Need for normalization: the non-standard reference standard for microvascular invasion diagnosis in hepatocellular carcinoma
© The Author(s). 2018
Received: 11 October 2017
Accepted: 26 February 2018
Published: 7 March 2018
Preoperative microvascular invasion (MVI) assessment in hepatocellular carcinoma (HCC) is one of the current research focuses, with studies reporting controversial results regarding MVI-associated risk factors. As a possible source of bias, reported MVI rate (percentage of MVI-positive patients) varies a lot among studies. Pathological examination should have been the golden criteria of MVI diagnosis, but no standard and generally adopted pathological examination protocol exists.
Methods and results
It is highly possible that underestimated pathological diagnosis of MVI exists. We present two likely examples to stress the problem and indicate the root of the problem partially being an unreliable pathological examination. Results of studies basing on unreliable reference standard can be less convincing and even misleading, which is the most basic and fundamental problem in this research field.
There is an urgent need to settle the disputes regarding pathological sampling, microscopy, and reporting, in order to promote future academic exchange and consensus development on MVI assessment. Several concerns about pathological MVI assessment should be focused on in the future research as we put up in the review.
MVI refers to tumor invasion of vessels lined by endothelial cells, which substantially worsens the prognoses of HCC patients . Successful preoperative assessment of MVI may change how the patient is managed and improve survival . Factors reported to be associated with MVI risk in the previous studies are mixed and even controversial, tumor size for example [3–6]. As a possible source of bias, MVI rate varied significantly among studies, which is from 7.8 to 74.4% [3, 4]. The most frequently reported MVI-associated risk factors are alpha-fetoprotein (AFP), tumor size, tumor number, and histological differentiation [1, 4, 7, 8]. And preoperative anti-tumoral treatment such as trans-arterial-chemoembolization (TACE) or systemic chemotherapy may also have impact on MVI presence, which has not yet been thoroughly studied. All these factors considered, what would have caused the varied MVI rate?
Pathological examination is the reference standard for MVI diagnosis. However, in our experience, this gold standard is not always reliable in the clinical practice. The overall MVI rate of the retrospective and prospective database in our center is approximately 26 and 50%, respectively. And the previous negative results can also be altered by re-sampling of preserved surgical specimens, or intended for re-microscopy by a more experienced pathologist.
Kim et al.  revealed that MVI rate differed significantly between anatomical and un-anatomical surgical resection specimen. MVI was also reported to be detected more often in the site of tumor “protruding”  or capsule absence . Thus, intended sampling considering these aspects should make a difference in pathological MVI detection rate.
Most previous studies were observational and retrospective, in which MVI status could have been less routinely reported, let alone with specific staining methods and intended microscope inspection. Special immunohistochemistry is needed to differentiate vascular nature of which MVI invades, for example CD34 (vascular endothelium), smooth muscle α-actin (vascular smooth muscle), elastic fibers of vessel wall and D2–40 (lymphatic endothelium) [10, 13]. Iguchi et al.  revealed that MVI cell count of more than 50 and multiple-invaded vessels were indicators of poor prognosis. Relationship of tumor cells nests and the vascular wall was also reported to be associated with tumor recurrence and overall survival in patients with HCC after R0 liver resection . Thus, invaded vessel type, tumor cell count, invaded vessel count, and relationship of tumor cells nests and the vascular wall should be recorded intentionally, which was less often the routine procedure in clinical practice.
Comparisons between the two cohorts reported by Huang et al.
Age (year, median (range))
Liver cirrhosis (n/N)
BCLC stage (B/N)
AFP > 200 ng/dL (n/N)
Mean tumor size (cm, mean ± SD)
5.3 ± 3.5
5.5 ± 3.7
Patients with multiple tumors (n/N)
Tumor differentiation (III–IV/N)
Types of resection (anatomic/non-anatomic)
Comparisons between the two cohorts reported by Cucchetti et al. and Qiao et al.
Cohort in Cucchetti et al.’s
External validation cohort in Qiao et al.’s
Origin of patients
University of Bologna (1999 to 2008)
University of Bologna (2000 to 2011)
Age (years, mean ± SD)
62.8 ± 9.9
63.5 ± 9.4
Mean tumor size (cm, mean ± SD)
3.7 ± 1.8
3.9 ± 2.1
Moderate or poor tumor differentiation (n/N)
Patients with multiple tumors (n/N)
Liver cirrhosis (n/N)
CTP score (class C) (%)
AFP > 400 ng/ml (n/N)
It is highly possible that underestimated MVI rate does exist; if not, there should be factors impacting on MVI risk to an extent greater than any of the crucial factors already known. Otherwise, selection bias would have been a major limitation of related studies. We think the most convincing way to put this issue into a verdict, as a suggestion for future research, is to conduct studies on how much intended pathological sampling and microscopy can improve MVI detection rate, in an effort to develop a standardized pathological examination and reporting protocol.
Considering the diverse size, shape and location of HCC nodes, (1) Is MVI clinically important when tumor size or number exceeds a particular threshold (for example, 5 cm in diameter or 3 nodes)? (2) What is the optimal sampling distance from the tumor boundary: 1 cm or 2 cm? (3) Which section to choose for sampling: transversal or longitudinal? (4) Is multiple-section sampling necessary? If it is, what is the optimal number of sampling sections? and (5) Is particular site sampling decisive, for example, site of tumor “protruding” or capsule absence?
As for microscopy and reporting, (1) What is the optimal number of slices per block for inspection? (2) What vessels invaded by MVI are significantly associated with prognosis: hepatic vein branches, portal vein branches, hepatic artery branches, or bile ducts? (3) How to record invaded vessel count: sum or average count in a slice serious, or the one with the most vessels invaded? (4) How to record tumor cell account: count in one vessel section, sum count in the vessel of a slice serious, or average count by vessel sectional area? and (5) further inspection into the association of relationship between MVI cell nests and the vascular wall with tumor prognosis.
With the disputes of pathological MVI diagnosis settled, academic exchange between studies on preoperative MVI assessment would be practicable and clinically valuable, and related guideline and consensus development would be greatly promoted, in hope of better management of HCC patients and improving survival.
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HTH and ZW prepared the manuscript. Conception and manuscript revision was done by MK and WW. All authors read and approved the final manuscript.
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