Histology of Hepatocellular Carcinoma: Association with Clinical Features, Radiological Findings, and Locoregional Therapy Outcomes

INTRODUCTION

Histological grades and cytological subtypes of hepatocellular carcinoma (HCC) have been established.^[1] Previous studies suggest that high tumor grade is associated with tumor recurrence and decreased survival after orthotopic liver transplantation (OLT) among patients within Milan criteria and higher local tumor progression rates after radiofrequency ablation (RFA) among Barcelona clinic liver cancer (BCLC) Stage A patients.^[2-4] High tumor grade on explant is also associated with lack of odds ratio (OR) to transcatheter arterial chemoembolization (TACE).^[5] In regard to cytological subtype, the clear-cell variant has been associated with improved survival after surgical resection^[6-8] though data conflicts.^[9,10] However, aforementioned studies predominantly include patients with early-stage disease and are not fully reflective of HCC populations treated with locoregional therapy (LRT). Further, tumor grade on explant after LRT may differ from before therapy. There is also a paucity of literature on the prognostic value of cytological subtype in patients treated with LRT. This study was undertaken to assess the relationship between baseline histological features of HCC and clinical stage, computed tomographic (CT) and magnetic resonance imaging (MRI) features, and outcomes in patients treated with LRT.

SUBJECTS AND METHODS

RESULTS

TTP and TFS by univariate and stratified analysis

Median TTP was 178 days (IQR 81–311 days), with overall disease progression rate of 59.8% with median follow-up of 183 days (range: 12–1799 days).

On univariate analysis [Table 4], tumor burden exceeding Milan criteria (hazard ratio [HR] 2.6, 95% CI 1.6–4.4; P < 0.001), baseline serum AFP level >50 ng/ml (HR 2.5, 95% CI 1.4–4.4; P = 0.002), BCLC Stage B (HR 3.0, 95% CI 1.6–5.7; P < 0.001), infiltrative appearance (HR 2.4, 95% CI 1.2–4.8; P = 0.01), and macrovascular invasion (HR 2.9, 95% CI 1.4–6.2; P = 0.006) were associated with disease progression. A trend was observed of a higher hazard of progression in tumors with microvascular invasion (HR 2.5, 95% CI 0.9–6.9; P = 0.08). High ES grade (HR 1.5, 95% CI 0.7–3.1; P = 0.33) [Figure 1a] and 100% clear-cell variant (HR 1.4, 95% CI 0.8–2.6; P = 0.28) [Figure 2a] were not associated with disease progression by univariate analysis. High ES grade or 100% clear cell also were not associated with progression when stratified by LRT modality or BCLC stage [Supplemental Table 1].

Table 4: Time to progression and transplant-free survival by Univariate analysis.

