Handout for the USCAP Symposium Cytomorphology of …/102nd/pdf/companion24h02.pdf · Handout for the USCAP Symposium ... Diagnostic Criteria with Emphasis on Diagnostic Pitfalls

Handout for the USCAP Symposium

Cytomorphology of Bile Duct Lesions: Diagnostic Criteria with Emphasis on Diagnostic Pitfalls

Introduction

Abnormalities of the extrahepatic biliary tract and pancreatic ductal system may come to clinical

attention following patient presentation with unexplained jaundice, abdominal pain or weight loss.

These abnormalities may be secondary to a variety of processes including auto-immune disease,

inflammatory lesions, malignancy and calculus disease. While most benign strictures are managed by

ductal dillitation and/or stenting, malignant strictures when operable are treated by Whipple resection

or bile duct resection. In patients with unresectable disease, simple stenting may be undertaken or

neoadjugant chemo and radiation therapy performed to shrink the tumor to an operable size. Thus,

confirmation of the benign or malignant nature of the stricture is of utmost clinical importance.

Minimally invasive techniques for investigating extrahepatic biliary tract and pancreatic duct strictures

as well as pancreatic masses include endoscopic ultrasound guided (EUS) fine needle aspiration (FNA)

and duct brushing cytology. Unfortunately, the majority of biliary and pancreatic duct lesions are not

accessible to forceps or clamshell biopsy. Brushing cytology is the predominant initial technique for

obtaining a tissue diagnosis for strictures. This, despite the fact that diagnostic sensitivity of brushing

cytology is disappointingly low.

A large number of authors have reported their experience with the diagnostic sensitivity, specificity and

accuracy of brushing cytology. The sensitivity of the technique has varied from approximately 25% to

90% with most studies revealing a sensitivity of between 40% and 60%.1-36 Specificity is superior to

sensitivity and has been reported to range between 80% and 100%.1-36 A number of clinical conditions

including inflammation and primary sclerosing cholangitis are known to decrease diagnostic

accuracy.16,37,38 A variety of ancillary techniques have been utilized in an attempt to improve overall

diagnostic sensitivity, specificity and accuracy. 39-49

While clinicians treating pancreatical biliary strictures favor a definitive classification into benign or

malignant, most cytopatholigists favor a diagnostic scheme utilizing one or more intermediate

categories.16,18,31,35 Terminology for these intermediate categories has varied and includes the terms

dysplasia, atypical cells, atypical cells suspicious for malignancy, atypia not otherwise specified and

atypia favor reactive.16,18,31 The Papanicolau Society of Cytopathology is in the process of developing a

set of guidelines for diagnostic techniques, diagnostic criteria and categories along with

recommendations for the use of ancillary studies and post-FNA follow-up and therapy. The proposed

categorization for the diagnosis of pancreatic and biliary lesions contains the categories: 1) non-

diagnostic, 2) benign, 3) atypical (including reactive), 4) suspicious for malignancy and 5) malignant. A

number of studies have investigated the diagnostic criteria for the separation of benign from malignant

pancreatico-biliary duct brushings.2,16,18,19,31,32,50-55 Many of these studies have utilized linear regression

analysis to determine which criteria are most accurate in separating benign from malignant lesions.

Some studies have established primary and secondary criteria for this separation.2,18,53 Primary criteria

have included nuclear moulding, chromatin clumping and increased nuclear cytoplasmic ratio.

Secondary criteria included anisonucleosis, irregular nuclear membranes, loss of polarity, chromatin

clearing, nuclear enlargement, macronucleoli and disruption of the normal honeycomb pattern. The

sensitivity and specificity of these criteria have varied.31 In many of these studies proposals have been

made to use multiple diagnostic categories because many times the identified criteria are difficult to

recognize or apply uniformly. Hence, the inclusion of atypical and suspicious for malignancy categories

in many diagnostic schemes. This is similar to the Bethesda system for squamous lesions of the cervix

and the system for thyroid FNA. Characteristics of benign biliary epithelium, atypical, suspicious for

malignancy and malignant are listed in tables I through IV. In the diagnostic categorization used in the

PSC guidelines, major criteria for malignancy include increased nuclear cytoplasmic ratio, chromatin

clumping, nuclear moulding, loss of honeycomb pattern or polarity, cell-in-cell arrangements and a

fourfold or greater variability of nuclear size within a single cell group or cluster.

