Next-Generation Sequencing for Detection of Clonal TRG Gene
Rearrangements Shows Improved Specificity and Positive Predictive Value Compared
to Fragment Analysis Using BIOMED-2 Primers and Capillary Electrophoresis
Mark D. Ewalt1,2, Michael Klass3, Jeff Panganiban3, Ying Huang3, Tim Stenzel3, Kasey Hutt3, Lisa Ma1, Daniel A. Arber1, Jason D. Merker1, Iris Schrijver1, James L. Zehnder1
1Department of Pathology, Stanford University Medical Center, Stanford, CA USA; 2Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, CA USA; 3Invivoscribe® Technologies, Inc., San Diego, CA USAResults

During T cell development, somatic rearrangements of T cell receptor gamma(TRG) genes generate unique V-J rearrangements within each cell. Overrepresented TRG rearrangements can be identified in the majority of T cell andsome B cell malignancies, but are generally not seen in benign reactive processes.
PCR-based capillary electrophoresis (PCR-CE) assays are the current gold
standard for detecting clonal rearrangements. In this study, we evaluated the®
®
®
®
Invivoscribe LymphoTrack TRG clonality assay using the Illumina MiSeq tocompare the performance of Targeted Next-Generation Sequencing (T-NGS) to thegold standard PCR-CE assay. We also sought to compare the performance of the TNGS assay in two separate laboratories to assess inter-laboratory reproducibility.

Stanford

Introduction
LabPMM
Positive Negative
Positive
Negative

19
5NOT
EVALUABLENo≥ 10,000 reads
Combine sequences with≤ 2 bases different
Depth based cutoff≥ 5% for 10,000 reads≥ 2.5% for 50,000 reads

NoLN	  w/CLL/SLL
LN	  w/FL

NONCLONAL
PTCL,	  NOS

Cutaneous	  CD4+	  small/medium	  TCLSPTCL

T-­‐LGL	  leukemia

Skin,	  negative	  for	  breast	  cancer
LN,	  negative	  for	  breast	  cancer
Tonsil,	  reactive

Test
LymphoTrack

Spleen,	  unremarkable
LN,	  reactive
LN,	  negative	  for	  breast	  cancer
Tonsil,	  reactive

Results

Results for the 58 samples were evaluated for sensitivity, specificity, concordance,
positive predictive value (PPV), and negative predictive value (NPV). T-NGS
analysis was compared between Stanford and LabPMM and showed 91%
concordance. 5 cases were discordant; however, 4 of 5 cases identified many of thesame clonal sequences with slight differences in frequency.
Separately, histopathologic diagnosis was considered the reference and PCR-CE
was compared to T-NGS. PCR-CE as compared to T-NGS showed similarsensitivity (86% vs. 79-89%), concordance (85% vs. 90-93%), and NPV (86% vs.
83-90%). In contrast, PCR-CE showed lower specificity (83% vs. 97-100%) andPPV (83% vs. 96-100%).

BIOMED-2

®

0.7
0.6
0.5
0.4

Average
LymphoTrack

0.3
0.2

CD30+	  large	  cell	  lymphoma

LN	  /FLunremarkableSpleen,	  LN,	  wreactive

Tonsil,	  reactive

CLONAL

ALCL,	  ALK-­‐positive

LN	  /FLunremarkable
Spleen,	  LN,	  wreactive
LN	  w/CLL/SLL
Skin,	  negative	  for	  breast	  cancer
LN,	  negative	  for	  breast	  cancer
Tonsil,	  reactive

LN,	  reactive

Yes

MFLN	  
/FLunremarkable
Spleen,	  LN,	  wreactive
LN	  wn/CLL/SLL
Skin,	  negative	  for	  breast	  cancer
LN,	  egative	  for	  breast	  cancer
Tonsil,	  reactive

0.8

Most frequent sequence ≥twice third most frequentALCL,	  ALK-­‐negativeLN	  wn/CLL/SLL
Skin,	  negative	  for	  breast	  cancer
LN,	  egative	  for	  breast	  cancerTonsil,	  reactive

0.9

1 or 2 sequences above cutoff

NoAITLLN	  wn/CLL/SLL
Skin,	  negative	  for	  breast	  cancer
LN,	  egative	  for	  breast	  cancer
Tonsil,	  reactive
LN	  wreactive/FLunremarkable
Spleen,	  LN,	  No1Sensitivity

58 FFPE Samples

LN	  /FLunremarkable
Spleen,	  LN,	  wreactive

031

Interpretation Criteria

DNA was isolated from 58 FFPE samples that had previously been evaluated in thePathology Department at Stanford University Medical Center and evaluated byPCR-CE for TRG gene rearrangement (29 T cell lymphoproliferative disorders, 26
reactive tissues, and 3 lymph nodes involved by B cell lymphoma). T-NGS wasperformed using the Invivoscribe LymphoTrack TRG – MiSeq assay according to
the manufacturer’s instructions at Stanford University Medical Center and®
LabPMM , a subsidiary of Invivoscribe. T-NGS data was analyzed by anInvivoscribe-developed bioinformatics pipeline and results were interpreted usingnumerical criteria, blinded to results of PCR-CE and histopathologic diagnosis.

LN	  w/FLunremarkable
Spleen,	  LN	  wn/CLL/SLLSkin,	  negative	  for	  breast	  cancer
LN,	  egative	  for	  breast	  cancer

Sample Negative Case

Concordance Rate = 91%

Methods

LN	  w/CLL/SLL
Skin,	  negative	  for	  breast	  cancer

Sample Positive CaseLabSensitivity

Specificity

Stanford

79%

100%

LabPMM

89%

97%

86%

83%

BIOMED-2

0.10®

00.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 - Specificity

Conclusions
The Invivoscribe® LymphoTrack® TRG assay shows good inter-laboratory reproducibility and similar sensitivity, concordance, and NPV to PCR-CE whenusing histopathologic diagnosis as a reference. In contrast, T-NGS shows a higher specificity and PPV than PCR-CE. In addition, T-NGS offers the potential to follow
specific clonal sequences for monitoring of minimal residual disease in T cell malignancies. Given this potential benefit and the superior assay performance
demonstrated by our data, T-NGS represents an exciting advance in the diagnosis of clonal lymphoproliferative disorders.

Disclosures
MK, JP, YH, TS, and KH are employees of Invivoscribe®.
ME has received an honorarium and travel expenses from Invivoscribe®.
LymphoTrack® TRG Assays are for research use only (RUO). Not for use in diagnostic procedures.