Pancreatic cancer is a lethal and challenging disease worldwide.¹ Fewer than 20% of the patients were diagnosed early while still resectable and curable, resulting in most of the patients with grave prognosis. Neoadjuvant therapy became a standard of care with improved overall survival and R0 resection rate in patients with borderline resectable disease.^2,3 In resectable disease, neoadjuvant therapy is also a choice of treatment with debated benefit in overall survival.⁴ Besides, gene profiling is suggested in patients with locally advanced or metastatic disease for the purpose of individualized therapy.^3,5 Therefore, obtaining naïve tissue prior to treatment for analyses such as next-generation sequencing can help understand genetic mutations of the tumor and tailor drugs to individual needs.

Endoscopic ultrasonography (EUS) tissue acquisition has evolved to be an important diagnostic tool in pancreato-biliary malignancy since the first EUS-guided fine needle aspiration performed in 1992 by Vilmann et al.⁶ EUS-guided tissue acquisition became the standard of care today as it reduces risk of needle tract seeding, decreased complication rate, and its comparable diagnostic performance.^7,8 However, needle preparation and biopsy technique still remain controversial.⁹ Wet-suction method provides higher vacuum pressure,¹⁰ resulting in higher cellularity and tissue adequacy.¹¹ However, higher vacuum pressure results in tissue injury with blood contamination that can be problematic and interfere with pathological diagnosis.^12,13 Heparin is a thrombin inhibitor that is clinically used to prevent blood from clotting. A novel heparinized wet-suction method was developed in EUS-guided liver biopsy, which showed better tissue acquisition in comparison with the dry-suction method in terms of tissue length and number of portal triad.¹⁴ However, the efficacy of this technique in pancreatic mass biopsy is yet to be determined.

Precision medicine is an emerging concept in treating cancer patients with individualized therapy tailored to each patients’ specific needs that may offer a chance to improve outcome. Individualized therapy depends tremendously on genetic sequencing of the tumor.¹⁵ The quality of acquired tissue and the amount of extracted DNA are all independent factors for successful genomic sequencing.¹⁶ Preexisting data suggests the utilization of a larger needle gauge in EUS-guided tissue acquisition for pancreatic tumor, resulting in a higher successful rate of genome sequencing.^17,18 These data suggest that the acquired tumor volume is important for successful next-generation sequencing. However, whether the acquired white tissue length measured by macroscopic on-site evaluation (MOSE) can be correlated with the amount of extracted DNA has yet to be determined.

The objective of this study was to evaluate if tissue acquired via heparinized wet-suction method will yield a longer white tissue core compared to that with dry-suction method, and to analyze the correlation between white tissue length and the amount of DNA extracted.

1. Patients enrollment and study design

This was a prospective, randomized, crossover clinical trial conducted at a tertiary medical center in Taiwan. The study was approved by the Investigational Review Board of National Cheng Kung University Hospital (IRB number: B-BR-108-070). Informed consent was obtained from all patients before the procedure. This trial was also registered on clinicaltrials.gov (NCT04707560).

Patients with a solid pancreatic mass who had not undergone tissue-proof biopsy were enrolled into the trial. Subjects were excluded if they met one or more of the following: (1) age <20 years, (2) pregnancy, (3) inability to give informed consent, (4) coagulopathy (platelet <50,000, prothrombin time prolonged for more than 3 seconds), and (5) the active use of anticoagulant or antiplatelet agents without adequate cessation.

2. Randomization

Patients were randomly distributed into either dry-suction first group (group A) or heparinized wet-suction first group (group B) by using block randomization method. A block size of four was chosen and six possible balanced combinations within block were got. The sequence of block was determined randomly by throwing the dice. After randomization, each patient first received two needle passes of their designated group, then had two needle passes of the other method, undergoing a total of four needle passes (Fig. 1).

Figure 1. Flow diagram of the study participants.

