APPROACHES IN DIAGNOSIS

1. INDETERMINATE BILIARY STRICTURES- DIAGNOSTIC APPROACHES
• MODERATOR: PROF DR.ABHIJIT CHANDRA
• PRESENTER: DR. MAHESH. R

2. Definition
• No overt mass on noninvasive imaging
• Cannot be distinguished as malignant or benign after standard
diagnostic procedures.

3. • 15% to 24% - Benign diagnosis on pathology.
Clayton & Clarke et al Surgeon 1(1):32–38, 2003.
• Diagnosis of malignancies at early stage allow curative surgical resection or even
liver transplantation for early stage CCA.
• Challenge is obtaining a histological diagnosis because of cellular yield
Ustundag Y et al, World J Gastroenterol 14(42):6458–6466, 2008.

4. • The first major pathologic factor is tumor cellularity .
• Pancreatic carcinoma- Desmoplastic and fibrotic reaction, makes tumor very
dense and of low cellularity.
Logrono R et al,Arch Pathol Lab Med 124(3):387–392, 2000.
• CCA  Desmoplastic nature  Infiltrative pattern  early detection difficult.
• Host of benign causes radiographically mimic CCA.
Mizumoto R, et al,Hepatogastroenterology 40(1):69–77, 1993.

5. • Large specimens are often necessary to differentiate from normal tissue in very
well differentiated tumor.
• Explains why no biopsy technique, even open surgical wedge biopsy, has a 100%
yield.
Ohara H et al J Hepatobiliary Pancreat Sci 19(5):536–542, 2012.

6. • Maximizing yield requires repeated, deep, or large specimen sampling.
• Immune response or relative ischemia-obscure cell recovered
• Diagnosis of biliary stricture in the setting of a pancreatic mass may go
undetected on cross-sectional imaging, and thorough evaluation of the
pancreatic head in such strictures is a necessary part of the evaluation.

8. LABORATORY EVALUATION
• Direct hyperbilirubinemia more commonly with malignant obstruction
• Hyperbilirubinemia higher likelihood of malignancy than elevations in alkaline
phosphatase.
La Greca G et al,World J Gastroenterol 18(31):4150–4155, 2012.
• CEA sensitivity and specificity range from 33% to 84% and 50% to 87.8%.
• CA19-9 also has sensitivity and specificity: 38% to 93% and 67% to 98%.
• variable diagnostic accuracies of CA19-9, therefore, limit its role in screening, and
its greatest value may be in the surveillance of patients with PSC.
Tanaka H, et al, Ann Surg Oncol 15(2):583–589, 2008.

9. • IL-6, demonstrated sensitivity as high as 100% in diagnosing CCA.
Goydos JS et al,Ann Surg 227(3):398–404, 1998
• CCA-specific miRNA-31,31,32 or, alternatively, upregulation of some miRNA’s
such as miR-21.33
Lumachi F et al Anticancer Res 34(11):6663–6667, 2014.
• Elevated CYFRA 21-1 expression in CCA
Liu CZ, et al, Chin Med Sci J 27(2): 65–72, 2012.
• Sperm-specific protein 411 in bile of CCA patients and use in distinguishing CCA
from choledocholithiasis.
Shen J et al, PLoS ONE 7(10):e47476, 2012.

10. ERCP
• Goals are to first obtain definite tissue diagnosis
• Second, to provide palliation of biliary obstruction with stent placement.
• Limitations
1.Technical difficulty
2.Time consideration
3.Patient restlessness
4.Need to proceed with the goal of biliary drainage
Sugimoto S et al,Endosc Int Open 3:E323–E328, 2015.

