Objective: Endoscopic Ultrasound (EUS) guided Fine Needle Aspiration (FNA) biopsy of the pancreas is a standard practice for diagnosis and staging of pancreatic malignancy. The material obtained by EUS guided FNA forms the basis of therapeutic decisions.

Methods: 111 cases of EUS guided FNA biopsy of the pancreas performed during the year January 2008 to December 2009 having solid pancreatic mass/lesion on the USG/CT and suspicious for the malignancy or malignant on clinical and radiological investigations were reviewed in August 2011 at the Department of Pathology and Laboratory Medicine, Hospital of University of Pennsylvania, Philadelphia U.S.A

Results: There was an 83.9% correlation between Diff-Quick diagnosis and the final cytological diagnosis. The overall diagnostic accuracy for the malignancy was 89.7%, sensitivity 90.6%, and specificity 100% of the cases where the cytological diagnosis was correlated with histological diagnosis and the other investigations. The positive predictive value for the malignancy was 100%. The false negative diagnosis was encountered in 10.3% cases. 51% of the cases showed intranuclear vacuoles, in the malignant cases on Diff-Quik Stain.

Conclusions: EUS FNA of the pancreas is a safe and reliable technique with high diagnostic accuracy, sensitivity, and specificity. The Diff-Quik stain smear is a useful technique for the rapid on-site cytological evaluation for the detection of malignancy of the pancreas. Thin Prep was found superior to the PAP / Diff-Quick stain for the diagnosis of the malignancy. The EUS FNA samples processed by multiple staining techniques help to improve the diagnostic accuracy and sensitivity.

Lad LKV, Jhala NC, Samsudin AT (2016) Endoscopic Ultrasound (EUS) Guided Fine Needle Aspiration (FNA) Biopsy of Solid Pancreatic Lesions: A Review of 111 Cases and Comparative Study of Diff-Quik/PAP/Thin Prep Staining Techniques. Ann Clin Cytol Pathol 2(1): 1014.

•    EUS guided FNA
•    Pancreatic malignancy
•    Diff-Quik
•    Thin Prep
•    PAP

EUS: Endoscopic Ultrasound; FNA: Fine Needle Aspiration; PAP: Papanicolau

Pancreatic cancer is the fourth leading cause of cancerassociated deaths in the United States and ranks ninth in its incidence [1]. EUS Guided FNA cytology has emerged as an important diagnostic tool since Vilman and Grimm (1992) made the first reports of endoscopic guided fine needle aspiration. In the last two decades, EUS-guided FNA has become popular in the clinical field, especially Western Countries [2]. Currently, EUS- guided FNA biopsy of the pancreas is a standard practice for diagnosis and staging of pancreatic malignancy [3]. Various published series on EUS guided FNA of Pancreas report diagnostic accuracy between 79-97%, sensitivity 77-95% and specificity 96-100%. False negative diagnosis range from 3.5-15% and false positive diagnosis range from 0-1% [4-13].

The present study was conducted for a short term research project of the Union International for Cancer control. The present study was carried out with the following objectives.

1. To find out the diagnostic accuracy, sensitivity, and specificity of EUS- guided FNA of the pancreatic malignancy by correlating cytological diagnosis with histological diagnosis and other investigations.

2. To know the diagnostic accuracy of air-dried smears using Diff-Quik Stain.

3. To compare the diagnosis obtained by Diff-Quik, conventional PAP Stain smears/ Cytospin Preparation and Thin Prep (Liquid Based Media).

Inclusion criteria: The cases with solid or solid cystic lesions suspicious or malignant on clinical and radiological investigations were selected for this study.

Exclusion criteria: Cystic lesions not suspicious or malignant on clinical and radiological investigations or pancreatitis were excluded from the study.

