ECP 2017 - Abstract Book

permit post-sequencing error correction, reducing background noise and

increasing analytical sensitivity.

Results:

This assay demonstrates 100 % detection sensitivity for 1 % AF

variants using 10 ng DNA input and 71.9 % detection sensitivity for

0.1 % AF variants using 50 ng DNA input. Post-sequencing error correc-

tion with MBC adapters results in 91.7 % specificity. Finally, mutations

detected from liquid biopsy-derived ctDNA show cancer type-dependent

concordance with tissue biopsy findings, and reveal additional oncogenic

driver mutations.

Conclusion:

These results indicate that AMP-based NGS is a powerful tool

for sensitive and specific NGS-based detection of variants in liquid biopsies.

OFP-13-011

Characterisation of B- and T-cell immune repertoires using anchored

multiplex PCR and next-generation sequencing

J. Eberlein

, T. Harrison, J. Sims, I. McKittrick, M. Wemmer, M. Bessette,

K. Trifilo, H. Wang, L. Griffin, B. Culver, L. Johnson, B. Kudlow

ArcherDX, Boulder, USA

Objective:

The immune repertoire (IR) provides a means to monitor

adaptive immune responses to disease, vaccination and therapeutic inter-

ventions. NGS-based IR characterization usually requires large primer

panels to capture its extensive combinatorial diversity and a complex

system of synthetic controls to account for differential amplification effi-

ciency across segment combinations. Here, we describe an Anchored

Multiplex PCR (AMP

™

)-based NGS assay to analyze the IR, employing

a minimal set of gene-specific primers in conjunction with molecular

barcodes (MBCs) to reduce amplification bias.

Method:

AMP is a library preparation method for NGS that uses MBC

adapters and gene-specific primers for amplification, enabling immune

chain mRNA interrogation from a single side. This eliminates the need for

opposing primers that bind within the highly variable V-segment and

facilitates CDR3 sequence capture from highly fragmented RNA inputs.

Results:

This assay demonstrates high reproducibility between replicates

and quantitative clone tracking down to 0.01 %, with the ability to deter-

mine IGHV mutational status. Our data indicate that clonal diversity in

sequencing data is primarily driven by input quantity and total T-cell

number.

Conclusion:

AMP-based NGS with MBC quantification and error-

correction is a powerful method to characterize the immune repertoire.

OFP-13-012

Evaluating overlap of circulating and tumour-infiltrating T-cells

using AmpliSeq-based Ion Torrent TCRB immune repertoire

sequencing

T. Looney

, E. Linch, L. Miller, D. Topacio-Hall, L. Lin, A. Pankov, J.

Zheng, K. Bergefall, A. Mongan, G. Lowman, F. Hyland

Thermo Fisher Scientific, Clinical Sequencing Division, South San

Francisco, USA

Objective:

TCR

immune repertoire analysis by next-generation se-

quencing is emerging as a valuable tool for research studies of the tumour

microenvironment and potential immune responses to cancer immuno-

therapy. Here we describe a multiplex PCR-based TCR

sequencing

assay that takes advantage of the exceptionally low base-call error rate

and long read capability of the Ion S5 530 chip.

Method:

We evaluated assay performance by 1) sequencing TCR

rear-

rangements from donor peripheral blood leukocyte (PBL) cDNA that had

been spiked with 30 reference rearrangements taken from literature and 2)

deeply sequencing libraries prepared from 10 ng to 1 ug of PBL cDNA.

We then evaluated the extent of clonal overlap between matched tumour

infiltrating lymphocyte (TIL) and peripheral blood leukocyte repertoires

in an individual with squamous cell carcinoma of lung.

Results:

Results from sequencing of spike-in reference rearrangements

indicate that the assay is accurate and sensitive over 5 logs of input

template amount while showing minimal amplification bias.

Rarefaction analysis of deeply sequenced libraries revealed libraries pre-

pared from <100 ng PBL template to approach saturation at <15 M reads

depth. Sequencing of matched PBL and TIL repertoires showed that a

subset of the PBL repertoire (8 %) consisted of clones also found in TIL.

Technical replicates showed high concordance (r > .96) in the frequency

of detected clones, indicating that the results were reproducible and sam-

ples were sequenced to an appropriate depth.

Conclusion:

In summary, these data suggest that AmpliSeq-based mul-

tiplex PCR and Ion Torrent sequencing provide unbiased, reproducible,

scalable, complete, and accurate information for immune repertoire re-

search sequencing applications.

OFP-14-001

Restoration of 50+ year old pathology museum specimens from the

University of Papua New Guinea (UPNG)

R. Cooke

Princess Alexandra Hospital, Anatomical Pathology, Brisbane, Australia

Objective:

This poster demonstrates how a 50 year old teaching pathol-

ogy museum in a poor country, Papua New Guinea is being rejuvenated

so that it can continue to be used for teaching. Similar advice was given to

the Curator of the museum in the University of Indonesia. This Museum

consists of specimens that were mounted before 1940. The Curator has

managed to remount most of the old specimens in newly, custom made

perspex display cases.

Method:

Pathology museums in many more affluent countries have been

neglected, but there is now a resurgence in interest in teaching pathology

to doctors and other health professionals. These museum specimens are

irreplaceable and they need to be rejuvenated so that they can be used

again for teaching.

Results:

This poster illustrates how it was done in PNGwith seed funding

from the Royal Australasian College of Surgeons. The remounting pro-

cess has continued during the 18 months since it was started. New display

cases have been purchased and the Curator has added photographs of the

specimen descriptions that allow students to study at their leisure.

Conclusion:

The PNG collection contains examples of Tropical Diseases

that occurred in the stone age population at the time of first contact with

Western Medicine. These are important historical records. Photographs of

clinical cases from that era, and photographs of the microscopic appear-

ances are being added to the display of potted specimens to further en-

hance the teaching value of the museum specimens.

OFP-14-002

Quain's fatty heart

R. Henriques de Gouveia

, T. Ferreira, M. J. Aguiar, A. Lopes, L.

Carvalho, F. Corte Real

INMLCF, Pathology, Coimbra, Portugal

Objective:

Obesity, visceral/epicardial fat, heart steatosis and their influ-

ence in cardiovascular disease are under international scientific investi-

gation spotlights. Yet, this issue

’

s concern goes back for centuries, namely

to the Irish physician Sir Richard Quain, whose surname was used to coin

a cor adiposum synonymous. The authors intend to know the old and the

new about

‘

fatty heart

’

Wednesday, 6 September 2017, 14:00

–

16:00, G104-105

OFP-14 Joint Session: History of Pathology /

Haematopathology

Virchows Arch

(

2017

)

471

(

Suppl 1

–

S352

S42