11. • Cholangiographic features suggestive of a malignant stricture
1. Length > 14 mm,
2. Irregularity
3. Abrupt shelf-like borders
4. Presence of intraductal polypoid or nodular areas
5. Simultaneous CBD and PD dilation (i.e., double duct sign)
Bain VG et al,J Gastroenterol 2000; 14: 397-402
A “dominant stricture” is a subtype of IDBS that arises in the setting of underlying
primary sclerosing cholangitis (PSC) or other fibrosing cholangiopathies and may be
loosely defined as a CBD stenosis of ≤ 1.5mm or hepatic duct stenosis ≤ 1 mm in
diameter
Chapman R et al,Hepatology 2010

12. Brush Cytology Sampling Methods
• Initially, nonwire guided endoscopic brushes were inserted,after sphincterotomy;
• Currently use brushes which could be inserted over a guidewire.
• Protection of the acquired cells by withdrawing the brush back into the catheter
before leaving the area of the stricture
• simultaneous aspiration of bile immediately after brushing, thereby collecting
loose cells
• overall sensitivities using these devices range from 8% to 57%
Stewart CJ et al,J Clin Pathol 54(6):449–455, 2001.

15. • A 2013 review of ERCP brush cytology covered 16 studies over 10 years from
2002 to 2012, which included a total of 1586 patients.
• The combined yield of brush cytology ranged from 6% to 64% with an overall
sensitivity of only 41.6% +/− 3.2%, and did not appear to vary across new devices
and techniques. Burnett AS et al,10-y review of the literature, J Surg Res 184:304–311, 2013
• Identified 16 studies reported an overall biliary brush cytology sensitivity of 42%
with a negative predictive value (NPV) of 58%
Burnett AS et al,J Surg Res 2013; 184: 304-311

16. • A probable pathologic explanation for these varied yields relates to interiors of
malignant strictures are composed of benign epithelium compressed by
surrounding neoplastic tissue
• This fact explains the low yield of simple bile aspiration for cytology
• When the stricture is traumatized by dilation, removing the benign epithelium,
the yield of aspirating bile increases
• One would predict the lowest yield is in metastatic malignancy, followed by
pancreatic cancer, with a much higher yield with primary CCA.
• The type of brush bristles, the overall brush length, and the amount of time spent
brushing all affect yield.
Stewart CJ, et al,J Clin Pathol 54(6):449–455, 2001

17. • Rabinovitz et al (1990) used three separate brushes at each ERCP and repeated
the procedure with three new brushes when suspicious strictures were initially
negative.
• Positive yield continued to increase until diagnoses were eventually made by
brushing alone in 62% of their patients.
• Two additional ERCP brushing studies
1. Long cytology brush with stiff angulated bristles was compared with the
standard-length brushes. The true-positive yields were only 27% to 30%—and no
advantage was observed with the new brush.
Fogel EL et al,Gastrointest Endosc 63(1):71–77, 2006.

18. 2. Second study compared brushing with a more traumatic technique of inserting a
grasping basket through the suspicious stricture.
Of 50 malignant strictures, the basket technique had a near doubling of yield to
80% compared with a brush yield of 48% (p = 0.018).
The unexpected high yield of the brushing suggests some selection bias, and this
technique requires additional study
Dumonceau JM et al,Am J Gastroenterol 103(2):333–340, 2008.

19. ERCP Needle Biopsy
• Howell et al (1992) developed a ball-tipped catheter with a retractable 22-gauge
Chiba-type biopsy needle .
• The needle extends 7 mm beyond the ball tip when the catheter is placed within
the duct, and permits deeper sampling than afforded by brushing
• Traverses only tissue to be resected en bloc
• The technique requires sphincterotomy
• Technically challenging.
• The initial relatively high yield of 62% (positive and suspicious samples) has not
been reproduced.
Farrell RJ et al,Gastrointest Endosc 54(5):587–594, 2001.

