Keywords Endoscopic ultrasonography - Pancreas - Tissue diagnosis - Fine-needle aspiration/biopsy
Introduction
Despite improvements in therapy, the 5-year survival rate for pancreatic ductal adenocarcinoma
(PDAC) remains poor at <8% [1 ]
[2 ]. Because it is mainly diagnosed in advanced stages, only 15% to 30% of patients
with PDAC are eligible for resection, the only potentially curative treatment [3 ]
[4 ]. Moreover, PDAC is resistant to many conventional therapeutic modalities and rapidly
metastasizes to other organs [5 ]. In view of this fact, it is necessary to develop a more profound understanding
of the underlying biology of PDACs and also to address potential therapeutic targets.
So-called precision medicine aims to deliver treatment options to patients based on
genetic profiles, specific biomarkers, and bioinformatics [6 ]. In the case of PDAC, pancreatic cancer patient-derived organoids (PDOs) might be
able to make a significant contribution to precision medicine in the future. Organoids
are cells growing in a 3-dimensional (3D) structure, generated from primary tissues
and with the capacity to expand into ex vivo organ-like structures [7 ]
[8 ], thereby allowing for evaluation of potential diagnostic biomarkers, drug testing,
and identification of therapeutic vulnerabilities [9 ]
[10 ]. Organoids can primarily be obtained from surgical specimens and then have the potential
to be used for personalized treatment.
A major problem regarding a clinical benefit is that, as mentioned previously, the
majority of patients with PDAC are not eligible for surgical resection because they
are usually in a palliative or neoadjuvant setting at the time of diagnosis. Organoids
at the time of initial tumor diagnosis, therefore, are crucial.
Our group as well as others were able to show that successful organoid creation is
also possible with endoscopic ultrasound (EUS) material [8 ]
[10 ]
[11 ]. Recent studies have shown superior results with EUS-fine-needle biopsy (EUS-FNB)
in comparison with EUS-fine-needle aspiration (EUS-FNA) regarding histological tissue
acquisition and diagnostic accuracy with fewer needle passes [12 ]
[13 ]
[14 ]
[15 ]. However, with regard to the generation of PDOs, there are very little data about
which needle type has potential to produce the best results. The aim of this study
was to compare the extent to which the use of FNA needles (22G) or FNB needles (22G)
influences the generation of pancreatic cancer PDOs to establish endoscopic standards
for organoid technology.
Patients and methods
Patient selection
Eligible patients for this study were those aged >18 years with written informed consent
and pancreatic masses highly suspicious for PDAC (detected by computed tomography
and/or magnetic resonance imaging) and an indication for EUS-guided puncture of these
lesions such as planned neoadjuvant or palliative chemotherapy. Exclusion criteria
were patients without written informed consent, patients with pregnancy, an international
normalized ratio >1.5, a platelet count <50 × 109 /L or who were medically insufficiently stable to undergo sedation for EUS. The study
was approved by the Ethics Committee of the University Hospital of the Technical University
of Munich, Klinikum rechts der Isar. The study was performed in accordance with the
principles of the Declaration of Helsinki.
Study design and outcomes
This prospective study was conducted at Klinikum rechts der Isar, a high-volume university
endoscopy center with >1000 EUS procedures per year by experienced endosonographers
(U.M., M.T., G.F., M.A., C.S.). The primary outcome was successful generation of pancreatic
organoids, defined as reaching passage 5 (P5). Secondary outcomes included diagnostic
performance (sensitivity, specificity, and accuracy of FNA/FNB).
Procedure details
EUS punctures were carried out under sedation with propofol (Braun, Melsungen, Germany).
All procedures were performed by using a linear array echoendoscope (PENTAX, EG-3870UTK,
PENTAX Medical, Tokyo, Japan). Each patient received EUS-FNA (Beacon 22G, Medtronic,
Minneapolis, Minnesota, United States) and EUS-FNB (SharkCore 22G, Medtronic, Minneapolis,
Minnesota, United States) in a randomized order for histology and PDOs without the
need to exchange the needle shaft (only the inner needle [FNA/-B] was exchanged) between
the passes. All punctures were carried out using the “slow pull” method. Per needle,
at least one passage for histology and one passage for PDOs were performed, with a
limit of two passages for each needle and purpose (PDOs or histology). Therefore,
a maximum of eight passages in total were possible. The adequacy of the specimen was
estimated by the endosonographer after each passage and a second passage was only
performed in case of non-adequate material. Obtained specimens for PDOs were immediately
transferred into a minimal organoid media (10 mL DMEM-F12 cell culture media containing
1% penicillin/streptomycin and 0.2% Primocin) and transported to the organoid facility
for further processing and organoid generation. The specimens for histology were placed
into formalin and paraffin embedded for standard histological analysis in the Pathology
Department.
