chapter 33 - premalignant hepatocellular lesions

nomenclaturDysplastic Foci Dysplastic Nod

Dysplastic noPrecursors 4natural HistoHepatic PremSampling: cyBasic HistopaParenchymal CNonparenchym

Diagnostic isLow-Grade DysDysplastic NodHigh-Grade DysCarcinoma 4Biomarkers 4

AbbreviationAASLD AmeCEUS contCK cytoCT comDN dyspEASL EuroHCC hepaHGDN highHSP70 heatLCC larg

*Although the prefix cytokeratins, a new coPA, et al. New consenrecommends the repla

33

Massimo Roncalli, MD, PhD, Luca Di tommaso, MD, Mauro Borzio, MD, and Young nyun Park, MD, PhD

Premalignant Hepatocellular Lesions

e 458458

ules 460

dules as Hepatocellular carcinoma 60

ry of Premalignant Lesions 461alignant Lesions in Daily clinical Practice 463tologic and Histologic Samples 464thologic Features (elementary Lesions) 464

hanges 464al Changes 465

sues and new tools 466plastic Nodules versus Regenerative/High-Grade ules/Hepatocellular Carcinoma 466plastic Nodules versus Well-Differentiated Hepatocellular

6767

srican Association for the Study of Liver Diseasesrast-enhanced ultrasonographykeratin*

puted tomographylastic nodulepean Association for the Study of the Livertocellular carcinoma-grade dysplastic nodule shock protein 70e cell change

CK (for cytokeratin) is widely used in surgical pathology to designate human nsensus nomenclature published in 2006 (Schweizer J, Bowden PE, Coulombe sus nomenclature for mammalian keratins. J Cell Biol. 2006;174[2]:169–174) cement of “cytokeratin” with “keratin” and the prefix “CK” with “K.”

LGDN low-grade dysplastic noduleLRN large regenerative noduleNMR nuclear magnetic resonanceSCC small cell change

Most patients with compensated cirrhosis with de novo hepatocellular carcinoma (HCC) at an early stage (single nodule <5 cm, or 3 nodules each ≤3 cm) may expect 5-year survival rates exceeding 50%. This sig-nificant improvement in prognosis of HCC over recent decades is a reflection of three major factors:

1. Protocols for regular screening of cirrhotic patients to detect early HCC

2. Availability of successful modalities of therapy such as orthotopic liver transplantation, resection, and percutaneous ablation1,2 for early HCC

3. Internationally recognized criteria for accurate selection of patients based on precise definitions of number and size of HCC nodules3

Regular ultrasound surveillance of patients with cirrhosis leads to fre-quent detection of small 0.5- to 3-cm lesions as hypoechoic or, less frequently, hyperechoic nodules arising in the cirrhotic parenchyma. The diagnostic approach to these small nodular lesions is a chal-lenge. Difficulties arise from the fact that 10% to 40% of these small lesions are represented by nonmalignant or premalignant nodules (Table 33-1). These nodules are radiologically indistinguishable from early HCC; however, their distinction is crucial for proper patient selection.

The size of a radiologically detected nodule is highly predictive of the presence of malignancy (Table 33-2). Almost half of all lesions less than 1 cm are nonmalignant, whereas the large majority of lesions exceeding 2 cm are HCC; this association is so strong that diagnosis of nonmalignancy in a nodule greater than 2 cm should raise the possibil-ity of a diagnostic error.

It is well established that, in cirrhosis, progression from nonmalig-nant nodules to dysplastic nodules (DNs) to early and advanced HCC is characterized by progressive loss of portal venous blood and develop-ment of de novo arterial vascularization (neoangiogenesis).8 This arterial

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Practical Hepatic Pathology

458

Reference

time of observation (yr)

no. of Lesions

Hcc

nM-Hn

other

Kondo et al7 5 161 132 (82%) 28 (17.5%) 1 (0.5%)

Borzio et al4 10 317 184 (58%) 125 (39.5%) 8 (2.5%)

Bolondi et al1 3 72 60 (83%) 12 (17%) 0

Caturelli et al5 10 294 258 (87%) 31 (11%) 5 (2%)

Forner et al6 3 89* 60 (67%) 24 (27%) 5 (6%)

table 33-1. Prevalence of Ultrasonography Detected Focal Liver Lesions in Cirrhosis

Selected criteria: diameter <20 mm.HCC, hepatocellular carcinoma; NM-HN, nonmalignant hepatocellular nodule.

Dysplastic Foci

Box 33-2. Nomenclature for Premalignant Hepatocellular Lesions

• Toselectpatientsharboringregenerative/low-gradedysplasticnodulesforarecall policy

• Toselectpatientsathigherriskofmalignanttransformation(thosewithhigh-grade dysplastic nodules) for optimal management

• Toguidepatientswithmalignantnodulestoappropriatetherapy:surgicalresection/waiting list for transplantation/ablative therapy according to international guidelines

• Todecreasethelikelihoodofprematurelytransplantingpatientswhodonothave hepatocellular carcinoma and, conversely, to avoid the exclusion of patients with multiple nodules, some of which may be nonmalignant

• Toaccuratelystagepatientswithmalignantnodules,particularlythoseamenable to hepatic resection

• Tomakeanindividualprognosisbasedonaninterdisciplinaryapproach(histology, radiology, clinical parameters) and not on the false belief that every nodule in cirrhosis is (or is likely to become) malignant, thus necessitating “prophylactic” ablation

• Toobtainaccuratecorrelativedataformolecular/geneticexpressionstudiestocharacterize individual lesions for individual therapy