3. EUS tissue acquisition

All the procedures were performed by single experienced endosonographer with more than 250 EUS tissue acquisition experiences (M.Y.L.). All patients received EUS tissue acquisition by linear echoendoscope (GF-UCT260; Olympus, Tokyo, Japan) adapted to either EU-ME1 or EU-ME2 system and a 20-gauge EchoTip ProCore needle (Cook Endoscopy, Inc., Bloomington, IN, USA). Depending on the location of the tumor, the needle was punctured into the pancreatic mass either via the stomach or the duodenum after checking for the absence of surrounding vessels. Fanning technique with 16 to 20 times of door knocking strokes under direct endosonographic visualization of needle tip was done with each needle pass. Patients were kept in hospital for one day after the EUS procedure to monitor for acute adverse events and came back 7 and 30 days after discharging to report any delayed adverse events.

4. Heparinized wet-suction method

The heparinized wet-suction method was adapted from the previous modified wet-suction method suggested for liver biopsy.¹⁹ We removed the stylet and flushed 1 mL heparin (5,000 U/mL) into needle until droplets spurted out from needle tip before starting the procedure. A 10-mL negative pressure syringe was attached after puncturing the target mass. After the acquired tissue was ejected, heparin was flushed through the same biopsy needle before the second pass.

5. Dry-suction method

The stylet was also removed before puncture and a 10-mL negative pressure syringe was connected after needle punctured the target mass. If heparinized wet-suction was done prior to dry-suction method, the needle chamber was irrigated with 50 mL of normal saline and then forcefully flushed with air until no droplets pass through the needle tip.

6. Specimen processing and histological interpretation

After tissue acquisition, we gently ejected tissue from needle tip onto a sterile plate with 10 mL saline, 10 mL air, and the stylet in an orderly fashion. A standardized MOSE was then performed which included separating white tissue core from pasta-like red material. The white tissue core was then straightened and lined-up to measure the total length with a ruler.²⁰ After MOSE, all tissue was picked up and fixated into a formalin bottle which was marked according to pass sequence. The formalin-fixed tissue specimen was processed according to pathology routine and embedded in paraffin. The paraffin-embedded tissues were cut into 3 μm slices. Only sections containing the most tissue were processed into slides. The tissue sections were stained with hematoxylin and eosin for evaluation. Immunohistochemical procedures were performed only if necessary. One gastrointestinal pathologist received no information regarding each patient’s clinical condition and procedure detail was assigned for histological assessment. The pathologist would record the score of blood contamination and the final diagnosis. Blood contamination was scored by a 1 to 4 scale, defined as 1: minimal (<25%), 2: mild (25%–50%), 3: moderate (50%–75%) and 4: high (>75%) blood occupied area on that slide microscopically.²¹ The histological diagnosis was routinely confirmed by another pathologist in our hospital.

7. DNA extraction

Subsequent patients with the same inclusion and exclusion criteria were further enrolled into another trial that was approved by the National Cheng Kung University Hospital Institution’s Investigational Review Board (A-ER-109-073). These patients underwent EUS tissue acquisition with heparinized wet-suction method by the same endosonographer. For each patient, small fraction of white tissue separated by using MOSE method was embedded in optimal cutting temperature compound without formalin fixation and stored in –80°C freezers. Genomic DNA was extracted from those frozen tissues by using a DNA isolation kit (Favorgen, Ping-Tung, Taiwan) with standard protocol provided by the user’s operation manual. After mixing and vortexing the white tissue with cell lysis buffer and proteinase K., protein was lysed, and DNA was freed and isolated. After centrifugation, the extracted DNA was then rinsed with ethanol. Finally, the pellet was then air-dried and re-suspended in water and processed for DNA concentration by NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer. The used tissue length for DNA extraction and total DNA amount in nanogram were recorded.

8. Outcomes

The primary outcome was the total length of aggregate white tissue assessed by MOSE. The degree of blood contamination on histological evaluation and the diagnostic performance including sensitivity and accuracy of each pass and suction method were listed as secondary outcome. The performance of each suction method was defined as either first or second pass yielding diagnosis. An additional outcome validating the correlation between white tissue length and extracted DNA amount was analyzed in another subset of patients.