Organoid generation
The isolation of organoids out of FNA/FNB was performed as previously described [16 ]. Briefly, the sample was centrifuged (1000 rpm, 4°C, 5 minutes) and the supernatant
was discarded. The biopsy was cut into small pieces and transferred into a new 15-mL
falcon filled with cold phosphate-buffered saline (PBS) (#14190144 Thermo Fisher Scientific)
supplemented with 0.1% bovine serum albumin (#11930 Serva). After a second centrifugation
and discarding of the supernatant, the sample was incubated with red blood cell lysis
buffer (#A1049201 Thermo Fisher Scientific) for 3 to 15 minutes. PBS was added and
the flask was centrifuged. The supernatant was discarded and the sample was digested
with TrypLE (#12604039 Thermo Fisher Scientific) for 5 to 10 minutes at 37°C. The
flask was filled up with PBS and centrifuged again. After discarding the supernatant,
the sample was finally resuspended in 50 µL of Matrigel/well (#354230 Corning Life
Sciences). (One FNA or FNB sample was usually used to generate two to four wells.)
After 20 minutes, 500 µL of organoid media (DMEM-F12 (#11320033 Thermo Fisher), 5
mg/mL D-glucose (#G8270 Sigma-Aldrich), 0.5% ITS Premix (#354350 Fisher Scientific),
5 nM 3,3,5-Triiodo-L-thyronine (#T0821 Sigma-Aldrich), 1 µM dexamethasone (#D175 Sigma-Aldrich),
100 ng/mL cholera toxin (#C9903 Sigma-Aldrich), 1% penicillin/streptomycin (#15140122
Thermo Fisher Scientific), 5% NU-Serum IV (#355500 Fisher Scientific), 25 µg/mL bovine
pituitary extract (#P1167 Sigma-Aldrich), 10 mM nicotinamide (#N3376 Sigma-Aldrich),
100 µg/mL Primocin (#ant-pm05 Invivogen), 0.5 µm A83–01 (#2939 Tocris), 10% RSPO1-conditioned
medium (R-spondin-1 overexpressing cell line HEK293T, provided by the Hubrecht Institute
(Uppsalalaan 8, 3584 CT Utrecht, Netherlands), 100 ng/mL recombinant human geregulin-1
(#100–03 Peprotech), and 10 µM Rho kinase inhibitor (#TB1254-GMP Tocris) were added
per well.
Statistical analysis and sample size calculation
Statistical analyses were performed using Prism for MacOS (Version 9.1.1 GraphPad
Software, San Diego, California, United States). For diagnostic performance analysis
of needle types, Fisher´s exact test was used. Statistical significance was set at
P <0.05. We estimated a difference of at least 40% of growth rates of the organoid
depending on the needle type. To achieve a statistical power of 80% at a significance
level of P <0.05, at least 46 patients had to be included in the study. Block randomization was
used to ensure balanced group sizes. Thus, a total of 25 initial punctures were performed
using FNA and 25 using FNB.
Definitions
True positive was defined when malignancy was proven by histology. We considered a
lesion to be benign if there were no signs for malignancy on histology and interval
stability in radiological imaging.
Successful establishment of an organoid culture was defined by the ability to reach
P5 and sustain at least one freeze-thaw cycle. Any molecular characterization was
not the subject of the study.
Results
Between July 2019 and October 2020, 50 consecutive patients were included (26 men,
24 women) with a median age of 74.5 years (range 49–88 years). Median body mass index
was 23.9 kg/m2 (range 18.6–33.2 kg/m2 ). About one-fifth (22%) of the patients were diagnosed with type 2 diabetes mellitus.
The majority of the lesions were located in the pancreatic body region (60%), with
a median size of 32 mm (range 15–110 mm). [Table 1 ] shows the characteristics of the tumors. [Table 2 ] shows patient demographics. No serious adverse events were observed, and in particular,
no post-procedure pancreatitis or bleeding.
Table 1 Characteristics of newly diagnosed PDAs.
Tumors (n=50)
PDA, pancreatic ductal adenocarcinoma; IQR, interquartile range; FNA, fine-needle
Aspiration; FNB, fine-needle biopsy.
Localization, n (%)
Head/uncinate
15 (30)
Body
30 (60)
Tail
5 (10)
Size of mass, (IQR), mm
32 (15–110)
Average number of needle passes for positive histological diagnosis (range)
FNA
1.08 (1–2)
FNB
1.02 (1–2)
Table 2 Demographics of patients newly diagnosed with PDA.
Patient demographics (n=50)
PDA, pancreatic ductal adenocarcinoma; BMI, body mass index.