Box 33-1. Clinical Relevance of Accurate Classification of Small Hepatocellular Nodules in Cirrhosis

vascularization produces a characteristic pattern of “wash in” (contrast enhancement) and “wash out” (contrast dispersion) during arterial and early-late portal phases, respectively, on spiral computed tomography (CT), nuclear magnetic resonance (NMR), and contrast-enhanced ultra-sonography (CEUS). This pattern is distinctive enough to establish a firm diagnosis of malignancy on radiology alone and serves as the basis of the “noninvasive” criteria for diagnosis of HCC. These criteria, endorsed by both the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD),3,9 state that the diagnosis of HCC can be confidently accepted for lesions greater than 2 cm in diameter, which demonstrate unequivocal “wash in” and “wash out” during arterial and early-late portal phases by one of the previously mentioned imaging techniques. To avoid false-positive cases, two imaging techniques with unequivocal findings are required to docu-ment HCC in lesions less than 2 cm in diameter.9 A biopsy is required in all cases, irrespective of size, when imaging features are inconclusive or atypical. Several studies seeking to validate these “noninvasive” criteria in cohorts of cirrhotic patients with ultrasonographically detected nodules less than 3 cm have found sensitivity of less than 60% in lesions 1 to 2 cm in size, with either NMR or spiral CT along with CEUS. In this group, regenerative-dysplastic nodules accounted for one third of the cases, and about 20% of the HCCs were hypovascular; histology forms a mainstay for diagnosis and follow-up of these small lesions.1,6

This chapter discusses the pathobiology, natural history, and diagno-sis of premalignant nodules, the study and accurate diagnosis of which have great clinical relevance (Box 33-1). Additionally, the ample evi-dence linking DNs to HCC provides them with the potential to offer insights into the process of hepatocarcinogenesis—knowledge that will be invaluable for the diagnosis, prognosis, and treatment of HCC.

NomenclatureHepatic lesions thought to be able to transform into HCC are consid-ered premalignant. Several terms have been used in the past to describe

nM-Hn/Hcc

Reference total nM-Hn/Hcc <10 mm 10–20 mm >20 mm

Borzio M* 317 89/228 12/1 58/29 19/198

Bolondi et al1 72 12/60 NR 12/29 0/31

Forner et al6 89 29/60 11/2 18/58 NR

table 33-2. Prevalence of Focal Liver Lesions According to Size

NM-HN, nonmalignant hepatocellular nodule; NR, not reported.*Personal data.

these lesions, such as adenomatous hyperplasia and atypical adenoma-tous hyperplasia, but in 1995, the International Working Party10 rejected these terms as imprecise or misleading and proposed a new nomencla-ture (Box 33-2), which has gained wide acceptance and is in current use.

In practice, hepatocellular dysplastic lesions can be distinguished as those detectable only as microscopic foci (dysplastic foci) or as those form-ing grossly recognizable nodules (DNs) in cirrhosis (Fig. 33-1; see Box 33-2). The alternative terms, microscopic dysplasia and macroscopic dyspla-sia, are discouraged by the International Working Party nomenclature.

Dysplastic FociThese are small foci, less than 1 mm in size, of dysplastic hepatocytes rec-ognized within the liver parenchyma in chronic liver disease, particularly at advanced fibrotic stages (cirrhosis). Dysplastic foci are named accord-ing to the main cell type as large cell foci, small cell foci, or iron-free foci; the latter occur in hemochromatosis or any condition with significant parenchymal iron overload. In most cases, dysplastic foci are recognized incidentally in a liver biopsy; dysplastic hepatocytes showing large cell changes (LCCs) or small cell changes (SCCs) are also part of the spectrum of cytologic abnormalities occurring in DNs. Several studies have been conducted in the past to address the precancerous role played by these foci in the human liver, particularly as far as LCCs and SCCs, previously called large cell dysplasia and small cell dysplasia, respectively, are con-cerned. An extensive and detailed analysis of these lesions is beyond the scope of the present chapter; however, the main features are summarized

Cluster of hepatocytes, <1 mm in size, characterized by presence of either small cell change or large cell change

Dysplastic noduleA distinctly nodular lesion that differs from the surrounding liver parenchyma with regard to size, color, and texture and that bulges from the surrounding liver on cut surface (see Fig. 33-1A). It can be detected but not accurately diagnosed at imaging. Histologically it is distinguished into two categories:

• Low-grade:lesionswithfeaturessuggestiveofaclonalcellpopulationbutlacking architectural alterations (see Fig. 33-1B–D)

• High-grade:lesionsshowingacertaindegreeofcytologicatypiaandarchitectural alterations but which are insufficient for a diagnosis of malignancy (see Fig. 33-1E–G)


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33

A B

C D

E

Figure 33-1. A, Gross features of two dysplastic nodules (arrows), each measuring 1 cm in size. Both nodules stand out from the surrounding cirrhotic liver because of their bulg-ing cut surface and differences in color and consistency. B, LGDN manifesting, at low magnification, as a large and distinct hepatocellular nodule with expansile growth pattern and intranodular portal tracts. c, LGDN (below dotted line) showing a slight increase in cellularity compared with the adjacent cirrhotic nodule without architectural atypia. D, LGDN characterized by normal one- to two-cell–thick trabeculae and occasional large cell changes. e, HGDN showing, at low magnification, alternating light and dark eosinophilic areas. Intranodular portal tracts are noted (arrows).

Figure continues on following page.


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Figure 33-1—cont. F, HGDN (left) showing increased cell density, compared with the extranodular liver (right). G, HGDN at a higher magnification showing a thin trabecular pat-tern with two- to three-cell–thick plates, small cell change with high nucleocytoplasmic ratio, and clear and fatty cytoplasmic changes. (A, Reprinted with permission of Elsevier from Hytiroglou P, Park YN, Krinsky G, et al: Hepatic precancerous lesions and small hepatocellular carcinoma. Gastroenterol Clin North Am. 2007;36:867–887.)

