The successful application of endoscopic ultrasound guided fine needle biopsy to establish

pancreatic patient-derived tumour xenografts. Hermans E.1, Van der Merwe S. 2, Depreeuw J. 1,3, Dekervel J. 2, Radaelli E. 4, Van Pelt J. 2,

Thomas D. 1, Gommé E. 1, Lambrechts D. 3, Amant F.1* 1 Division of Oncology, KULeuven, Leuven, Belgium

² Division of Hepatology, KULeuven, Leuven, Belgium ³ Vesalius Research Center, VIB, Leuven, Belgium

4Center for the Biology of Disease, VIB, Leuven, Belgium

Methods

Introduction Results

Using the adapted protocol of EUS-derived FNB, we achieved an engraftment rate of 60% whereas all surgical samples were successfully transplanted. Despite a decrease in desmoplastic stromal reaction, the general morphology of EUS-derived FNB PDXs, as assessed by histology, was conserved compared to that of the primary tumor. Also at the genetic level, there was no obvious difference compared to surgical derived xenografts. In particular, despite some heterogeneity, somatic mutation and copy number profiles were largely shared between xenografts regardless of whether they were obtained by EUS or surgery.

We show that it is technically feasible to establish pancreatic PDXs from patients with locally advanced or metastatic disease by a minimal-invasive sampling technique such as EUS FNB. By acquiring a limited amount of tumour tissue, we demonstrated that tumour morphology, differentiation grade and genetic profile are largely maintained across the different passages, suggesting EUS-derived FNB xenografts may be useful to study pancreatic tumor biology and develop novel therapies in this patient population with a high clinical need. In addition, our approach could potentially be translated to establish PDXs in other unresectable tumor types.

Conclusion

Correspondence: Gynecologic Oncology, University Hospitals Leuven, Herestraat, 49, 3000 Leuven, Belgium. Tel.: +32 16 344252; E-mail address: frederic.amant@uzleuven.be (F. Amant).*

Biopsies of chemo-naive primary pancreatic carcinoma were collected at surgery and EUS. A FNB on patients (n=10) with suspected malignant pancreas tumour was performed using a 22G needle. Concentration of the EUS-derived FNB occurred by centrifugation of the sample prior to engraftment. Surgical samples (n=4) were obtained at the time of tumour resection. To asses preservation of general tumour morphology and tumour-specific characteristics, H&E and immunohistochemistry were performed. To determine conservation of the mutation profile and copy number alterations, whole exome sequencing (WES) and whole-genome low-coverage sequencing were executed.

Pancreatic cancer is one of the most lethal cancer types with an overall 5-year survival rate of less than 5%. The majority of the patients are diagnosed with already locally advanced disease or metastasis and are therefore excluded from surgery. At present, tumour biopsies obtained by invasive surgical approaches are still a prerequisite to successfully establish pancreatic PDXs thereby limiting this technique to patients that are eligible for surgery. The objective of the study was to establish pancreatic PDXs by transplanting tumour tissue acquired by endoscopic ultrasound (EUS) fine needle biopsies (FNB).

Figure 3: Diagram of somatic non-silent mutations of PAC005. Blue circle indicates mutations from primary tumor, red indicates mutations from the xenograft. Common mutations between primary tumor and xenograft are found in the center. Mutations found to be cancer consensus genes according to COSMIC are in blue.

Figure 1: Engrafted pancreatic ductal adenocarcinoma PDX models maintain the original tumor morphology irrespective of the biopsy technique. (H&E, 20x) A - Human FNB B - Third passage PDX FNB C - Third passage PDX Surgical derived sample

Figure 4: Post-transplant microenvironment adaptations are characterized by a general decrease in total amount of stroma and complete substitution of the original human stroma by a purely murine fibrovascular component. (A: Masson trichrome; B: human-vimentin; C: CD31; 20x)(Upper panel: Third passage Surgical derived sample; Lower panel: Third passage FNB PDX F3)

Figure 2: Copy number alterations in primary tumor and xenograft obtained from whole genome low coverage sequencing. PAC005 was found to be diploid in the primary tumor but tetraploid in the xenograft.

13 (0) 50 (4) 19 (0)

( ): Cancer Consensus genes COSMIC Primary tumor

Xenograft A B C

A B C