Median age, years (range)
74.5 (49–88)
Sex, n (%)
Female
24 (48)
Male
26 (52)
Median BMI kg/m2 (range)
23.9 (18.6–33.2)
Diabetes mellitus, type 2, n (%)
11 (22)
Smoker n, (%)
13 (26)
EUS-guided puncture of pancreatic lesions was followed by histological analysis and
PDO generation. Histologically, 42 lesions (84%) were malignant ([Table 3 ]). FNA had sensitivity, specificity, and accuracy of 61%, 100% and 61%, respectively,
whereas FNB had sensitivity, specificity, and accuracy of 84%, 100% and 84%, respectively
(P =0.18). The primary outcome, successful generation of PDOs, was achieved in 17 of
50 patients (34%; 95% confidence interval [CI], 21%–47%). Of these, nine PDOs were
generated by FNB only (53%), two by FNA only (12%), and six by both FNA and FNB (35%)
([Fig. 1 ] and [Fig. 2 ]). To summarize, 15 of 50 PDOs were generated by FNB (30%; 95% CI, 17%-43%) and eight
of 50 PDOs by FNA (16%; 95% CI 6%-26%), respectively.
Table 3 Diagnostic performance of needle types.
Diagnostic performance
FNA, fine-needle aspiration; CI, confidence interval; FNB, fine-needle biopsy.
Sensitivity
FNA
61% (95% CI 47%–73%)
FNB
84% (95% CI 71%–91%)
Specificity
FNA
100% (95% CI 5%–100%)
FNB
100% (95% CI 5%–100%)
Accuracy
FNA
61% (95% CI 51%–71%)
FNB
84% (95% CI 77%–91%)
Fig. 1 Schematic representation of the workflow and PDO generation. Created with
BioRender.com [rerif].
Fig. 2 Overview of established organoids per needle type.
Discussion
Because of the complex biology of pancreatic cancer with a high number of affected
genes and pathways [17 ], which vary in each case, there is a need for individualized treatment of each patient
with PDAC. Precision medicine describes the concept of using patient-specific information
(e.g. genomic or proteomic) to help clinicians make tailored diagnostic or therapeutic
decisions. Due to the limited treatment options for pancreatic cancer, this has particular
importance. Next-generation sequencing is a primary example of precision medicine
in pancreatic cancer. It allows the assessment of millions of segments of the genome
and detection of various genetic alterations or point mutations [18 ].
PDOs are a promising new tool for precision medicine and translational research in
pancreatic cancer. They represent a miniature tumor model of a human PDAC and can
be rapidly generated beside surgical resections from EUS-guided punctures [11 ]
[19 ]
[20 ]. EUS-guided sampling has a low risk of complications and can be repeatedly performed
at any stage of pancreatic cancer, therefore allowing for evaluation of the treatment
response to ongoing therapy.
Recently published data show that regarding pancreatic masses, FNB may be superior
to FNA in terms of histological core tissue [21 ], accuracy [14 ], and the number of passages required for an adequate specimen [16 ]. The accuracy of 84% in our study with FNB was similar to the current data for the
same FNB needle [14 ], as well as the FNA needle with inferior accuracy performance of 61%. Experience
with other FNB needles, such as the 22G Acquire needle (Boston Scientific Natick,
Massachusetts, United States) confirms similar accuracy (87%), and thus, the superiority
of the FNB needle [15 ].
Because the establishing organoids is a challenging and not-yet-standardized technique,
there are very little data regarding optimal endoscopic tissue acquisition for it.
To the best of our knowledge, this is the first prospective, randomized study comparing
EUS-FNA and EUS-FNB with respect to establish pancreatic cancer organoids. Tiriac
et al. [11 ] were able to show for the first time that establishing organoids through EUS-guided
tissue acquisition is generally possible. However, the organoids in their work came
exclusively from FNB after malignancy was proven by EUS-FNA using rapid on-site evaluation
(ROSE). Only patients from whom adequate cellular material was obtained were included
in their study. In the current study, we successfully created pancreatic cancer organoids
by means of EUS-FNA and EUS-FNB in patients at the time they were first diagnosed
with their tumor. Our results suggest that EUS-FNB is superior to EUS-FNA regarding
PDO generation and histologic specimen sampling of pancreatic tumors.
One limitation of our study is the relatively low rate of established organoids (34%).
Overall, culture and media conditions for PDOs are continuously improving; however,
to generate comparable results within our cohort, the original protocol was followed.
The studies by other groups with higher rates of established organoids ranging between
60% and 76% incorporated a step of ROSE to select for higher-quality biopsy material,
potentially influencing subsequent organoid culture [11 ]
[22 ]
[23 ]. For general diagnosis of pancreatic cancer and prerequisite for chemotherapy treatment,
histology is still the current diagnostic gold standard. Because FNB is already established
as having the best diagnostic performance, future research should be performed to
identify the FNB needle that offers the best histological as well as molecular analysis
capabilities. However, PDOs as functional biomarkers may complement diagnostics in
clinical practice in the future.
Conclusions
In conclusion, our study shows that EUS-FNB is superior to EUS-FNA in terms of successful
generation of PDOs as well as in diagnostic performance, although statistical significance
was not observed. EUS-FNB has already become the standard for histological assessment
and it also may be advantageous for PDO acquisition.