F G

in Box 33-311–19 and illustrated in Figure 33-2. From a practical point of view, in routine clinical practice, it is important to look for and mention dysplastic foci in the pathology report when they are present.

Dysplastic NodulesDNs are sizable lesions ranging between 1 and 2 cm, arising in cir-rhosis, detectable either at imaging or macroscopically in resection specimens and showing both parenchymal and nonparenchymal alter-ations. They are subdivided into low-grade DNs (LGDNs) and high-grade DNs (HGDNs) (see Box 33-2 and Fig. 33-1). HGDNs are closer to HCC in the spectrum of hepatocarcinogenesis.10 DNs have to be dif-ferentiated from large regenerative nodules (LRNs) on the one hand and from small well-differentiated HCC (usually up to 2 cm) on the other. Small well-differentiated HCC consists of two distinct entities: (1) that with indistinct margins, so-called “HCC of vaguely nodular type” or “small and early HCC,” and (2) that with distinct margins, so-called “HCC of distinctly nodular type” or “small and progressed HCC”

Small cell changes (see Fig. 33-2A)Abnormal hepatocytes, increased proliferation/apoptosis rate11; significant telomere shortening and loss of cell cycle checkpoint12

Common detection of morphophenotypic features of liver progenitor cells such as keratin 1913

Rare (5%) in cirrhotics under surveillance; frequent in high-grade dysplastic nodules and noncancerous liver adjacent to HCC

Less robust predictor of HCC development14

Accepted microscopic precursor of HCC15

Large cell changes (see Fig. 33-2B)Abnormal hepatocytes, possibly senescent or slowly proliferating;11 slight telomere shortening, altered cell cycle checkpoint control12,12a

Morphophenotypic features of more differentiated hepatocytesFrequent (20% to 30%) in cirrhotics under surveillance, dysplastic nodules

(low- and high-grade), and noncancerous liver adjacent to HCCRobust predictor of HCC development (i.e., liver more susceptible to malignant

transformation)16–18

Heterogeneous lesion likely including two populations: one reactive and terminally differentiated, the other as a very early precursor of HCC19

Box 33-3. Main Features of Small and Large Cell Changes

HCC, hepatocellular carcinoma.

(Table 33-3; Fig. 33-3).20 Although the former is the earliest, most indolent, and least angioinvasive type of HCC, the latter is thought to have already acquired an ability to invade vessels and metastasize.15,21–24 Therefore, small HCC with distinct margins represents a progressed cancer in spite of its small size.

Because the diagnosis of small and early HCC requires knowledge of its gross features (vaguely nodular and a replacing, not expansile, pat-tern of growth), which are not apparent in a liver biopsy, the term well-differentiated HCC is best used to indicate this entity in a biopsy report. Distinction between small and early HCC and the more common small and not early HCC is based on the well-preserved hepatic architec-ture of the former, mimicking a normal or steatotic liver, as opposed to the more obvious malignant changes of the latter with destruction of underlying hepatic architecture, trabecular or pseudoacinar pattern of growth, and well- to moderately differentiated histology.

Dysplastic Nodules as Hepatocellular Carcinoma PrecursorsEvidence supporting the premalignant nature of DNs includes the:

● Epidemiologic association of DNs with HCC in resected and explanted livers, particularly in end-stage cirrhotic patients17,21,23,25–28

● Morphologic detection of hepatocellular cytoarchitectural abnor-malities (dysplasia)10,15,24,27–30

● Morphologic continuity, detectable in some cases, between DNs and HCC, in the form of the so-called “nodule-in-nodule” appear-ance, where an inner malignant core seems to develop from an outer dysplastic hepatocellular precursor15,31

● Presence of nontriadal arteries (discussed later) in DNs, indicating evolving neoangiogenesis and arterialization of the nodule, as seen in HCC32–36

● Detection of both genetic and epigenetic changes similar to those seen in HCC. The incidence and extent of these changes is between those observed in nondysplastic cirrhotic nodules and those in HCC. In some cases, the very same molecular altera-tion has been reported in a precancerous lesion and the adjacent HCC.24,37–41

● Natural history of DNs showing an increased hazard risk of malig-nant transformation as compared with control patients with cirrhosis4,7,14,30,31,42,43


33

A

B

Figure 33-2. A, SCCs (left) characterized by decreased cytoplasmic volume, increased nuclear-to-cytoplasmic ratio, and mild nuclear pleomorphism and hyperchromasia, resulting in overall increased cell density. B, LCCs showing both nuclear and cytoplasmic enlargement with preserved nucleocytoplasmic ratio, nuclear pleomorphism and hyper-chromasia, and occasional multinucleation (arrows). (A, Reprinted with permission of Elsevier from Hytiroglou P, Park YN, Krinsky G, et al: Hepatic precancerous lesions and small hepatocellular carcinoma. Gastroenterol Clin North Am. 2007;36:867–887.)

Feature early Hcc (see Fig. 3A–E)

Synonyms Well-differentiated HCC of vaguely nodular type; well-differentHCC with indistinct margins

Capsule Replacing pattern at tumor/nontumor interface without fibrous cap

Differentiation and cytoarchitectural findings

Well-differentiated (G1) histology with little cellular and structuraatypia; increased cell density and nucleocytoplasmic ratio; irregularly thin trabeculae, some acinar pseudoglandular struct

Portal tracts Scattered (intratumoral) portal tracts

Unpaired arteries Scattered unpaired arteries

Steatosis Frequent (up to 40%) steatotic

Ease of diagnosis At low magnification, simulates normal or steatotic liver; at higmagnification, requires careful distinction from HGDN

table 33-3. Nomenclature and Comparative Features of Small (≤2 cm) Hepatocellular Carc

HCC, hepatocellular carcinoma; HGDN, high-grade dysplastic nodule.