9. Statistical analysis

All statistical analyses were performed by the SPSS software (version 20.0, IBM Corp., Armonk, NY, USA). Continuous variables were presented as the mean and standard deviation or the median and interquartile range as appropriate. Categorical variables were presented as frequency and percentage. While comparing the difference between heparinized wet-suction and dry-suction from the same patients, The Wilcoxon signed-rank and McNemar tests were applied for paired data set. The Spearman rank correlation was applied to analyze the relationship between white tissue length and extracted DNA amount. p-value less than 0.05 by a two-tailed test was recognized as significant.

10. Sample size calculation

Based on previous studies, a median of 8 mm white core tissue was anticipated by using dry-suction in EUS tissue acquisition.²⁰ Heparinized wet-suction may increase white core tissue length by at least 3 mm compared to that of the dry-suction in our preliminary data. The standard deviation of paired difference in our preliminary data was 7 mm. Based on this condition with a two side α-value of 0.05 and power of 80% (β=0.8), the total number required were 45 with an estimated dropout rate was 10%. Thus, a total of 50 patients were needed.

From January 2020 to November 2020, a total of 50 patients with a solid pancreatic mass were consecutively enrolled and randomly allocated to receive either heparinized wet-suction first or dry-suction first EUS fine needle biopsy. All patients received EUS tissue acquisition successfully and were followed completely (Fig. 1). The baseline characteristics, tumor features (tumor location, tumor size, and stage) and indications for EUS tissue acquisition were similar between groups (Table 1).

Table 1 . Baseline Characteristics of the Enrolled Patients.

Characteristics	Heparin wet-suction first (n=25)	Dry-suction first (n=25)	p-value
Age, mean±SD, yr	66.40±9.22	63.48±9.86	0.285
Sex			0.239
Female	7	11
Male	18	14
Carbohydrate antigen 19-9, mean±SD, U/mL	1,112±1,949	981±1,250	0.784
Tumor location			0.777
Head or uncinate process	12	13
Body or tail	13	12
Stage (I/II/III/IV)	0/2/10/11*	2/1/10/11*	0.615
Tumor size, mean±SD, mm	38.17±9.73	38.56±15.11	0.914
Final diagnosis			0.502
Pancreatic adenocarcinoma	22	23
Pancreatic neuroendocrine tumor	1
Chronic pancreatitis	2	1
Other diagnosis		1†
Adverse events	1		1.000

*Three patients were diagnosed as chronic pancreatitis and unable to be staged; ^†Lung small cell carcinoma with pancreatic metastasis.

1. Outcomes

All data were reported on a needle treatment basis and a combination of the same suction method. Table 2 shows aggregate white tissue length was significantly longer in the specimens acquired by heparinized wet-suction than that by dry-suction (mean, 11.07 mm vs 7.96 mm, p=0.001; median, 10.5 mm vs 7.5 mm, p<0.001). The degree of blood contamination on histology slides was less in the heparinized wet-suction specimens compared with the dry-suction ones (p=0.008). There were 21 specimens (21%) ranked as “high blood contamination” in the heparinized wet-suction method compared with 37 specimens (37%) in the dry-suction method (p<0.001) (Table 2). Moreover, the score of blood contamination significantly increased in the 2nd pass of dry-suction specimens (p=0.008), but the phenomena was reversed in the heparinized wet-suction specimens (p=0.035).

Table 2 . Tissue Quality Comparison between Different Suction Methods.

Overall pass order		White tissue length, mm					Score of blood contamination
		Mean±SD	p-value	Median	p-value		1	2	3	4	p-value
1st pass	HW (n=25)	13.72±8.20	0.007	15.00	0.006		9	2	8	6	0.243
	DS (n=25)	7.96±6.25		8.00			3	4	10	8
2nd pass	HW (n=25)	11.68±6.60	0.037	10.00	0.029		8	5	9	3	0.021
	DS (n=25)	7.16±8.18		5.00			3	4	5	13
3rd pass	HW (n=25)	9.68±7.76	0.707	8.00	0.830		8	4	5	8	0.372
	DS (n=25)	8.88±7.18		8.00			12	2	7	4
4th pass	HW (n=25)	10.40±9.07	0.477	10.00	0.444		17	0	4	4	<0.001
	DS (n=25)	8.64±8.26		6.00			3	4	6	12
Sum up	HW (n=100)	11.07±8.09	0.001	10.50	<0.001		42	11	26	21	0.008
	DS (n=100)	7.96±7.37		7.50			21	14	28	37

HW, heparin wet suction; DS, dry suction..

