The gross findings of hepatocellular carcinoma are important for staging and for guiding sampling for sections. The gross description should comment on the number of tumors, their size(s), the margin status (distance to closest margin) the percent of tumor necrosis (estimated to the nearest 10%), and the presence or absence of gross vascular invasion.
Most hepatocellular carcinomas are soft and bulge out from the cut surface of the liver, a helpful finding when grossing in a cirrhotic liver explant. Hepatocellular carcinomas also frequently have different colors than the background liver. Some hepatocellular carcinomas have a capsule of inflamed fibrotic tissue.
Hepatocellular carcinomas can be further classified into one of four growth patterns on gross examination: (1) a single distinct tumor nodule; (2) a large dominant nodule and multiple smaller “satellite” nodules, usually within 2 cm of the dominant nodule, a pattern that results from local spread of the large dominant nodule; (3) multiple distinct tumor nodules that are sufficiently distant from each other that they would not fit for satellite tumors, a pattern that reflects a field effect, with foci of independent hepatocellular carcinomas; (4) numerous small tumor nodules that are about the same size as a cirrhotic nodule, usually with 30 or more nodules, a pattern called diffuse” or “cirrhotomimetic.” The tumor burden with this last pattern is always greater than recognized on imaging studies and often by gross examination, with the extent of tumor only evident with histologic examination.
As another gross finding, up to 4% of hepatocellular carcinomas are pedunculated, protruding from the surface of the liver.85
Pedunculated hepatocellular carcinomas can mimic metastatic disease to the adrenal gland on imaging studies,86
but the gross findings are otherwise typical of hepatocellular carcinoma. In addition, tumors with this growth pattern do not
have any unique histologic findings.87
Clinically, some studies have suggested pedunculated tumors have a better prognosis, but overall the data is limited and this growth pattern is not entirely convincing as an independent prognostic factor.85
The optimal number of sections needed to evaluate a hepatocellular carcinoma specimen has not been well defined, but the standard of care is generally regarded as at least one section per cm of tumor. Some pathologists put two or three or more mini-sections into a single cassette, cleverly counting them toward the total section count, an approach that does not always seem to be within the spirit of striving for the best possible patient care. Tumor grade and microscopic vascular invasion are key prognostic findings, ones that depend on reasonably thorough sampling. The tumor-nontumor interface should be well sampled, being high yield for identifying vascular invasion.
Sections from the background liver are used to examine for ongoing liver disease and to determine the fibrosis stage. Sections should be taken as far away from the hepatocellular carcinoma as possible because the liver near the edge of the tumor commonly shows significant inflammation and fibrosis. The surgical resection margin section is often taken separate from the background liver section because the surgical margin commonly shows cautery effect.
These are the four major histologic growth patterns found in hepatocellular carcinomas (Figs. 28.17
, and 28.20
: trabecular (70%), solid (also known as compact, 20%), pseudoglandular (also known as pseudoacinar, 10%), and macrotrabecular (1%). All are defined by the H&E findings without use of special stains. Growth patterns are not the same as hepatocellular carcinoma subtypes,
which are described in their own section below. In fact, any of the growth patterns can be found in any of the hepatocellular carcinoma subtypes.
Figure 28.16 Hepatocellular carcinoma, Mallory-Denk bodies.
Figure 28.17 Hepatocellular carcinoma, trabecular growth pattern.
Figure 28.18 Hepatocellular carcinoma, solid growth pattern.
Figure 28.19 Hepatocellular carcinoma, psuedoglandular growth pattern.
Figure 28.20 Hepatocellular carcinoma, macrotrabecular growth pattern.
The solid growth pattern is just like it sounds: solid sheets of cells with no definite trabecular, pseudoacinar, or macrotrabecular growth. The trabecular variant has trabeculae of variable thickness, but less than 10 cells. In contrast, the macrotrabecular pattern is defined by trabeculae at least 10 cells in thickness on average. In the pseudoglandular pattern, the tumor cells form small gland- or rosette-like structures. In some cases, the psuedoglands are considerably large, often being filled with thin, granular material, a pattern of growth sometimes called acinar or pseudocyst of follicle-like (Fig. 28.21
). Additional changes can accompany any of the above growth patterns. These include bile accumulation (Fig. 28.22
), peliosis-like areas (Fig. 28.23
), or clusters of benign macrophages in the tumor sinusoids (Fig. 28.24
Figure 28.21 Hepatocellular carcinoma, large pseudocyst.