	TTP		TFS
	HR	Significance	HR	Significance
Age	0.97	P<0.05	1.0	P=0.79
Gender
Male	1.0		1.0
Female	0.7	P=0.18	1.0	P=0.93
Ethnicity
African-American	1.0		1.0
Caucasian	1.2	P=0.64	1.5	P=0.31
Other	0.7	P=0.35	0.9	P=0.78
DM
No	1.0		1.0
Yes	0.7	P=0.19	0.9	P=0.78
Cirrhosis etiology
HCV	1.0
HBV	1.5	P=0.45	0.7	P=0.68
ASH	0.8	P=0.52	0.5	P=0.33
NASH	0.4	P=0.11	0.6	P=0.42
Mixed	1.0	P=0.92	1.00	P=0.99
Other	1.6	P=0.36	0.7	P=0.59
Tumor diameter (cm)
≤5	1.0
>5	1.7	P=0.06	2.0	P<0.05
Within Milan criteria
Yes	1.0		1.0
No	2.6	P<0.001	3.1	P<0.001
AFP >50
No	1.0		1.0
Yes	2.5	P<0.005	1.4	P=0.36
ChildPugh class
A	1.0		1.0
B	1.5	P=0.14	1.3	P=0.41
C	2.1	P=0.13	2.9	P<0.05
BCLC stage
0/A	1.0		1.0
B	3.0	P<0.001	3.9	P<0.005
C	1.6	P=0.15	2.9	P<0.05
D	2.5	P=0.06	5.2	P<0.005
ES grade
Low	1.0		1.0
High	1.5	P=0.33	1.0	P=0.96
Architecture subtypes
Trabecular	1.0		1.0
Mixed trabecular	1.3	P=0.49	0.7	P=0.50
Pseudoglandular	1.0	P=0.89	1.3	P=0.55
Solid and mixed solid	1.2	P=0.64	1.5	P=0.37
Clearcell amount
None	1.0		1.0
Focal	1.0	P=0.91	1.0	P=0.97
100%	1.4	P=0.28	1.0	P=0.95
Microvascular invasion
No	1.0		1.0
Yes	2.5	P=0.08	1.6	P=0.47
Arterial phase hyperenhancement
Yes	1.0		1.0
No	0.7	P=0.19	1.1	P=0.75
Infiltrative HCC
No	1.0		1.0
Yes	2.4	P<0.05	1.7	P=0.30
Venous phase washout
No	1.0		1.0
Yes	1.6	P=0.18	1.5	P=0.41
Macrovascular invasion
No	1.0		1.0
Yes	2.9	P<0.01	3.0	P<0.05
Delayed capsular enhancement
No	1.0		1.0
Yes	1.1	P=0.81	0.7	P=0.37
OR after the first LRT
Yes	1.0		1.0
No	2.3	P<0.005	2.1	P<0.05
OR after all LRTs
Yes	1.0
No	2.7	P<0.001	2.4	P<0.05
Progression within 100 days
No	-		1.0
Yes	-	-	5.9	P<0.001

TTP: Time to progression, TFS: Transplantfree survival, HBV: HepatitiB virus, HCV: Hepatitis C virus, ASH: Alcoholic steatohepatitis, NASH: Nonalcoholic steatohepatitis, ethnicity other: Alaska native, Native Hawaiian, unavailable, cirrhosis etiology other: Primary, cryptogenic, sarcoidosis, DM: Diabetes mellitus, BCLC: Barcelona clinicliver cancer, ES grade: EdmondsonSteiner grade, LRT: Locoregional therapy, OR: Objective response

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Figure 1:

(a) Time to progression by Edmondson-Steiner (ES) grade after locoregional therapy. Kaplan–Meier curves of time to progression in patients with hepatocellular carcinoma of high ES grade (n = 13, median 118 days) or low ES grade (n = 93, median 182 days) treated with locoregional therapy. (b) Transplant-free survival (TFS) by ES grade after locoregional therapy. Kaplan–Meier curves of TFS in patients with hepatocellular carcinoma of high ES grade (n = 16, median 346 days) or low ES grade (n = 107, median 322 days) treated with locoregional therapy.

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Figure 2:

(a) Time to progression by clear-cell cytological composition. Kaplan–Meier curves of time to progression in patients with hepatocellular carcinoma (HCC) comprised 100% (n = 22, median 223 days), focal (n = 22, median 155 days), no (n = 55, median 178 days) clear cells treated with locoregional therapies. (b) Transplant-free survival by clear-cell cytological composition. Kaplan–Meier curves of TFS in patients with HCC comprised 100% (n = 24, median 350 days), focal (n = 23, median 429 days), no (n = 68, median 315 days) clear cells treated with locoregional therapies.

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Median TFS was 329 days (IQR 184–660 days). At the conclusion of the study period, 58 (47%) were alive, 40 (32%) were deceased, and 26 (21%) had undergone orthotopic liver transplant.

Decreased TFS was observed with baseline tumor diameter >5 cm (HR 2.0, 95% CI 1.0–3.8; P = 0.05), tumor burden exceeding Milan criteria (HR 3.1, 95% CI 1.6–6.0; P = 0.001), CP Class C (HR 2.9, 95% CI 1.1–7.6; P = 0.03), BCLC Stage B (HR 3.9, 95% CI 1.7–9.0; P = 0.002), Stage C (HR 2.9, 95% CI 1.2–6.9; P = 0.02), Stage D (HR 5.2, 95% CI 1.8–15.0; P = 0.002), and macrovascular invasion (HR 3.0, 95% CI 1.2–7.9; P = 0.02) [Table 4]. In addition, stable or progressive disease after LRT was associated with lower TFS (HR 2.4, 95% CI 1.2–4.9; P = 0.02).