111 cases of EUS guided FNA of Pancreas performed for the year January 2008 to December 2009 having a solid mass/lesion on Ultrasound/CT and suspicious for malignancy or malignant on clinical and radiological investigations were selected for the review in this study. The material for cytological examination was obtained from patients under conscious sedation by EUS guided FNA using 22 gauge needles by an experienced endosonographer in the endoscopic suite. An average 2 to 5 passes were used to obtain material for the cytological examination. The material obtained was processed for the Diff-Quik, PAP, Cytospin, Thin Prep and Cellblock as per institutional standard operating procedures. Direct smears were prepared on the glass slides for the Diff-Quik and the Pap staining from the samples obtained by EUS guided FNA. Remaining sample was poured into a normal saline container for Cytospin (49 cases) and Liquid-based media container for Thin Prep preparation. Diff-Quik stain slides were used for rapid on-site evaluation. PAP staining technique was used for the Cytospin preparation. The core biopsy and pancreatectomy specimen for histopathological examination were stained with Hematoxyline and Eosin stain. There were no major complications encountered. The cytology smears were evaluated by two pathologists independently with one having experience in EUS FNA for about two decades. The Cytological diagnosis was correlated with histological diagnosis and other investigations (Lymph node Aspirate, Liver Aspirate and Ascitic Fluid Cytology) to obtain the diagnostic accuracy, sensitivity, and specificity of the cytological diagnosis. EUS FNA samples were reported as

1. Unsatisfactory

2. Benign/Reactive

3. A typical

4. Suspicious

5. Malignant

Adequate cellularity was obtained in 111 cases for definitive final cytological diagnosis using a combination of multiple staining techniques Diff-Quik, PAP/Cytospin and Thin Prep. There were two unsatisfactory smears on the Diff-Quik stain and seven unsatisfactory smears on Pap/Cytospin and one on Thin Prep preparation. However, use of multiple staining techniques avoided unsatisfactory results on cytology.

Site within Pancreas

Head of the Pancreas was the commonest site affected, followed by the tail (Figure 1).

Sex Distribution

Males were more commonly affected than the females. Male: Female ratio was 1.13:1 (Figure 2)

Age Distribution

Pancreatic carcinoma was common in older age groups. Maximum age incidence was seen in the 7th decade (Figure 3) (Table 1).

Malignancy was the commonest diagnosis encountered followed by the suspicious for malignant and benign/reactive diagnosis. The diagnosis of malignancy was obtained in the 76.8% cases on Diff-Quik stain smears, 70.6% cases in the Thin Prep smears and 56.4% cases in the Pap/Cytospin stain smears (Figure 4,5) (Table 2).

The commonest diagnosis was adenocarcinoma in 36 (76.6%) out of 47 cases. Additional material was obtained from cell block and processed for immune histochemistry markers Thyroid Transcription Factor 1 to confirm metastases from the lung carcinoma (Table 3).

The final cytological diagnosis was malignancy in 83 cases (74.8%) followed by reactive/benign in 16 cases (14.4%) (Table 4).

There was 83.92% (47 out of 56 cases) correlation between the diagnosis of Diff-Quik and the final cytological diagnosis. If we include suspicious cases and malignant cases as malignant the correlation between the diagnoses improved further to 92.85% (52 out of 56 cases). The false negative diagnosis was encountered in 3.5% (2 out of 56 cases). 5 out of 6 cases in the suspicious group on the Diff-Quik stain smears were reported as malignant on the Pap /Thin Prep stain smears. This shows the superiority of the Pap/Cytospin and the Thin Prep smear staining over the Diff-Quik Stain (Table 5).

There was a 100 % correlation between the malignancy if malignant and suspicious of the malignancy cases were considered as malignant. In 3 out of 17 cases of the benign group, 2 out of 7 cases in A typical group and 4 out of 17 in a suspicious group on Pap/Cytospin smears were confidently reported as malignant on Thin prep smears. This shows the superiority of Thin Prep over Pap/Cytospin stain (Table 6).

In 39 patients where Cytological diagnosis was correlated with histopathology and other diagnostic investigations (Ascitic Fluid Cytology, Lymph node, and Liver FNA), the diagnostic accuracy of cytology were 89.75%, sensitivity 90.69%, and specificity 100% if suspicious for the malignancy were included as malignant. In the malignant group, there was a 100 % correlation and all the suspicious cases on cytology were found to be malignant on histopathology or other investigations. In the benign group on cytology, there were 4 (10.3%) false negative diagnosis and no false positive diagnosis was encountered.