Figure 28.22 Hepatocellular carcinoma with bile.
Figure 28.23 Hepatocellular carcinoma, peliosis-like changes.
Figure 28.24 Hepatocellular carcinoma, macrophages in sinusoids. Benign macrophages are seen within the tumor sinusoids.
Figure 28.25 Hepatocellular carcinoma, nodule within nodule. This hepatocellular carcinoma (top) had a distinct nodule within it that shows higher grade cytology (bottom).
The above patterns are straightforwardly defined, but not always so straightforward to apply. In some cases, compressed trabeculae can resemble a solid growth pattern. Likewise, the macrotrabecular pattern can also show areas of more compressed growth that resembles a solid growth pattern. In addition, about 50% of resection specimens have mixed patterns, most commonly trabecular plus one or two others. Finally, the results will vary, sometimes considerably, depending on the cutoff used to score a growth pattern as being present. Most studies use the reasonable cutoff of 5% but others use higher or lower cutoffs.
These growth patterns are significant for several reasons. First, familiarity with them can be useful when making a diagnosis of hepatocellular carcinoma. For example, recognizing the pseudoacinar growth pattern can help avoid a misdiagnosis of cholangiocarcinoma or combined hepatocellular-cholangiocarcinoma. Secondly, the macrotrabecular pattern has a worse prognosis when compared to the solid growth pattern.90
In particular, macrotrabecular hepatocellular carcinomas with small basophilic tumor cells are typically AFP-positive and have extensive angiolymphatic invasion.
Some hepatocellular carcinomas show a nodule-in-nodule growth pattern, with a main tumor mass showing one morphology and within it morphologically distinct nodule(s) with higher grade cytology that represent emergence of a more aggressive tumor clone (Fig. 28.25
). These higher grade nodules often show some morphologic similarities to the larger, lower grade nodule. In addition to this pattern, other hepatocellular carcinomas don’t show a dominant nodule with emergence of a higher grade nodule, but instead show multiple adjacent nodules that have distinctly different morphologies (Fig. 28.26
) often with the same overall cytologic grade, a finding of uncertain molecular genesis, but one that possibly reflects genomic instability.
Figure 28.26 Hepatocellular carcinoma, multiple morphologies. The nodules are adjacent to each other and show distinctly different morphologies.
Tumor grade is a strong predictor of overall patient survival and disease free survival. This prognostic power is seen after resections in cirrhotic livers,47
and after liver transplantation.47
Many (one-third) of hepatocellular carcinomas have two or more nuclear grades. In these cases, the predominant tumor grade and the worse tumor grade should be reported. The worse grade tends to drive prognosis.92
Tumor grade correlates with tumor size and with angiolymphatic invasion, but has independent prognostic value in most multivariate studies.
The modified Edmondson-Steiner grading system (Table 28.5
) is frequently used in research studies.89
In clinical practice, and many research studies, a threetier system is used: well differentiated, moderately differentiated, and poorly differentiated (Table 28.6
). In addition, some pathologists will use additional categories of very well differentiated and undifferentiated (Table 28.6
and Figs. 28.27
, and 28.31
Table 28.5 Modified Edmondson-Steiner grading system for hepatocellular carcinoma
Abundant cytoplasm; minimal nuclear atypia
Mild nuclear atypia with prominent nucleoli, hyperchromasia, and nuclear irregularity
Moderate nuclear atypia with greater hyperchromasia and nuclear irregularity
Marked nuclear pleomorphism, marked hyperchromasia, and anaplastic giant cells
Table 28.6 Grading hepatocellular carcinomas
Very well differentiated
Hepatic differentiation is readily evident on H&E
Mild nuclear atypia
N:C ratio is normal or increased
Additional stains needed to prove malignancy in most cases
Clearly malignant on H&E
Overall morphology strongly suggests hepatocellular origin
Moderate nuclear atypia
N:C ratio increased almost always
Stains are helpful to confirm hepatocyte differentiation
Clearly malignant on H&E
H&E morphology consistent with a range of poorly differentiated carcinomas
Marked nuclear atypia, or moderate nuclear atypia but minimal tumor cytoplasm with very high N:C ratio
Immunostains needed to confirm hepatocellular differentiation
No morphologic features of hepatic differentiation or glandular differentiation on the H&E
Stains are needed to prove the neoplasm is epithelial
H&E differential includes lymphoma, melanoma, or mesenchymal tumors
Keratin-positive, but keratin-negative for hepatic markers such as HepPar1, Arginase, and glypican 3
Metastatic disease excluded by immunostains, imaging, and clinical findings
Figure 28.27 Hepatocellular carcinoma, very well differentiated. This hepatocellular carcinoma (right, with normal liver on the left) cannot be distinguished from an adenoma without special stains.
