and Top 1 and/or 2 clonal rearrangements for the T-cell lymphoma samples are summarized in Table 5.

Four T-cell lymphoma samples (F19542.1a, F19539.1c, F19538.A1b and TS18-1512A) are detected with 2 clonal rearrangements. Data from incomplete rearrangement(s) such as D/J rearrangements is suitably discarded. Focus is on the complete V/J rearrangements. Thus for these samples the ‘Db1’ or ‘Db2’ rows of data are discarded due to being incomplete rearrangements. The Vb13, Vb29-1, Vb12-4 and Vb6-4 rows of data are retained.

Two samples (TS18-1508A and TS18-1499A) are detected with either none-J or D-J rearrangements, and/or the top % total reads less than 1.0% and are considered non-clonal. Data from remaining samples determined to be non-clonal or not-evaluable according to IFU 280410 of the LymphoTrack kit, more suitably according to Table 3 above, are discarded. Data which is non-clonal is suitably discarded. Focus is on data which is clonal.

Following discarded data as explained above, Table 5A is produced.

TABLE 5Top 1 and/or 2 clonal rearrangements for the T cell lymphoma samples10 T-cell Lymphoma FFPE Samples: Diluted to 10 ng/μLTCRCallDNADNAAmpliconbased onSampleConc.inputConc.Total% totalJ1 or J2ID(ng/μL)(ng)(nM)countLengthV-geneJ-genereadsClonality(C1 or C2)F19542.1a28.9501.527,344215Vb13Jb1-48.64ClonalC1208Db1Jb1-25.18F19539.1C52.0505.134,498216Vb29-1Jb2-25.38ClonalC2214Db1Jb1-14.26F45038.b58.05014.0104,194186Vb12-4Jb1-638.00ClonalC1F19538.A1b42.4507.142,097198Vb12-4Jb1-614.32ClonalC1196Db2Jb2-76.51FF6679.B1b45.8507.270,579146VB14Jb2-70.48Non-bC1, C2ClonalNegF45029.B50.582.9N/AN/AN/AN/AN/AN/AcNotcNotevaluableevaluableTS18-1508A29.9501.518,34760noneJb1-10.38Non-aC1, C2ClonalNegTS18-1513A51.0502.879,716214Db1Jb2-71.11Non-bC1, C2ClonalNegTS18-1512A53.0505.869,034192Vb6-4Jb2-323.09ClonalC2205Db1Jb1-618.00TS18-1499A36.1502.820,549189Db2Jb2-73.32Non-bC1, C2ClonalNegaThe top % total reads is less than the clonal cutoff 5.0% for samples with the total reads ≥10,000 reads and <20,000. According to IFU 280410 and/or Table 3, this sample is called “Non-clonal”.bThe top % total reads is less than the clonal cutoff 2.5% for samples with the total reads ≥20,000 reads. According to IFU 280410 and/or Table 3, this sample is called “Non-clonal”.cThe minimum DNA input requirement is 50 ng per sample. According to IFU 280410, this sample is considered as “Not evaluable”. Shading represents incomplete D-J rearrangements

TABLE 5ATop 1 and/or 2 clonal rearrangements for the T cell lymphoma samplesT-cell Lymphoma FFPE Samples: Diluted to 10 ng/μLTCRCallDNADNAAmpliconbased onSampleConc.inputConc.Total% totalJ1 or J2ID(ng/μL)(ng)(nM)countLengthV-geneJ-genereadsClonality(C1 or C2)F19542.1a28.9501.527,344215Vb13Jb1-48.64ClonalC1F19539.1C52.0505.134,498216Vb29-1Jb2-25.38ClonalC2F45038.b58.05014.0104,194186Vb12-4Jb1-638.00ClonalC1F19538.A1b42.4507.142,097198Vb12-4Jb1-614.32ClonalC1F45029.B50.582.9N/AN/AN/AN/AN/AN/AcNotcNotevaluableevaluableTS18-1512A53.0505.869,034192Vb6-4Jb2-323.09ClonalC2

TABLE 6Diagnostic and histology data for the T cell lymphoma samplesSample IDHistological Tumor TypeTissue TypeaTCR IHCF19542.1aPeripheral T cell lymphoma, nosLymph Node,+AxillaryF19539.1cPeripheral T cell lymphoma, nosLymph Node+F45038.bPeripheral T cell lymphoma, nosLymph Node+F19538.A1bAngioimmunoblastic T cell lymphomaLymph Node,+InguinalFF6679.B1bHodgkin's like, AnaplasticLymph Node,+Large T-cell (Ki-1+) lymphomaMediastinalF45029.B5Peripheral T cell lymphoma, nosLymph Node+TS18-1508ACutaneous, Anaplastic LargeLymph Node−T-cell lymphoma, ALK-positiveTS18-1513AAnaplastic Large T-cell lymphoma,Lymph Node,+ALK-positiveCervicalTS18-1512ACutaneous, Anaplastic LargeLymph Node,+T-cell lymphoma, ALK-positiveAxillaryTS18-1499AAnaplastic Large T-cell lymphoma,Lymph Node,−ALK-positiveCervicalaTCR Immunohistochemistry (IHC) staining was performed to determine the presence of T cell receptors in the samples. 8/10 samples were TCR positive and 2/10 samples were TCR negative by IHC.Based on J1-C1 and J2-C2 correlations (Table 5A), F1.9542.1a is C1, F19539.1C and TS18-1512A are C2.One sample (F45038.b) is detected with one V-J rearrangement (Vb12-4/Jb1-6). Based on J1-C1 and J2-C2 correlation, this sample is C1.

Based on J1-C1 and J2-C2 correlations (Table 5A), F19542.1a is C1, F19539.1c and TS18-1512A are C2.

One sample (F45038.b) is detected with one V-J rearrangement (Vb12-4/Jb1-6). Based on J1-C1 and J2-C2 correlation, this sample is C1.

In summary, a sample is identified as having TRBC1 (C1) or TRBC2 (C2) positivity if the sample is determined to be clonal by the LymphoTrack Dx TRB Assay—MiSeq; where the presence of a J1 sequence determines C1 positivity or the presence of a J2 sequence determines C2 positivity.

Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiment(s) shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

All publications mentioned in the above specification are herein incorporated by reference.