As shown in Table 3, the diagnostic sensitivity and accuracy were similar between the two suction method groups with two needle passes. Interestingly, there was a trend of decreasing diagnostic sensitivity and accuracy in the 2nd pass of dry-suction method (p=0.115 and p=0.119, respectively). These data suggested that the increased blood contamination in the 2nd pass of dry-suction method may hamper the performance of histological diagnosis.

Table 3 . Diagnostic Performance Comparison.

Overall pass order		Diagnostic sensitivity			Diagnostic accuracy
		% (n/n)	p-value		% (n/n)	p-value
1st pass	HW (n=25)	91.3 (21/23)	0.666		92.0 (23/25)	0.667
	DS (n=25)	83.3 (20/24)			84.0 (21/25)
2nd pass	HW (n=25)	91.3 (21/23)	0.017		92.0 (23/25)	0.018
	DS (n=25)	58.3 (14/24)			60.0 (15/25)
3rd pass	HW (n=25)	75.0 (18/24)	0.245		76.0 (19/25)	0.247
	DS (n=25)	91.3 (21/23)			92.0 (23/25)
4th pass	HW (n=25)	70.8 (17/24)	0.559		72.0 (18/25)	0.508
	DS (n=25)	78.3 (18/23)			80.0 (20/25)
Sum up	HW (n=50)	93.6 (44/47)	1.000		94.0 (47/50)	1.000
	DS (n=50)	93.6 (44/47)			94.0 (47/50)

HW, heparin wet suction; DS, dry suction..

2. Comparison of outcomes in the different subgroups

The patients were divided into several subgroups based on the factors that may impact study outcome. As shown in Table 4, heparin wet-suction obtained a significantly longer white tissue than dry-suction in the subgroups of trans-gastric needle route, tumor >2 cm, and unresectable disease; while a similar trend of increasing length was observed in the subgroups of trans-duodenal needle route, tumor ≤2 cm, and resectable disease, although not statistically significant due to small case number. Similarly, in the subgroups of tumor size >2 cm and unresectable disease, the heparin wet-suction group showed significantly less blood contamination than the dry-suction group; while a similar trend was observed in the subgroups of trans-gastric or trans-duodenal needle route, tumor <2 cm, and resectable disease.

Table 4 . Outcome Comparison between Suction Methods Stratified by Different Subgroups.

Subgroups	Heparin wet-suction	Dry-suction	p-value
White tissue length, mean±SD, mm
Needle route
Trans-gastric (n=25)	13.88±7.45	9.22±7.22	0.002
Trans-duodenum (n=25)	8.86±7.78	7.10±7.55	0.254
Tumor size
<2 cm (n=2)	12.00±9.42	3.75±3.50	0.179
>2 cm (n=48)	12.34±7.98	8.34±7.50	0.008
Resectability
Resectable (n=15)	8.40±6.62	6.47±6.79	0.269
Unresectable (n=35)	12.64±8.22	8.89±7.61	0.006
Blood contamination
Needle route
Trans-gastric (n=25)*	27/4/9/10†	14/8/14/14†	0.066
Trans-duodenum (n=25)*	15/7/17/11†	7/6/14/23†	0.057
Tumor size
<2 cm (n=2)*	1/0/3/0†	1/1/2/0†	0.549
>2 cm (n=48)*	41/11/23/21†	20/13/26/37†	0.007
Resectability
Resectable (n=15)*	8/4/10/8†	5/3/7/15†	0.321
Unresectable (n=35)*	34/7/16/13†	16/11/21/22†	0.016

*The number in the bracket is patient amount, two passes were done in each method; †The number listed here is the specimen amount with each degree of blood contamination (<25%/25%-50%/50%-75%/>75%)..