Figure 28.28 Hepatocellular carcinoma, well differentiated. The tumor cells have plenty of cytoplasm, and there is only mild nuclear atypia. Note, however, that the tumor nuclei lack the regular, orderly spacing of benign proliferations.
Changes after chemoembolization therapy
Chemoembolization or ablation therapy is used to treat hepatocellular carcinomas localized to the liver, both as primary therapy in unresectable tumors and to shrink tumors, allowing surgery in previously unresectable tumors. Not surprisingly, chemoembolization or ablation therapy can affect both the gross and microscopic findings.
Treated tumors are staged in the same fashion as nontreated tumors. The percent necrosis should be estimated to the nearest 10% using the gross and/or histologic findings. The percent necrosis after TACE
ranges from 0% to 100%, with an average of 50% to 70% necrosis in those tumors with necrosis.46
About 30% of tumors are completely necrotic.46
Hepatocellular carcinomas with strong and diffuse CD34 staining and negative VEGF staining appear to be more resistant to TACE.95
There is no reliable way to distinguish necrosis that resulted from treatment versus spontaneous tumor necrosis and all necrosis is included in the estimate.
Figure 28.29 Hepatocellular carcinoma, moderately differentiated. The tumor morphology strongly suggests hepatic differentiation and the tumor is clearly malignant.
Figure 28.30 Hepatocellular carcinoma, poorly differentiated. There is striking nuclear atypia. Immunostains demonstrated hepatic differentiation. Imaging studies reported no other tumor sites.
Treatment can lead to other histologic changes, such as intratumoral inflammation and intratumoral fibrosis. Treated tumors are also more likely to express CK19 and have areas with biliary and/or spindled cell morphology.96
The potential effect of treatment on tumor grade has not been well studied, but one study found a higher tumor grade in TACE-treated hepatocellular carcinomas.98
This same study also found an increase in multinucleated tumor cells and cytoplasmic hyaline inclusions following treatment.98
Embolic beads can be found in the tumor and in the adjacent nontumor liver. Embolic beads can also escape the liver, leading to damage to other organs, in particular the stomach (Fig. 28.1
Figure 28.31 Hepatocellular carcinoma, poorly differentiated. This tumor has scant basophilic cytoplasm with large pleomorphic nuclei.
Immunohistochemical stains and special stains
Immunohistochemical stains are used to (1) decide if a well-differentiated hepatic tumor is benign or malignant (Table 28.7
) and (2) decide if a clearly malignant tumor is hepatocellular carcinoma (Table 28.8
The differential for a well-differentiated hepatic tumor depends in part on the background liver. In noncirrhotic livers, the differential is focal nodular hyperplasia, hepatic adenoma, and hepatocellular carcinoma. In a cirrhotic liver, the differential is focal nodular hyperplasia-like lesion, macroregenerative nodule, dysplastic nodule, and hepatocellular carcinoma. Of note, some nodules in cirrhotic livers can express CRP or SAA,99
but at this point the term hepatic adenoma for these nodules is hard to justify, both because it leads to unnecessary confusion for the clinical team and because there is no evidence that such lesions behave clinically like an adenoma.
A wide variety of immunostains can be used when evaluating poorly differentiated tumors. Some of the more commonly used stains are shown in Table 28.9
along with the frequency of positive staining in hepatocellular carcinomas.