Table 1: Distribution of cases on Diff-Quik, Pap/Cytospin and Thin Prep.

Table 2: Distribution of cases on cellblock.

Table 3: Final cytological diagnosis.

Table 4: Comparison of Diff-Quik diagnosis with Pap and Thin Prep.

Table 5: Comparison of Pap/Cytospin diagnosis and Thin Prep.

Table 6: Correlation of cytological diagnosis with histopathology and other investigations.

Table 7: EUS FNA reported in the literature and present series.

Diff-Quik is a useful stain for preliminary on-site diagnosis as we reported malignancy in 43 (76.8%) out of 56 cases. The presence of the cytopathologist in endoscopic suit can avoid unsatisfactory aspirates by rapid on-site evaluation, obtain a preliminary diagnosis and collect additional samples for immunohistochemistry wherever needed [3]. The Cellblock was useful for confirming the cytological diagnosis and also confirmed metastases from the lung by additional immunohistochemical staining for the Thyroid Transcription Factor 1. In the present series final cytological diagnosis of malignancy was obtained in the 74.8% cases. Williams et al (1999) [4] reported malignancy by EUS-FNA in 62% of patients with clinically suspicious lesions, Shah SM, et al (2008) [10]: reported malignancy by EUS- FNA in 87.8% of patients with a pancreatic mass.

We found that there is an 83.92% correlation between the diagnosis of Diff-Quik and the final cytological diagnosis. If we include suspicious cases and the malignant cases as malignant the correlation between the Diff-Quik and final cytological diagnosis further improved to 92.85%. We found intranuclear vacuoles in 51% cases (22 out of 43 cases) on Diff-Quik stain, but were rare on Pap stain (3 cases only). False negative diagnosis of 3.5% was encountered when we compared Diff-Quik and PAP/Thin Prep diagnosis. False negative on Diff-Quik was due to sampling error.

When we compared PAP/Cytospin with the Thin Prep Smear, there was a 100 % correlation between the malignancy if malignant and suspicious for malignancy were considered as malignant. 3 benign, 2 atypical and 4 suspicious cases on Pap/ Cytospin smears, were confidently reported as malignant on Thin prep smears. Thin prep, therefore, improves diagnostic accuracy, sensitivity, and specificity due to better morphological features, improved cellularity, and clean background. This shows the superiority of Thin Prep preparation over Pap/Cytospin smears for the diagnosis of the malignancy. However, Lebalanc JK [11] et al in their prospective study found smear method more sensitive and accurate than Thin Prep in detecting malignancy from EUS FNA samples of the Pancreas. They reported sensitivity on Smear 98% and Thin Prep 62% and the diagnostic accuracy of the smear 98% and Thin Prep 64% (Table 7).

In 39 patients where the cytological diagnosis was correlated with the histological diagnosis and other investigations, the diagnostic accuracy of cytology was 89.7%, sensitivity 90.69%, specificity 100% and positive predictive value (PPV) 100% negative predictive value (NPV) 25% if suspicious for the malignancy is included as malignant. Our findings were compared with other reported series in the literature. The false negative diagnosis was encountered in 10.3% cases in the present series while it ranges between 3.5-15% in other reported series. The false negative diagnosis was encountered due to sampling error. No false positive diagnosis was encountered in the present series, which was comparable with other reported series.

A close interaction between endoscopist and the cytopathologist is essential to improve the diagnostic yield. The unsatisfactory sample is avoided if more than one pass is used for the cytological diagnosis and the material is obtained for the multiple staining techniques viz. Diff-Quik, Pap/Cytospin/Thin Prep and Cell Block [12,13]. The final diagnosis is always based on the clinical, EUS and the cytology features. Repeat EUS FNA is recommended in the cases where the diagnosis is suspicious for the malignancy or confirmed by the core biopsy.