3. Comparison of outcomes based on overall pass order

Considering the needle tip decay during puncture, we compared the outcome based on overall pass order as illustrated in Fig. 2. The white tissue length was significantly longer with heparin wet-suction in overall passes 1 and 2 while a similar trend of increasing length was observed in passes 3 and 4. Less blood contamination was noted by heparin wet-suction in overall passes 2 and 4 (corresponding to the second pass of each suction method) but showed no significant difference in overall passes 1 and 3 (corresponding to the first pass of each suction method). The per needle pass diagnostic sensitivity and accuracy was similar no matter which suction method used except for overall pass 2 which a better diagnostic sensitivity and accuracy was observed in the heparin wet-suction method.

Figure 2. Outcome comparison based on overall pass order. (A) The white tissue length was longer by applying heparin wet-suction especially in overall passes 1 and 2. (B) The blood contamination was significantly lesser in overall passes 2 and 4 if using heparin wet-suction. (C, D) Diagnostic sensitivity and accuracy were similar no matter which suction method used, except in overall pass 2. *p<0.05.

4. The relationship between white tissue length and DNA amount

Another 40 patients with the same inclusion/exclusion criteria receiving EUS tissue acquisition with the heparinized wet-suction were consecutively enrolled from November 2020 to February 2021. Among them, three cases were excluded due to insufficient white tissue to spare for undergoing DNA extraction. Fig. 3 shows the genomic DNA amount positively correlates to the white tissue length (p=0.001) with a moderate correlation coefficient (Spearman's ^ρ=0.568).

Figure 3. The scatter diagram and the trend line of white tissue length and extracted DNA amount. Each dot stood for a single specimen enrolled and the ρ value was done by Spearman rank correlation which showed a significantly positive correlation between white tissue length and DNA amount (ρ=0.568).

5. Adverse events

One patient reported tarry stool passage the day after the procedure. The esophagogastroduodenoscopy revealed an adherent clot at the puncture site and was successfully managed by diluted epinephrine injection. This patient was discharged smoothly after 2 days of proton pump inhibitor infusion. No other adverse event was further reported.

Heparinized wet-suction was proved to be safe and efficient for the diagnosis of pancreatic solid tumors. This study also demonstrated that heparinized wet-suction facilitates obtaining longer white tissue core and decreases blood contamination. To date, this is the first prospective randomized study in evaluating the impact of heparinized wet-suction method for EUS tissue acquisition of solid pancreatic mass. We are also the first to demonstrate the positive correlation between white tissue length and the extracted DNA amount.

A recent study by Mok et al.¹⁴ demonstrated how utilizing heparinized wet-suction in EUS-guided liver biopsy outperformed its opponent in the acquisition of tissue length and numbers of complete portal triad. We again confirmed its benefit in solid pancreatic tumors. Concerning the anti-coagulating effects of heparin, increased adverse events or tissue blood contamination were expected but had been proven to be safe and equal in the amount of blood contaminants in samples.^14,22,23 In fact, due to the capillary phenomenon, heparin was retained inside the needle chamber and did not seep into the target tissue. This study further proved that heparinized wet-suction could decrease blood contamination. High degree of blood contaminants in tissue samples has a negative effect on the performance of histological diagnosis.^24,25 In this study, the diagnostic accuracy and sensitivity both decreased from more than 90% to less than 50% if a sample was graded as high blood contaminants (p<0.001).

There is a vast array of different tissue acquisition methods out there, each with its own set of pros and cons. First, the wet-suction method was reported to be superior to dry-suction method for EUS-guided fine needle aspiration of solid lesion in terms of diagnostic accuracy, specimen adequacy, and blood contamination.²⁶ However, 87% of the wet-suction specimens in that study contained >50% of blood upon inspection. We aimed to decrease the rate of blood contamination by adding heparin in wet-suction. As a result, the numbers of moderate to high (>50%) blood contamination decreased to only 47% in our heparinized wet-suction group. Second, stylet slow-pull method is a novel way in EUS tissue acquisition. Theoretically, because of its use of “slow pull,” it creates less suction pressure yielding less blood contaminated tissue in some previous studies.^12,13,27 We did not choose stylet slow-pull as the control group, because dry-suction uses a fixed suction pressure which can reduce the variability of manual pulling. Future studies are warranted to determine whether heparinized wet-suction is superior to normal saline wet-suction or stylet slow pull method in EUS tissue acquisition.