Table 28.7 Stains used to distinguish benign from malignant hepatic lesions
To be helpful, staining should be significantly higher than background liver
Of note, not all hepatocellular carcinomas will have a higher proliferative rate
Not widely used as there are better stains
Strong diffuse staining is commonly but not always found in hepatocellular carcinoma. Also, some hepatic adenomas can show strong and diffuse CD34 staining
Positive in about 50% of well-differentiated hepatocellular carcinomas
Also stains lipofuschin and benign hepatocytes with significant inflammation
Focal staining also found in some dysplastic nodules
Universal rules governing the use of immunohistochemistry
Four fundamental, immutable laws govern the use of immunohistochemistry in tumors.
Law 1. All special stains should be interpreted in conjunction with the H&E findings. For example, if a tumor’s morphology is inconsistent with hepatocellular carcinoma, immunostain findings will not change that. Cross checking positive immunostains with the H&E is also important to avoid mistaking the positive staining of entrapped hepatocytes for positive staining of a tumor.
Law 2. The sensitivity and specificity of immunostains invariably get worse as more studies are published.
The first one is (almost) always the best, but the sensitivity and specificity will fall as more data accumulates. This is because the performances of stains depend on a variety of factors including those specific to laboratory methods and those that reflect biology, such as the strong correlation that can be seen between stain sensitivity and tumor grade, underlying liver disease, and/or tumor subtype. For these
reasons, most expert liver pathologists will choose from a panel of top performing stains because a wisely chosen panel of stains will help mitigate individual weaknesses of different immunostains.
Table 28.8 Stains used to demonstrate hepatocellular differentiation
Positive in 80%-90% of hepatocellular carcinomas
Performs poorly in well-differentiated hepatocellular carcinomas, where only 50% are positive
In situ hybridization for albumin
Polyclonal CEA (canalicular pattern)
Positive in 60%-80% of hepatocellular carcinomas
Canalicular staining indicates hepatic differentiation
Membranous or cytoplasmic staining is compatible with hepatocellular carcinoma but not specific
There are better stains as this one tends to be challenging to interpret in poorly differentiated carcinomas
CD10 (canalicular pattern)
Positive in about one-third of hepatocellular carcinomas
Can be useful in poorly differentiated tumors, which may lose expression of other markers such as HepPar and Arginase
None of these stains are 100% specific and they are best used in combination with clinical findings and H&E morphology
Table 28.9 Frequency of positive staining for nonhepatocellular markers in hepatocellular carcinoma
Positive tumors have a worse prognosis. Positive cases typically coexpress CK7
Hepatocellular carcinomas that are cholestatic or occur in younger individuals (<40) are positive in the majority of cases
Positive cases typically coexpress CK7
Stains for site of origin
Staining is often focal in hepatocellular carcinoma. Also, 20%-30% of cholangiocarcinomas are CDX2-positive
TTF1 can show cytoplasmic staining with some antibodies, but not nuclear staining
May be a marker of “stem cellness”
Staining is typically strong and cytoplasmic and can have a membranous accentuation
Usually patchy. Strong diffuse staining is seen in many cholangiocarcinomas
Stains used to subtype hepatic adenomas
Stain is positive in normal liver; lost staining is informative
Should be strong and fairly diffuse to be informative; some clones show considerable background staining
Can be very patchy
Should be strong and diffuse to be informative
Nonneoplastic hepatocytes are also positive. Membranous staining is seen in both tumor and nontumor
About 10% of conventional hepatocellular carcinomas are positive. Sarcomatoid hepatocellular carcinomas are positive
Law 3. If there is a discrepancy between the morphology and immunohistochemical findings, additional studies must be performed. Examples of additional studies include submitting more sections on resection specimens, repeating discrepant stains, and performing an additional round of immunostains that focuses on clarify the differential(s) raised by the discrepant findings.
Law 4. A difficult case is the wrong time to first use a stain with which you’re not familiar. The temptation can be very strong to use an unfamiliar stain in the hopes of getting yourself out of a diagnostic jam. However, this often leads to diagnostic misadventures. If you cannot resist temptation, at a minimum, the results of unfamiliar stains should be reviewed (not just verbally, but actually reviewed) by somebody with skill in interpreting the stain.
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