EUS FNA of the pancreas is a safe and reliable technique with high diagnostic accuracy, sensitivity, and specificity. The Diff-Quik stain smear is a useful technique for the rapid on-site cytological evaluation for the detection of malignancy of the pancreas. We have reported intranuclear vacuoles as additional morphological criteria on the air-dried Diff-Quik smears for the diagnosis of malignancy which needs to be confirmed by the larger studies. Thin Prep was superior to the PAP/Diff-Quick stain and improved the diagnosis of the malignancy and also reduced the incidence of suspicious diagnosis in the malignant cases. We, therefore, recommend samples obtained by EUS FNA to be processed by multiple staining techniques to improve the diagnostic accuracy and sensitivity.

The above research project was supported by grants from Union for International for Cancer Control (UICC), Geneva, Switzerland for International Technology Transfer Fellowship (ICRETT).

Our sincere thanks to Dr. Prabodh Gupta, head department of Cytopathology, all faculty members, fellows, residents, Cytotechnologist and supporting staff, Department of Pathology, hospital of the university of Pennsylvania, Philadelphia, USA.

The research project was sponsored by Union for International Cancer Control (UICC) for International Cancer Technology Transfer Fellowship. The grants covered travel and living costs only.

1. Jhala DN, Jhala NC. Endoscopic Ultrasound-Guided Fine Needle Aspiration of the Pancreas. Adv Exp Med Biol. 2005; 563: 93-103.

2. KidaM, Itoi T. current status and future perspective of interventional endoscopic ultrasound in Japan. Diag Endosc. 2009; 21: 50-52.

3. Jhala NC, Jhala DN, Chhiieng DC, Eloubeidi MA, Eltaum IA: Endoscopic ultrasound -guided fine needle aspiration: A cytologist perspective AM J Clin Pathology. 2003; 120: 351-367.

4. Williams DB, Sahai AV, Aabakken L, Penman ID, Velse A Van, Webb J, et al. Endoscopic ultrasound-guided fine needle aspiration biopsy: a large single center experience. Gut. 1999; 44: 720-726.

5. Chang KJ, Katz KD, Durbin TE. Endoscopic ultrasound-guided fine needle aspiration. Gastrointest Endosc. 1994; 40: 694-699.

6. Giovannini M, Seitz JF, Monges G, Perrier H, Rabbia I. Fine needle aspiration cytology guided by endoscopic ultrasonography in 141 patients. Endoscopy. 1995; 27: 171-177.

7. Wiersema MJ, Vilman P, Giovannini M, Chang KJ, Wiersema LM. Endosonography-guided fine needle aspiration biopsy: diagnostic accuracy and complication assessment. Gastroenterology. 1997; 112: 1087-1095.

8. Bentz JS, Kochman ML, Faigel DO, Ginsberg GG, Smith DB, Gupta PK. Endoscopic ultrasound-guided real-time, fine needle aspiration: clinicopathologic features of 60 patients. Diagn Cytopathol. 1998; 18: 98-109.

9. Shin HJ, Lahoti S, Sneige N. Endoscopic ultrasound –guided fine needle aspiration in 179 cases. Cancer. 2002; 96: 174-180.

10. Shah SM, Riebero A, Levi J, Jorda M, Lima CR, Sleeman D, et al. EUS –Guided Fine Needle Aspiration with and without Trucut Biopsy of Pancreatic Masses. JOP. 2008; 9: 422-430.

11. Leblanc JK, Emerson RE, Dewitt J, Symms M, Cramer HM, Mc Henry L, et al. A prospective study comparing rapid assessment of smears and Thin Prep® for endoscopic ultrasound - guided fine needle aspirates. Endoscopy. 2010; 42: 389-394.

12. Gress FG, Savides TJ, Sandler A, Kesler K, Conces D, Cummings O, et al. Endoscopic ultrasonography, fine needle aspiration biopsy guided by endoscopic ultrasonography, and computed tomography in the preoperative staging of non-small-cell lung cancer: a comparison study. Ann Intern Med. 1997: 127: 604-612.

13. Chhieng DC, Jhala D, Jhala N, Eltoum I, Chen VK, Vickers S, et al. Endoscopic ultrasound –guided fine needle aspiration biopsy: a study of 103 cases. Cancer. 2002; 96: 232-239.