20. Forceps Biopsy
• Initially, there was only one method known as fluoroscopically guided biopsies.
• Initially used gastroscopic forceps then flexible-tipped duodenoscopic forceps
over the elevator were developed.Large sphincterotomy required
• Cholangioscopy systems and accessories helped.
• Technique of forceps biopsy involves insertion of the device to the lower edge of
the stricture. Using fluoroscopy, an accurate biopsy specimen can be obtained
from the lower edge of the apparent tumor. Several passes of the forceps are
required to produce an optimal yield.
• Ponchon et al (1995) suggested a minimum of three forceps bites.

21. • The guidewire-based device currently in use,is the Howell biliary introducer
• The 10-Fr device goes over a 0.035-inch or smaller guidewire while permitting
the passage of a specially designed reusable 5-Fr long forceps.
• Multiple passes of the forceps and other sampling devices can be quickly
accomplished once the introducer is in position

22. • New scraping device was developed and trialed in Japan.
• In 123 indeterminate stricture cases, 119 were eventually proven to be
malignant.
• This device involves a three-leaf clover–type design of three nitinol loops, rather
than bristles, that are compressed in guide wire-compatible catheters.
• Stricture is roughly scraped and bile is aspirated using the device’s side angled
port.
• Yield was compared to a transpapillary forceps biopsy.
• The yields of forceps biopsy, the new scraping device, and both were 51%, 65%,
and 75%, respectively.
• 67% of their 119 cases had CCA and only 32% had pancreatic cancer.
Sakuma Y et al,Gastrointest Endosc 85:371–379, 2017

23. • In a review published in ASGE, de Bellis et al (2002) tabulated all reports for three
major techniques since 1989.
• Among the 502 patients ,1 major bleeding requiring transfusion,1 perforation of
a benign stricture required surgery
• Pediatric forceps produce a smaller specimen, but no complications were
encountered in more than 200 cases.
Barkun A et al,Gastrointest Endosc 63(6):741–745, 2006.

24. Combining Multiple Sampling Techniques
• Takes more time than a single technique,
• Improved yields.
• Brushing had a sensitivity of 43% and forceps biopsy had a sensitivity
of 30%, their combined yield was increased to 63% (a 20% overall
gain).

25. • Not become a standard practice during ERCP.
1. Technically difficult
2. Time-consuming
3. Ancillary to the main goal of the therapeutic procedure.
4. Availability of EUS-guided FNA cytology with its high sensitivity and safety
Puli SR et al,Pancreas 42:20–26, 2013.

26. • A comparative trial of the HBI device against standard brushing was reported.
• Considered any “positive,” “suspicious,” or “atypical or suggestive of malignancy”
to be true-positive samples.
• Used only the HBIN 22-gauge needle and HBI brush and reported an 85% yield
compared with a sensitivity of 57% for brushing alone.
Wiersema MJ et al,Gastrointest Endosc 56(3): 463–464, author reply 464-465, 2002.

27. Other Methods of ERCP
• Leung et al (1989) examined indwelling plastic biliary stents on their removal may
produce a positive cytologic specimen
• Since 1989, only one series has approached the initial 70% yield of Leung .
• Most centers report only 11% to 44% positive specimens
Schoefl R et al,J Gastroenterol 32(4):363–368, 1997
• Truepositive yield from pancreatic stents was 25% compared with only 11% from
biliary stents.
Simsir A et al,Biliary stent replacement cytology, Diagn Cytopathol 16(3):233–237, 1997.

28. • Specimens from the adjacent stricture of the pancreatic duct.
• More recent reports (1994–96) emphasize that yields increase very little when a
biliary stricture is also present and can be sampled
• Postprocedural pancreatitis a concern
Nakaizumi A et al,, Cancer 70(11):2610–2614, 1998.
• Vandervoort et al (1999) noted 21.5% pancreatitis in both benign and malignant
cases, but noted a marked decrease in risk if pancreatic temporary plastic stents
were placed

29. • Collection of pancreatic juice.
• Yields may increase to greater than 50% with the infusion of secretin.
Vandervoort J et al,Gastrointest Endosc 49(3):322–327, 1999
• Pugliese et al (1995) concluded that pancreatic juice collection did not add to
positive diagnosis when pancreatic duct strictures were directly sampled by
brushing