Natural History of Premalignant LesionsProspective studies conducted in large series of histologically proven nonneoplastic nodules detected by ultrasonography during surveillance programs in cirrhosis demonstrated a wide evolutionary fate of these lesions, as shown in Table 33-4.4,7,14,43 Borzio and colleagues4 followed 90 patients with cirrhosis with histologically proven LRNs and DNs and found that the presence of HGDNs or extranodular LCCs was associ-ated with an increased hazard risk of malignant transformation—2.4 and 3.1, respectively, over a mean follow-up period of 33 months. On the other hand, LRNs and LGDNs were associated with a lower pro-pensity for HCC development. In another study, Kobayashi and asso-ciates42 followed 154 patients who had histologically proven LRN (n = 99), LGDN (n = 42), and HGDN (n = 13) for a median period of 2.8 years. The hazard ratios of HGDN, LGDN, and LRN for transforma-tion to HCC were 16.8, 2.96, and 1.0, respectively. Thus, only a minority of regenerative-dysplastic nodules became malignant; those transform-ing were mostly HGDNs, and they did so in a relatively short interval. Forty percent to 60% stabilized, and a few definitely disappeared during follow-up. Because most of these nodules are 1 to 2 cm in size, they sel-dom display a typical diagnostic pattern at imaging; histologic assess-ment is therefore necessary at initial detection and should be repeated, at least once, when sampling is not adequate. If the nodule is classified definitively as nonmalignant, follow-up should involve regular ultra-sonography at 3-month intervals. According to international criteria,3,9 transformation to HCC should be suspected and histologic confirmation sought if there is an increase in size or a change in echogenicity of the nodule.3 However, it is still unclear whether, in individual patients, HCC regularly develops from DNs along the sequence of low-grade to high-grade to small and early HCC to small and not early HCC.

The features of early HCC and the so-called “nodule-in-nodule” form have been reported mainly in Eastern literature and seem to be less commonly encountered in Western clinical experience.23,44–47 Because most of the experience in the East comes from surgically resected cases and that in the West from explanted livers, it is likely that the former conditions simply represent early cirrhosis and therefore an earlier stage of HCC in contrast to more advanced cirrhosis and late HCC in the explants. The more advanced stage might explain the dif-ference in growth features, such as indistinct versus distinct margins, because the fibrous septa surrounding the nodule make the nodule dis-tinct. Interestingly, there is evidence that small and not early HCC with distinct margins may also develop de novo, having skipped the gradual

461

Progressed Hcc (see Fig. 3F–I)

iated Well-differentiated HCC of distinctly nodular type; well-differentiated HCC with distinct margins

sule Peripheral fibrous capsule (>50%)

l

ures

Well- (G1) to moderately (G2) differentiated histology; mostly trabecular/pseudoglandular pattern of growth

No portal tracts

Numerous unpaired arteries

Rare fatty change

her Malignancy easily recognized in most lesions; may arise within early HCC or within HGDN, thus growing, radiologically and macroscopically, as a “nodule-in-nodule”

inoma


462

Figure 33-3. A, Gross features of small and early HCC manifesting as a 1.3-cm, uncapsulated, vaguely nodular lesion with indistinct margins (arrows). B, A microscopic view of small and early HCC (top) and adjacent liver parenchyma (bottom). A certain degree of architectural disorder and increased cell density can be appreciated, even at low magnification; clonal changes such as fatty and clear cells can be seen in the upper field. Notice that there is no compression of the surrounding liver parenchyma. c, A microscopic view of small and early HCC showing well-differentiated histology and small neoplastic cells with a high nucleocytoplasmic ratio and thin trabeculae. Macrovesicular fatty change is noted (left), where the nucleocytoplasmic ratio is lower. D, Small and early HCC showing well-differentiated histology characterized by pseudoglands and a thin trabecular pattern. Cholestasis is not detectable. e, Periphery of a small and early HCC (right) gradually substituting the adjacent non-neoplastic hepatic cords (replacing pattern of growth) without altering the preexisting architecture (arrows). F, Gross features of small and not early HCC manifesting as a 1.4-cm encapsulated nodule with distinct margins. The cut surface is more compact, homogeneous, and fleshy compared with that of dysplastic nodules (see Fig. 33-1A) or small and early HCC (see Fig. 33-3A).

A B

C

E

D

F

A

F

A


33

G

I

H

Figure 33-3—cont. G, Microscopic features of small and not early HCC at low magnification. Malignancy is obvious at first glance due to moderate differentia-tion and the trabecular pattern. H, Small and not early HCC with obvious malignant features such as extensive clear cell changes, expansile pattern of growth, and the invasion of tumor capsule (arrows). i, A higher magnification of small and not early HCC showing moderate degree of tumor differentiation. (F and G, Reprinted with permission of Elsevier from Hytiroglou P, Park YN, Krinsky G, et al: Hepatic precan-cerous lesions and small hepatocellular carcinoma. Gastroenterol Clin North Am. 2007;36:867–887.)

and sequential transition from dysplasia to carcinoma. Along the same lines, HCC has also been found to originate from outside DNs during surveillance of patients with cirrhosis.4

Hepatic Premalignant Lesions in Daily Clinical PracticeIn clinical practice, pathologists are faced with diagnosis and classifica-tion of dysplastic liver nodules and small HCC in three clinical settings:

1. Grossly distinct nodules, in surgically resected specimens for HCC2. Grossly distinct nodules, in explanted liver for end-stage cirrhosis

with or without HCC

Reference

type

no.