High ES grade (HR 1.0, 95% CI 0.3–2.8, P = 0.96) and 100% clear-cell variant (HR 1.02, 95% CI 0.5–2.2, P = 0.95) were not associated with TFS on univariate analysis [Figures 1b and 2b]. High ES grade and 100% clear cell also were not associated with survival stratified by LRT modality or BCLC stage [Supplemental Table 1]. When stratified by BCLC stage, there was a trend toward longer TFS with 100% clear cell in comparison to no clear cell in BCLC Stage B patients (HR 3.2, 95% CI 0.9–11.5; P = 0.08).

DISCUSSION

The baseline histological features of HCC underlying clinical presentation, radiologic features, and clinical outcomes following LRT are not fully understood. Prior studies that have demonstrated an association between histological subtype of HCC on recurrence and survival after resection, RFA, and transplant predominantly represent early-stage disease and histological assessment on surgical resection specimens or explant.^[2-5] In this study, histological information was assessed on percutaneous biopsy before LRT, and most patients (61%) were BCLC Stage B or greater. High tumor grade was significantly associated with aggressive tumor features such as large tumor diameter, elevated baseline serum AFP, and infiltrative appearance. In addition, the clear-cell variant was significantly associated with non-viral etiologies of cirrhosis and atypical contrast enhancement patterns on CT or MR images. While these findings suggest that tumor grade and cytological subtype in part underlie differences in baseline clinical and imaging features, such conventional histological information on percutaneous biopsy did not impart additional prognostic information from tumor stage and high-risk imaging features.^[16]

High ES has been associated with lack of objective response after drug-eluting embolic TACE in a retrospective cohort study of 93 patients who subsequently underwent OLT; however, the majority of patients were early stage, and histological differentiation was determined after TACE on explant.^[5] In this study, no association was observed between high ES grade on percutaneous biopsy before LRT and objective response (OR 2.3, 95% CI 0.7–7.4; P = 0.15), suggesting that the predictive value of ES grade on post- therapy explant cannot be extrapolated to percutaneous biopsy specimens before LRT or patient populations with intermediate- or advanced-stage disease.

Poor histologic differentiation was associated with tumor diameter >5 cm, infiltrative appearance, and elevated serum AFP, supportive of prior studies. AFP is considered to be secreted by dedifferentiated HCC,^[17] and infiltrative appearance on imaging is known to be associated with higher AFP levels and worse survival after intra-arterial therapy.^[18] While tumor size, infiltrative appearance, and elevated AFP were associated with reduced TTP and TFS in this cohort, no such association was observed with histological features on univariate or stratified analysis. The predictive value of histologic differentiation on prognosis was limited by the fact that 70% of tumors with elevated AFP (>50 ng/ml), 75% of infiltrative HCC tumors, and 76% of tumors >5 cm were histologically low grade on percutaneous biopsy in this study. This is consistent with a retrospective study of 63 patients showing that tumor grade and microvascular invasion did not predict clinical response or overall survival after transarterial embolization or TACE in 50 patients with prior percutaneous core biopsy.^[19] While poor tumor differentiation has been associated with local tumor progression in a study of 95 BCLC Stage 0/A patients treated with RFA, these findings are confounded by high-grade tumors being larger than low-grade tumors in the cohort and more frequent use of internally cooled electrodes with a higher- powered generator in the low-grade group.^[4]

Clear-cell HCC is distinguished from other variants by clear cytoplasm attributed to increased glycogen or lipid contents secondary to metabolic changes.^[9] Such morphologic and metabolic changes may result from differential oncogenic process between chronic viral hepatitis and non-viral etiologies of cirrhosis such as alcohol and metabolic syndrome.^[20] Furthermore, clear-cell variant of HCC is known to have decreased number of intratumoral arteries in association with fatty changes; this is supportive of its association with atypical enhancement on CT or MRI in this study.^[21] A significant association was observed between clear-cell variants and the solid architectural subtype, but its biological and clinical significance remains to be determined in future study.