30. Specimen Handling and Analysis
• Air-drying artifact after thin smear.
• Excessive blood in Thick smears
• Intermediate cytologic abnormalities such as “atypical,” in which mild cellular
abnormalities are usually associated with inflammation and reparative changes,
and “suspicious,” in which there are rare cells exhibiting cytologic features of
malignancy, but they are present in insufficient numbers to render a definitive
diagnosis of malignancy.
• In patients with PSC or postbiliary irradiation, cytologic changes can result in
occasional false-positive samples.
• In-Suite Cytopathologist
• Transport media

31. Intraprocedural Techniques at ERCP
• Termed squash prep, this technique evolved because frozen sections cannot be
done on fat-rich brain tissue.
• Small 5-Fr or 6-Fr forceps biopsy specimens by vigorously smashing them
between two dry glass slides to attempt to create a monolayer.
• Immediately stained by rapid Papanicolaou and read in suite.
• This new technique is termed SMASH protocol

32. • When in-suite cytopathology is not available five rapidly obtained specimens
from the lower edge of an indeterminate stricture using only fluoroscopic
guidewire.
• If negative, an additional five specimens could be sent.
• In the specific setting of CCA of the CBD,CHD or bifurcation, established an overall
true positive diagnosis in 87% of consecutive cases
Wright ER et al,Am J Gastroenterol 106(2):294–299, 2011

33. Advanced Specimen Analysis
• FISH
• Fluorescently labeled chromosome-specific DNA probes to identify cells with an
abnormal number of chromosomes or mutations.
• Fluorescence microscopy is then used to quantify cells containing nuclei with
abnormal probe signal numbers
• The presence of ≥ 5 such cells showing gains of ≥ 2 of probes
• Chromosomes 3 (CEP3), 7 (CEP7), 17 (CEP17), and 9p21 locus of chromosome 9
• Can be performed on cells obtained from routine brush cytology samples
• Same brush can even be used to provide both FISH results and cytology results
with the use of a multipart brush, such as a brush with 3 separate clusters

34. • Levy et al (2008), using CEP3, CEP7, and CEP 17 probes found that, in previously
cytology-negative strictures, the sensitivity of FISH was 62%, with specificity of
79% for malignancy.
• When a fourth probe to the 9p21 loci of chromosome 9 added,sensitivity
improved from 47% to 84%, with preserved specificity of 97%.
Levy MJ et al,Am J Gastroenterol 103(5):1263–1273, 2008.
• Nanda et al (2007) demonstrated that triple modality sampling with brush
cytology, fluoroscopically guided biopsies, and FISH resulted in significantly higher
sensitivity of 82% versus 42% for brush cytology alone.
• Cost analysis in a study population, in which the respective sensitivity of cytology
and FISH was 42% and 70% respectively, suggested that FISH testing be used as a
second-line evaluation if cytology samples were negative given the significant
additional cost for FISH analysis. oldorini R et al,J Clin Pathol 68(4):283–287, 2015.

35. The main limitation of FISH
• Relative disadvantage: Centers that analyze FISH samples are limited
• Analysis typically can take up to 3 weeks to return
• Reduced specificity in the setting of chronic inflammatory
• Other molecular-based techniques that have been examined to improve the
diagnostic yield include bile aspirate analysis for p53 and KRAS mutations
• Gonda TA et al, has evaluated the value of combination testing of FISH and
genetic analysis (KRAS mutation, LOH, tumor suppressor genes at 10 loci) and
found that adding both FISH and molecular profiling to cytology can increase
sensitivity from 32% to 73%
Gastroenterol Hepatol 2016