Malignant changes

Unchanged

Disappeared

Kondo et al7 LRN 17 — 13 (76%) 4 (24%)

Terasaky et al43 LRN/DN 34 5 (15%) 4 (12%) 25 (73%)

Seki et al14 DN 33 4 (9%) 14 (42%) 15 (49%)

Borzio et al4 LRN/DN 90 28 (31%) 44 (49%) 18 (20%)

table 33-4. Natural Outcome of Regenerative/Dysplastic Nodules in Cirrhosis

DN, dysplastic nodule; LRN, large regenerative nodule.

3. Ultrasonographically detected nodules, during surveillance of patients with cirrhosis for biopsy diagnosis. As mentioned previ-ously, international guidelines recommend biopsies of the following radiographically detected hepatocellular nodules:● Nodules less than 2 cm with equivocal radiologic features on two

imaging techniques● Nodules greater than 2 cm without diagnostic radiologic features

of HCC at imaging (one technique)When imaging is diagnostic for HCC, biopsy is not recommended, making HCC the only malignancy where a tissue diagnosis is not always mandated.

Although diagnosis is straightforward in moderately or poorly dif-ferentiated HCC, the main diagnostic difficulty lies in the distinction of HGDN from well-differentiated HCC. Diagnosis has to be made on tiny and often fragmented material; in this limited material, par-ticularly on low magnification, HGDNs can be easily misinterpreted as malignant, whereas well-differentiated HCC may simulate a nor-mal or steatotic liver. The remaining chapter will focus on the main morphologic, histochemical, and immunocytochemical tools used to distinguish, in the liver biopsy, malignant from nonmalignant nodules within the group of well- differentiated hepatocellular lesions. This dis-tinction requires the evaluation of features related to both the paren-chymal (hepatocytes) and the nonparenchymal structural components (stromal framework and vessels).

463


464

LRn LGDn HGDn WD Hcc

Parenchymal changes

Cytologic Alterations

Small cell change – – + – Large cell change ± ± ± – Clonelike foci (clear, fatty) – ± + +

Architectural Alterations

Plate thickening ± ± + + Cell density – – + + Pseudoglands – – ± + Perisinusoidal nuclear alignment – – ± + Nodule-in-nodule – – – ± Stromal invasion – – – ±

nonparenchymal changes

Unpaired arteries – ± ± + Capillarized sinusoids – ± ± + Reticulin framework + + + ± Portal tract + + + ±

table 33-5. Elementary Features of Hepatocellular Nodules in Biopsied Liver

–, absent; ±, may be present but not necessarily detectable in biopsy; +, usually present and detectable in biopsy. HGDN, high-grade dysplastic nodule; LGDN, low-grade dysplastic nodule; LRN, large regenerative nodule; WD HCC, well-differentiated hepatocellular carcinoma.

Sampling: Cytologic and Histologic SamplesIt is recommended that sampling include both intra- and extralesional tissue, because the architectural and cytologic abnormalities are better appreciated by comparing these two regions with each other (Fig. 33-4). Cytology alone is not recommended for the distinction of well- differentiated hepatocellular nodules; however, histologic microsamples carefully retrieved from the fine needle and embedded in paraffin may be sufficient to analyze a number of basic architectural features. We believe that cytology alone is best used as a confirmatory technique for clinically obvious HCC (<3 cm), tumor relapse, metastasis, or tissue collection for molecular studies. An important practical issue to be taken into account is when the patient harbors more than one nodule. Given that the diag-nostic uncertainty is greater in small lesions, we recommend sampling the smaller nodule, assuming the larger to be already malignant.

Basic Histopathologic Features (Elementary Lesions)Several histopathologic features help in the distinction between regen-erative nodule, dysplastic nodule, and well-differentiated HCC in a liver biopsy.44–47 These features can be categorized as parenchymal and nonparenchymal. The former consist of cytologic and/or architectural changes (Table 33-5).12,37–41,48–67

Parenchymal ChangesSCC is a feature of HGDNs (see Fig. 33-2A).When present extensively, SCC appears as areas of increased cell density and plate thickening, simulating a well-differentiated HCC.

LCC may be detected in cirrhotic regenerative nodules as well as in both LGDNs (see Fig. 33-2B) and HGDNs. Because LCC is a cytologic change, basic liver architecture is retained and diagnostic confusion with HCC is unlikely.

Clonelike foci are clusters of hepatocytes with homogeneous cyto-plasmic features that appear distinct from surrounding hepatocytes, suggesting a clonal expansion of cells.48,49 Clonelike foci may appear clear, fatty, eosinophilic, siderotic, iron-free, or inclusion- containing as a result of the accumulation of glycogen, fat, mitochondria, hemosiderin, or Mallory bodies. These foci may grow by compressing the

HOW TO CHECK SA

Extralesion

Intralesional biopsy

Not adequate

Figure 33-4. This schematic figure illustrates how to determine if sampling of a hepatocelleven if incompletely sampled, seems to stand out in terms of size over the surrounding nod

surrounding parenchyma (bulging type of growth) (Fig. 33-5A and B) or without compression of the adjacent liver (maplike type of growth). The term irregular regeneration of hepatocytes has been also proposed for these types of growth,68 particularly in association with cytologic fea-tures such as anisonucleosis and an increased nucleocytoplasmic ratio. The most common examples are clonelike foci of clear or steatotic (see Fig. 33-5C) cells or cells with Mallory bodies (see Fig. 33-5D). Clonelike foci are a hallmark of DNs but are also extensively present in HCC where different clonal cell populations may be found, indicating tumor heterogeneity. Clonelike foci do not occur in LRNs.