While poor tumor differentiation is associated with recurrence and poor survival after OLT or surgical resection,^[2,3] the impact of clear-cell HCC on prognosis is controversial: Some studies suggest that clear-cell variant is associated with longer overall survival,^[6-8] while other studies found similar survival between clear and non-clear- cell variant tumors after surgical resection.^[9,10] It is postulated that longer survival in clear-cell variant is associated with relatively low histological grade and capsule formation that are favorable for surgery.^[7] However, neither tumor grade nor clear-cell variant of HCC was associated with disease progression or TFS in our cohort after LRT.

Microvascular invasion on explant is strongly correlated with recurrence after OLT or surgical resection.^[22,23] In this study, a trend was observed toward a higher hazard of progression in tumors with microvascular invasion but not TFS. The lack of observed association in this study may be due to different methods to acquire HCC specimens: Percutaneous biopsy versus explant or surgical resection. In this cohort, only 13% of tumors were high ES grade and 9.1% demonstrated microvascular invasion on percutaneous core biopsy. In contrast, a study comprised 76% BCLC 0/A patients reported 53% were high ES grade and 51% had microvascular invasion on resection.^[24] Thus, the lack of observed association between ES grade and microvascular invasion in the current study with treatment response, TTP, and TFS may be attributable to the intrinsic limitation of percutaneous biopsy, where high-grade tumors are frequently misclassified as low grade due to sampling error of heterogeneous tumors. Prior studies have demonstrated that percutaneous biopsy underestimates tumor grade and microvascular invasion compared to resection.^[25]

This study demonstrates that histological analysis of tumor grade can stratify HCC given its association with aggressive clinical and radiologic features. However, this study also supports the current practice of deferring conventional histological analysis before LRT in tumors diagnosed by imaging criteria^[12] given the lack of independent prognostic information imparted by tumor grade and microvascular invasion on percutaneous biopsy. Although the prognostic value of conventional histological analysis before LRT may be limited, immunohistochemistry and genomic sequencing analysis of percutaneous biopsy specimens are under active investigation and percutaneous biopsy may yet prove critical to treatment allocation in the emerging era of precision medicine. Certain genetic mutations involved in Wnt/β- catenin and hypoxia stress response have been associated with objective response after transarterial embolization in primary and metastatic liver tumors.^[26] Furthermore, high expression of programmed death ligand-1 on resected HCC specimens is associated with poor tumor differentiation, elevated AFP, microvascular invasion,^[24] and poor disease- free survival and overall survival.^[27,28]

Our study bears several limitations. First, the retrospective single-institution design renders potential sampling bias in our cohort. Although the majority of tumors in this study demonstrated typical arterial-phase hyperenhancement and washout, a retrospective study of percutaneously biopsied HCC study will be inevitably enriched with HCC with non-diagnostic imaging features. Our cohort consisted of predominantly Caucasian and African-American patients with viral etiologies of HCC, and extrapolation of findings to other populations should be performed with caution. The relatively low number of patients with high ES grade (13%) in this cohort might have increased our susceptibility to a type II error. Similarly, while our study is unique in including BCLC B, C, and D patients, the variability in BCLC stage and LRT modality may have introduced confounding and reduced our ability to observe associations between histological features and outcomes. However, numerous tumor and clinical features, such as tumor size and BCLC stage, were significantly associated with TFS in this study; although a larger study may detect an association between histologic features on percutaneous biopsy on LRT outcomes, it is unlikely that histological features are superior prognostic indicators compared to macroscopic tumor features such as size and portal vein invasion, and clinical factors including BCLC stage. As discussed, studies utilizing percutaneous biopsy specimens are prone to histological misclassification bias due to non-representative tissue sampling of histologically heterogeneous tumors.

CONCLUSION

High histologic grade of HCC on percutaneous biopsy is associated with poor prognostic indicators such as larger tumors, infiltrative appearance, and advanced BCLC stage. The clear-cell variant of HCC was associated with non-viral cirrhosis, and the paucity of arteries in this subtype may underlie the atypical enhancement associated with these tumors. Percutaneous biopsy, therefore, may be used to diagnose HCC, but histological stratification did not provide independent prognostic information. The findings support deferral of percutaneous biopsy and histological assessment of HCC diagnosed by imaging criteria before LRT.