Priming heparin in blood contaminated sample is commonly used and does not interfere with histological diagnosis.²² Administration of heparin inside the EUS needle chamber theoretically avoids blood clot formation. Thus, it may help maintain needle patency during tissue acquisition. We found that the saline used to flush out tissue sample became more reddish when using heparinized wet-suction method (Fig. 4A), indicating that blood clot was lysed and spontaneously separated from the acquired tumor tissue. This increases the efficacy of separating white tissue (tumor tissue) from the red tissue (red blood cell aggregation) (Fig. 4B).²⁰ In contrast, this phenomenon was not observed in the dry-suction specimen (Fig. 4C and 4D). In concordance with the increased white tissue length and decreased red tissue aggregation macroscopically, the microscopic blood contamination in the histological slides was significantly lower in the heparinized samples. For tumor DNA sequencing, it is important to acquire bigger, purer tumor tissue with less blood contamination.

Figure 4. Macroscopic view of acquired tissue and the result of on-site evaluation. More red blood cells (RBCs) dissociated from the specimen got by heparinized wet-suction method and precipitated on the plate (A) and more white tissue was separated from the acquired tissue (B). Only small number of RBCs dissociated from the specimen got by dry-suction method and precipitated on the plate (C), which resulted in shorter white tissue been separated (D).

We found the diagnostic performance including sensitivity and accuracy was similar between the two suction methods. Interestingly, heparinized wet-suction method showed higher diagnostic sensitivity/accuracy and less blood contamination than the dry-suction method in pass 2 (Table 3, Fig. 2). We suppose that the decreased blood contamination in pass 2 of heparinized wet-suction group may improve the diagnostic performance. However, as long as the white tissue core is longer than 4 mm, the diagnostic performance can be equally good.²⁰ In this study, comparably high percentage of cases (86% with dry suction and 96% with heparin suction, p=0.160) achieved more than 4 mm white tissue. This may explain why the overall diagnostic performance is comparable between the two suction methods.

A few limitations were noted in this study. First, we did not use a new needle when switching suction methods. Instead, we flushed the needle chamber thoroughly with a fixed, large amount of normal saline and checked for cleanliness by flushing the chamber again with air until nothing is flushed out. Previous studies have shown that heparin-primed dry-suction had the same performance compared to that with dry-suction alone. Thus, even if heparin is not completely cleaned, it should not affect the outcome.¹⁴ Second, without changing new needle in every needle pass, the needle tip deformity after previous puncture was concerned. Therefore, we proved that the superiority of heparin wet-suction in obtaining longer white tissue was observed mainly in overall passes 1 and 2. Third, heparin wet-suction showed a non-significant trend of better outcome in the subgroups of small tumor (<2 cm), trans-duodenal route, and resectable disease due to small case numbers. Future study with a larger case number is warranted to confirm the superiority of heparinized wet-suction in these subpopulations. Fourth, the correlation between tissue length and extracted DNA amount, was carried out in subsequent cohort. It is simply because separating tissue from original cohort may interfere with the diagnostic performance. An extra needle pass for collecting tissue in original cohort resulted in at least five needle passes in every patient which may increase complications. Furthermore, the subsequent cohort was collected with the same criteria and method by the same doctor. Therefore, reproducibility can be expected.

In conclusion, EUS-guided biopsy of pancreatic mass with heparinized wet-suction method can obtain longer white tissues with less blood contamination, while maintaining a comparable diagnostic performance. The longer the white tissue, the greater the amount of DNA extracted, which is important to personalized tailored medicine.

This study was funded by National Cheng Kung University Hospital (NCKUH-10904003 & NCKUH-10909044).

No potential conflict of interest relevant to this article was reported.

Study concept and design: M.Y.L., W.L.C. Data acquisition: M.Y.L., Y.Y.S., C.J.H. Data analysis and interpretation: M.Y.L., C.L.W., W.L.C. Statistical analysis: M.Y.L., W.L.C. Study supervision: W.L.C., B.S.S. Drafting of the manuscript: M.Y.L., W.L.C. Writing - review & editing: M.Y.L., Y.Y.S., W.L.C. Approval of final manuscript: all authors.