MPLE ADEQUACY

al biopsy

Adequate

HN

Intralesional biopsy

ular nodule is adequate. Adequacy in the biopsy is when a nodule containing portal tracts, ules.


33

A B

C D

Figure 33-5. Clonal changes in a dysplastic nodule on liver biopsy. A, A cluster of clear hepatocytes slightly compressing the surrounding parenchyma (bulging type of growth). B, A hepatocellular nodule with clear and steatotic clonal changes (bulging type of growth). Notice the presence of an intralesional portal tract (arrow). c, A cluster of hepatocytes show-ing fatty change. Notice a peripherally located portal tract (arrow). D, A cluster of ballooned hepatocytes containing Mallory bodies.

Architectural changes are characterized by focal or diffuse paren-chymal disarrangement and are best appreciated at low magnification by comparing lesional and extralesional tissue. These changes include the following:

● Increased cell density (1.5× to 2× compared with surroundings) (Fig. 33-6A)

● Thickening of cell plates (up to three cells thick compared with surroundings)

● Formation of pseudoglands (see Fig. 33-6B)● Perisinusoidal alignment of hepatocyte nuclei (see Fig. 33-6C)● Subnodular growth (see Fig. 33-6D), the last due to the bulging

growth of clonal hepatocytes in a “nodule-in-nodule” arrangement

Architectural changes are the main features of HGDNs, helping to distinguish them from LGDNs. Architectural changes are also pres-ent in HCC, in which they are much more extensive than in HGDN. The presence of a “nodule-in-nodule” pattern and stromal invasion is definite evidence of well-differentiated HCC and allows its distinction from HGDN (see Table 33-5).

Nonparenchymal ChangesUnpaired arteries, so called because they have no accompanying bile ducts, are small nontriadal arteries occurring outside the original portal tracts; they are also devoid of a venous counterpart. Unpaired arteries may not be easily recognizable on a hematoxylin and eosin stain (Fig. 33-7A) and are better visualized by immunohistochemi-cal stains for smooth muscle actin. These arteries are indicative of ongoing neovascularization. Given that hepatocarcinogenesis is characterized by progressive increase of a neoarterial supply and concomitant decrease in the original venous and arterial vasculari-ties, the presence of unpaired arteries helps in the distinction of regenerative nodules from dysplastic ones. The number of unpaired arteries increases with increasing grades of dysplasia, and they are more numerous in HCC; however, threshold values have not been established to separate LGDNs from HGDNs or HGDNs from HCC. Along the same note, because small and early HCC is character-ized by incomplete arterial neovascularization, it does not produce the diagnostic hypervascular pattern of more advanced HCC at imaging.

465


466

A B

C D

Figure 33-6. Architectural changes in a dysplastic nodule on liver biopsy. A, Increased cell density (1.5× surroundings) in the left upper field, marked by a dotted line. B, Pseudoglandular arrangement of hepatocytes without cholestasis (arrows). c, Perisinusoidal nuclear arrangement showing basal polarization toward the sinusoids. D, A nodule-in-nodule pattern of growth. The inner nodule in the upper field, marked by three arrows, is a well-differentiated hepatocellular carcinoma, and the outer nodule is a high-grade dysplastic nodule. (Reprinted with permission of Elsevier from Roncalli M, Borzio M, Di Tommaso L: Hepatocellular dysplastic nodules. Hepatol Res. 2007[suppl 2]:S125–S134.)

Sinusoidal capillarization is the progressive increase in the num-ber of capillarized sinusoids, a feature indicative of altered blood flow and vascular remodeling during hepatocarcinogenesis. Capillarized sinusoids are characterized by uniform staining of the sinusoidal lining cells by endothelial markers such as CD34 and CD31 (see Fig. 33-7B and C). The concepts described previously for the detec-tion and significance of unpaired arteries also apply to capillarized sinusoids.

Portal tracts are retained and may be seen in regenerative and dys-plastic nodules. However, because they are also retained in small and early HCC, their presence does not help in distinction of DNs from early and small HCC. On the other hand, portal tracts are the site of earliest invasion by malignant cells, so-called “stromal invasion” (discussed later), which provides definitive evidence of malignancy. The sensitivity of detecting stromal invasion within a portal tract is enhanced by histochemical stains such as Victoria blue, which help dis-tinguish neoplastic desmoplasia surrounding invading cells from nor-mal portal fibrous tissue.69

Stromal framework detectable by reticulin staining (Gomori) is usu-ally well preserved in both regenerative and dysplastic nodules. It is decreased or lost in advanced HCC; however, in early well- differentiated HCC, the reticulin framework may be retained or only slightly decreased. An extensively retained reticulin framework is therefore evidence against malignancy; conversely, malignancy should be con-sidered whenever the reticulin framework is reduced, even if it is focal.

Diagnostic Issues and New ToolsLow-Grade Dysplastic Nodules versus Regenerative/High-Grade Dysplastic Nodules/Hepatocellular CarcinomaAlthough several parenchymal and nonparenchymal features are available to distinguish HGDNs and HCC from LGDNs, the latter share a number of features with regenerative nodules, making the distinction between the two lesions particularly difficult. A consen-sus panel, which met to evaluate interobserver agreement for diagno-sis of hepatocellular nodules, found that although there is no serious