36. Endoscopic Ultrasound-FNA
• In setting of suspected proximal CCA remains controversial.
• Transplant centers adopted protocols in which tissue sampling with EUS FNA of
the primary lesion is a contraindication to liver transplantation of hilar CCA.
• For patients not considered for transplant or who have extrahepatic disease and
are being considered for resection only, the reported overall sensitivity of EUS
FNA is 43% to 86%
• EUS FNA is generally less reliable in the evaluation of proximal CCA, as its
sensitivity decreases to 59% compared to 81% in distal CCA.
• Presence of a previously placed biliary stent can also decrease its sensitivity

37. Intraductal Ultrasound
• Employs a thin (2.0-3.1mm), high frequency (12-30 MHz) wire-guided radial
ultrasound probe that is passed through the working channel of a duodenoscope
• Does not require a sphincterotomy,
• Radiopaque
• Provides high-quality imaging of the periductal tissue along with limited tumor
staging, such as mass size and periportal vascular invasion (full lymph node
staging still requires EUS).
• Two to three mural layers are visualized during IDUS:
(1) Inner hypoechoic layer representing mucosa, muscularis propria, and the
fibrous layer of serosa;
(2) Outer hyperechoic layer representing subserosal adipose tissue and serosa;
(3) Interface layer between bile and the inner hypoechoic layer
Tamada K, et al, Gastrointest Endosc 47(5):341– 349, 1998.

38. • Differentiating benign from malignant strictures
1.Disruption of the normal triple layer wall architecture,
2.Eccentric wall thickening,
3.Presence of a hypoechoic mass with irregular margins
4.Invasion of adjacent structures,
5.Papillary surface
6.Malignant-appearing periductal lymph nodes
• Disadvantage
1.Does not provide histopathology.
2.Fragile
3.Not disposable, and requires a separate processor
Farrell RJ et al,Gastrointest Endosc 56(5):681–687, 2002

39. Optical Coherence Tomography
• Analogous to ultrasound but relies on low-intensity infrared light (700 to 1500
nm wavelength range) instead of sound waves
• Delay in time of light back-scattered by the various tissues is measured using a
technique known as low coherence interferometry, which has a depth of
penetration of 1-3 mm and lateral and axial resolution down to 10 μm.
Kobayashi K et al,Gastrointest Endosc 47(6):515–523, 1998
• OCT findings that correlated to the three ductal wall layers,
single layer epithelium- Inner hyporeflective layer
deeper fibromuscular layer- Homogenous hyperreflective layer
outer smooth muscle layer- hyporeflective layer.
Testoni PA et al,Dig Liver Dis 38(9):688–695, 2006.

40. • Using two OCT criteria
1.Unrecognizable layer architecture
2.Presence of large, nonreflective areas compatible with tumor vessels,
• Arvanitakis et al (2009) evaluated the feasibility of OCT in detecting malignant
biliary strictures in 35 patients.
Malignant strictures were diagnosed in 54% of patients compared to tissue as gold
standard.
The sensitivity of at least one or both OCT was 79% and 53%, respectively, and
accuracy was 70% for both categories.
When combining brushings and biopsies to at least one criteria, sensitivity
increased to 84%.

41. Cholangioscopy
• Small-caliber,flexible endoscope to directly inspect the biliary epithelium and
facilitate targeted sampling.
• Cholangioscope (daughter scope) is typically passed either through the working
channel of a therapeutic (mother) scope during ERCP or via direct peroral
cholangioscopy following endoscopic papillotomy and percutaneous transhepatic
cholangioscopy.
• Widespread adoption was limited due to the need for two operators, scope
fragility, limited tip maneuverability, and prolonged procedures.