Major diagnostic pitfalls exist for the cytologic diagnosis of pancreatico-biliary brushings. These include

sampling, special tumors types, smear background, dysplasia, primary sclerosing cholangitis, the

presence of stones or stents, cloaking of malignant epithelium by overlying benign epithelium and

degeneration of the cellular content due to bile or duodenum contamination.14 Well differentiated

adenocarcinomas of the pancreatico-biliary system also represent a problematic area for the cytologic

diagnosis of strictures. Biliary tract stones and stents may be associated with marked reactive atypia.

Helpful clues in differential diagnosis include the fact that atypical cells often lack abnormal chromatin

patterns and membrane irregularities are usually mild. Importantly, the nuclear cytoplasmic ratio

remains near normal. Acute inflammation involving the biliary tract is also associated with nuclear

atypia which can be distinguished from malignancy by recognizing the presence of acute inflammation.

Other helpful features in separating the atypia of inflammation from true neoplasia are that reactive

atypia due to acute or chronic inflammation maintains a near-normal nuclear-cytoplasmic ratio.

Additionally, the nuclear membrane changes seen with inflammatory atypia are usually less than those

seen in true malignancy. Moreover, in reactive atypias two distinct cell populations are not seen but

there is a merging of features between cells with significant atypia and clearly benign cells. This range of

morphologies favors benign while two distinctly separate populations (atypical versus benign) favors

malignancy.

As described above the sensitivity and specificity of pancreatico-biliary brushing cytology is well known

and published a large number of studies.1-37 Follow-up for the atypical and suspicious for malignancy

categories is less well described. In a study of a thousand cases, Volmar, et al35 documented that clinical

or pathologic follow-up of cases designated “suspicious for malignancy” revealed a malignancy in 79.2%.

For the category “atypical/inconclusive” follow-up revealed malignancy in 43.6% of cases. Our personal

experience demonstrates that atypical/favor reactive is associated with an absolute risk of 0.20 and a

relative risk of 0.8 for malignancy. The atypical/suspicious in our experience has an absolute risk of 0.74

and a relative risk of 3.0 for malignancy. From the results of these two studies the risk of malignancy in

the “suspicious for malignancy” category closely approaches that of a definitive diagnosis of malignancy.

The “atypical (favor reactive)” category has a risk of malignancy which varies between studies. In the

study by Volmar, the atypical/inconclusive category had a significant risk of malignancy (43.6%) and was

similar to the risk of malignancy in the benign category reported in a number of studies. In the studies

at the University of Utah, the “atypical (favor reactive)” category had an absolute risk and relative risk of

malignancy less than that seen for the benign category. The absolute risk and relative risk for the benign

category were 0.248 and 1.0 respectively. From this data it appears that the category “atypical (favor

reactive)” can be followed/treated similarly to the benign category while the “suspicious for

malignancy” category should be followed/treated like the malignant period.

Because of the poor diagnostic sensitivity of pancreatic biliary brushing cytology a number of ancillary

testing methods have been utilized including mutational analysis for KRAS mutations, p53 mutations as

well as ploidy analysis and loss of heterozygosity. More recently, ancillary testing has included

fluorescence in-situ hybridization for changes in chromosomal numbers.39 Evaluation of current

ancillary testing appears to indicate that fluorescence in-situ hybridization (FISH) is the most

diagnostically useful in separating benign from malignant pancreatico-biliary strictures. This technique

should be utilized on all equivocal cytologies to improve diagnostic sensitivity without loss of

specificity.39

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Table I - Morphologic Characteristics of Benign Biliary Epithelium

Regular “honeycomb” sheets

Retention of cell polarity

Smooth nuclear membranes

Even chromatin pattern

Small or indistinct nucleoli

Mitotic figures and necrosis absent

Low nuclear/cytoplasmic ratio

Variability in nuclear size less than threefold in any single group

Table II – Morphologic Characteristics of “Atypia”

Mild variation in nuclear size less than threefold in any single group

Nuclear hyperchromasia

Distinct nucleoli

Normal mitotic figures may be present

Relatively normal n/c ratio

Mild nuclear membrane irregularities

Squamous metaplasia

Table III – Morphologic Characteristics of “Suspicious for Malignancy” Category

Loss of “honeycomb” pattern

Loss of polarity

Nuclear crowding and overlap

Coarse chromatin

Enlarged nucleoli

Increased n/c ratio

Nuclear membrane irregularities

Three to fourfold variability in nuclear size within a single cell cluster

*some but not all the features of malignancy present

Table IV – Morphologic Characteristics of “Malignant”

Cell groups with prominent nuclear overlap and crowding

Coarse chromatin

Enlarged nucleoli

Increased n/c ratio

Irregular nuclear membranes

Three to four or more fold variability in nuclear size within a single cell group