33

A

B i

C iii

i ii

iv

ii iii

Figure 33-7. Nonparenchymal changes in a dysplastic nodule on liver biopsy. A, Two unpaired (nontriadal) arteries (arrows) without accompanying bile ducts. B, Progressive sinusoidal capillarization evidenced by CD34 expression from (i) low-grade dysplastic nodule (LGDN), to (ii) high-grade dysplastic nodule (HGDN), and to (iii) overt hepatocel-lular carcinoma (HCC) (CD34 immunostaining). c, This increasing capillarization mirrors what is seen in a surgically resected specimen: (i) LGDN, (ii) HGDN, (iii) HCC arising within an HGDN, and (iv) overt HCC (CD34 immunostaining). (B, Reprinted with permis-sion of Elsevier from Roncalli M, Borzio M, Di Tommaso L: Hepatocellular dysplastic nod-ules. Hepatol Res. 2007[suppl 2]:S125–S134; c, Reprinted with permission of Elsevier from Oh B-K, Chae KJ, Park C, et al: Telomere shortening and telomerase reactivation in dysplastic nodules of human hepatocarcinogenesis. J Hepatol. 2003;39:786–792.)

difficulty in differentiating LGDNs from early HCC, at least on resec-tion specimens, at the opposite end of the spectrum, the distinction between LGDNs and LRNs is often difficult or impossible.20 It is there-fore recommended that distinction between LGDNs and LRNs should not be attempted on a biopsy.

High-Grade Dysplastic Nodules versus Well-Differentiated Hepatocellular CarcinomaStromal InvasionThe presence of stromal invasion separates a well-differentiated HCC from an HGDN20; unfortunately, this histologic feature is not always present, especially on a biopsy. In the absence of stromal invasion, the distinction of well-differentiated HCC from HGDN is challenging because most histologic features useful in this distinction are present in both lesions, albeit to a variable extent: however, semiquantitative evaluation is not very helpful because no definite cutoff values have been validated for the various histologic parameters. Stromal invasion is defined as the presence of tumor cells in portal tracts or fibrous septa within or outside the lesion under examination (Fig. 33-8A and B). Stromal invasion needs to be distinguished from clusters of regener-ating hepatocytes that may be present in portal tracts or fibrous septa (see Fig. 33-8C). These can be distinguished by immunostaining for cyto keratins (CKs) 7 and 19, which highlight the presence of a ductu-lar reaction around reactive and regenerating clusters; a ductular reac-tion is absent around true stromal invasion (see Fig. 33-8D and E).70 Evaluation of stromal invasion is difficult in a biopsy because the latter samples only a minority of the tumor-nontumor interface and intral-esional portal tracts. In our experience with liver biopsies, a negative or focal (>10% of stromal-parenchymal interface) ductular reaction in a desmoplastic area adjacent to abnormal-looking hepatocytes suggests stromal invasion and a malignant nodule. In contrast, the presence of a CK7/19-positive ductular reaction around abnormal-looking hepato-cytes located in portal/periportal areas represents pseudoinvasion and is evidence against malignancy.

BiomarkersSeveral genetic and epigenetic events have been described in hepato-carcinogenesis (Fig. 33-9). Mutations of p53 and β-catenin, which seem to play a pivotal role in hepatocarcinogenesis, occur late in the dys-plasia-carcinoma sequence, being detectable only in established and well-recognized HCC. The two pathways are mutually exclusive; it has been suggested that p53 mutation mainly occurs in hepatitis B–related HCC and that β-catenin mutation is largely a feature of nonhepatitis B–related HCC.58 Mutations in p53 and β-catenin can be detected by nuclear positivity on immunohistochemistry, which provides confir-matory evidence of malignancy in doubtful cases. However, although β-catenin mutation has been shown to occur as a relatively early event in both well- and poorly differentiated HCC,71,72 p53 mutations are a feature of less-differentiated HCC.58,73

Other genetic changes such as loss of heterozygosity, chromosomal imbalance, telomere shortening, and telomerase reactivation,63 as well as epigenetic changes such as DNA methylation, occur at an earlier stage, being detectable in DNs, albeit at a level lower than in HCC. The dosage of these abnormalities has been proposed as a potentially help-ful diagnostic tool both in tissues and in sera, but cutoff levels have not been defined. It is likely that loss of heterozygosity, DNA methyla-tion, telomere shortening, and telomerase reactivation63 give rise to an abnormal molecular background favoring the emergence of preneo-plastic hepatocyte clones. It can thus be theorized that HCC arises from the progressive accumulation of these events or from abrupt mutations in a few critical carcinogenic genes such as p53 and β-catenin.

Because the number of abnormally regulated genes in the sequen-tial steps of hepatocarcinogenesis is expected to be much higher than that shown by genetic and epigenetic studies, the quest is on to identify sets of genes that may serve as fingerprints or signatures for premalig-nant nodules and early HCC (Fig. 33-10).74–78 Such studies have high-lighted the differential expression of heat shock protein 70 (HSP70)74

467


468

A

B

C

E

D

Figure 33-8. Stromal invasion by tumor cells. Invasive tumor nests within fibrous septa and portal tracts in surgical resection (A) and liver biopsy (B). Stromal invasion should be considered when isolated nests of abnormal-looking hepatocytes are noted within a portal tract or fibrous septa (arrows). Invasive tumor nests are not surrounded by a ductular reac-tion as shown in Fig. 33-8A (right) after CK7/19 immunostaining. (c) High-grade dysplastic nodule bordered by a florid CK7/19-positive ductular reaction. (D) Invasive tumor nests in liver biopsy without ductular reaction (CK7/19 immunostaining).(e) In this ambiguous biopsy field, ductular reaction is documented by CK7/19 immunostaining and is diagnostically helpful to exclude stromal invasion.

and glypican 376,77 in well-differentiated HCC compared with non-malignant nodules. Wurmbach and coworkers78 have also tentatively defined a set of genes peculiar to nonmalignant nodules and cirrhosis as opposed to malignant ones. Taken together, these data suggest that novel biomarkers can be used effectively to distinguish between well-differentiated malignant nodules and their precursors.