42. • Spyglass Direct Visualization System (Boston Scientific, Marlborough, MA) in 2005
• capability for 4-way tip deflection, a channel for insertion of a reusable fiberoptic
probe, and irrigation and working channels
• Recent modifications, includes the use of a video chip
• 10-Fr access catheter (SpyScope) inserted through the standard 4.2-mm working
channel of a therapeutic duodenoscope, a reusable optical probe (SpyGlass) that fits
through the SpyScope catheter, and disposable 3-Fr biliary biopsy forceps to allow
visually-directed biopsies (SpyBite).
Tischendorf JJ et al,Endoscopy 38(7):665–669, 2006

44. • Evaluating the role of performing rapid on-site evaluation of touch imprints of DV
biopsy specimens (ROSE-TIC), Varadarajulu et al (2016) studied 31 patients and
with a mean of 3.3 biopsies, were able to achieve a sensitivity of 100% and
accuracy of 93.5% in diagnosing malignancy in IDBS
• Strongest feature suggestive of malignancy has been the presence of dilated and
tortuous vessels with a reported specificity and positive predictive value of 100%
Nimura Y, Kamiya J: Cholangioscopy, Endoscopy 30(2):182–188, 1998.

45. • Chen et al (2011) included 226 patients with biliary strictures with a sensitivity,
specificity, positive predictive value, and negative predictive value for malignancy
of 78%, 82%, 80%, and 80%, respectively, based on the visual impression criteria.
• Visual impression had a higher sensitivity compared to visually targeted biopsies,
which was only 47%, although biopsy specificity was much higher at 98%, with a
positive predictive value of 100%.

46. • Ramchandani et al (2011) involving 36 patients found sensitivity of the SOC visual
impression to be 95%, and specificity of 79%, while sensitivity and specificity for
SpyBite biopsies were lower at 82%.
• A comparison study of directly visualized (DV) biopsies versus both brush
cytology and fluoroscopically guided biopsies in 26 patients demonstrated that
despite adequate tissue quantity with all three modalities, the accuracy of the DV
biopsies was 84.6%, which was significantly higher than brush cytology (38.5%)
and standard forceps (53.8%)
Draganov PV et al,Gastrointest Endosc 75(2):347–353, 2012.

47. Confocal Laser Endomicroscopy
• Based upon the principle of illuminating a tissue with a low-power laser and then
detecting reflected fluorescent light depth 40 to 70 μm below the tissue surface.
• Laser is focused at a specific depth,and only light which is reflected back from
that plane is refocused and able to pass through the pinhole confocal aperture
• “Confocal” hence refers to the fact that the reflected light is refocused onto the
detection system by the same lens through which the laser light was initially
emitted. As a result, scattered light from above and below the plane of interest is
not detected,thereby increasing spatial resolution.
• A focused, scanning light source (i.e., laser) and processor then generate
reconstructed grayscale images of the target area, enabling epithelial and
subepithelial visualization.
• Requires administration of iv or topical contrast (typically fluorescein) to highlight
tissue features and better differentiate normal architecture or inflammatory
changes from neoplastic tissue

48. • In the first study of pCLE, Meining et al reported that the visualization of irregular,
dilated (“angiogenic”) vessels predicted malignancy with a sensitivity of 83%
specificity of 88%,and accuracy of 86% among 14 patients.
• Miami classification proposed consisting of:
Blinded consensus review provided a sensitivity, specificity, positive predictive
value (PPV), and NPV of 97%, 33%, 80%, and 80%, respectively, compared with
48%, 100%, 100%, and 41% for standard tissue acquisition

52. • Recognizing the limited ability of the Miami Classification to differentiate
between malignant and inflammatory strictures,
1.Attempt was made to distinguish specific criteria for the benign inflammatory
conditions:
2.Multiple thin white bands (vascular congestion),
3.Dark granular patterns with scales,
4. Increased spaces between scales ([20 um), (thickened reticular structures)
• Addition of these criteria did in fact improve specificity from 61% to 81.2%
• Slivka et al (2015) in the FOCUS trial demonstrated that the additive value of all
three diagnosis (pCLE, ERCP, and sampling) could provide an accuracy of 88% in
the diagnosis of malignancy in indeterminate strictures