We have tested the diagnostic yield of glypican 3, HSP70, and glutamine synthetase in both surgical resection specimens and liver biopsy specimens. In surgically resected specimens,79 we have identi-fied two clinically relevant phenotypes. The first, with all three markers negative, is seen in 100% of LRNs/LGDNs, 73% of HGDNs, and 3% of well-differentiated HCC, including small and early forms. The second,

with all three markers positive, is a feature detected in less than 50% of well-differentiated HCC. Using this three-marker panel, the sensitiv-ity and specificity for HCC detection, when at least two markers were positive, were 72% and 100%, respectively. We are currently testing the diagnostic accuracy of these markers in a large set of liver biopsy spec-imens, and data accumulated so far indicate that LRNs and LGDNs do not stain for these three markers, whereas obvious moderate to poorly differentiated HCC show at least one or two immunoreactive mark-ers in 97% and 74% of the cases, respectively. One fourth to one fifth (22%) of HGDNs are positive for one marker (22%) but never for two or three. In well-differentiated HCC, two markers are detectable in 49% of the cases, with 100% specificity. Figure 33-11 summarizes the

469

33

Cirrhosis/regenerative nodule

Dysplastic nodule

LGDN HGDN

HCC

G1 G2–3

Chuma et al74

Paradis et al75

Nam et al76

Llovet et al77

Wurmbach et al78

GE

NE

SIG

NAT

UR

E

CH/LRN vs e-HCC:95 genes,

HSP70 selected

CH/LRN vs.HCC:

13 genes

LGDN vs.HGDN:

40 genes

e-HCC vs.HCC:

92 genes

HGDN vs.e-HCC:

40 genese-HCC vs.

G2–3 HCC:80 genes

DN vs. e-HCC:13 genes,

GPC3, LYVE-1,and survivin

selected

e-HCC vs.HCC:

9 genes

CH/LRN vs DN:24 genes,

DN: GREM2,EPO, and NRG1

selected

DN vs. e-HCC:93 genes,

e-HCC: ASPM,PRIM1, HMMR,

and IRAK1 selected

Figure 33-10. Gene expression profile in multistep hepatocarcinogenesis. CH, chronic hepatitis; DN, dysplastic nodule; EPO, erythropoietin; G1, well differentiated; G2–3, moderately–poorly differentiated; HCC, hepatocellular carcinoma; HGDN, high-grade dysplastic nodule; LGDN, low-grade dysplastic nodule; LRN, large regenerative nodule.

Figure 33-9. Genetic and epigenetic features in multistep carcinogenesis. G1, well differentiated; G2–3, moderately–poorly differentiated; HCC, hepatocellular carcinoma; HGDN, high-grade dysplastic nodule; LGDN, low-grade dysplastic nodule; LOH, loss of heterozygosity.

Cirrhosis/regenerative nodule

Dysplastic nodule

LGDN HGDN

HCC

G1 G2–3

MU

TAT

ION

S

GE

NE

TIC

S

DN

A m

ethy

latio

n

EP

IGE

NE

TIC

S

LOH37–40,48–51

p1 4ARF/p53 pathway40,50–53

Wnt/β-catenin pathway• p53 (up to 30% of HCC)

• β-catenin (up to 40% of HCC)40,54,55

• Axin (up to 10% of HCC)56,57

Other pathways• PIK3CA58,59

• TCF-158,60

• M6 p6/IG F2 R61

• Telomere shortening and telomerase reactivation12,62,63

APC, GSTP1, RASS F1 A, p 16,Cox-2, E-cadherin, etc.41,64–67


470

Negative forthree markers

Positive forone marker

Diagnostic significance of HSP70, GPC3, and GS in small andwell-differentiated hepatocellular nodules

Positive fortwo or three markers

Does not supportdiagnosis ofWD HCC

Suggestive ofHGDN

•

•

Suggestive ofWD HCC

HGDN cannot beruled out

•

•

Highly suggestiveof WD HCC

Does not supportdiagnosis ofHGDN

•

•

Figure 33-11. Diagnostic scheme for application of glypican 3 (GPC3), heat shock protein 70 (HSP70), and glutamine synthetase (GS) in the liver biopsy of small and well-differentiated hepatocellular nodules. HGDN, high-grade dysplastic nodule; WD HCC, well-differentiated hepatocellular carcinoma.

A

C

Figure 33-12. A, Well-differentiated hepatocellular carcinoma with steatosis showing immsynthetase (D).

immunohistochemical profile and diagnostic significance of glypican 3, HSP70, and glutamine synthetase in small and well-differentiated hepatocellular nodules as detected in a liver biopsy.

A panel of biomarkers, therefore, seems a very promising tool to be added to the pathologist’s armamentarium for the differential diagnosis of well-differentiated hepatocellular nodules in a liver biopsy. Specifically, positivity for at least two immunoreactive markers is specific and sensi-tive for diagnosis of HCC. Recently Abdul-Al and colleagues80 reported glypican 3 immunoreactivity in non-neoplastic hepatocytes in hepatitis C infection with high inflammatory activity. This finding underscores the importance of documenting intralesional marker overexpression, as compared to the adjacent cirrhotic liver, before drawing diagnostic con-clusions. Additionally, special care should be taken in the interpretation of immunocytochemical results when only one marker is positive. We believe that the application of a panel of makers of hepatocellular malig-nancy, while awaiting further validation, increases the diagnostic power of the liver biopsy for detection of small and/or well-differentiated HCC in daily practice (Fig. 33-12).

D

B

unohistochemical positivity for heat shock protein 70 (B), glypican 3 (c), and glutamine


33
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chapter 33 - premalignant hepatocellular lesions

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