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\begin{document}

\title{A Case of Poorly Differentiated Hepatocellular Carcinoma,
Lymphocyte-rich.}



\author[1]{Ginji Omori}%
\author[1]{Takahiro Osuga}%
\author[1]{Koji Miyanishi}%
\author[1]{Kota Hamaguchi}%
\author[1]{Shingo Tanaka}%
\author[1]{Hiroyuki Ohnuma}%
\author[1]{Kazuyuki Murase}%
\author[1]{Kohichi Takada}%
\author[1]{Minoru Nagayama}%
\author[1]{Yasutoshi Kimura}%
\author[1]{Ichiro Takemasa}%
\author[1]{Yasuhiro  Kikuchi}%
\author[1]{Toshihiko  Torigoe}%
\author[1]{Junji Kato}%
\affil[1]{Sapporo Medical University}%


\vspace{-1em}



  
  \date{May 12, 2024}


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\selectlanguage{english}
\begin{abstract}
A 70-year-old woman was diagnosed poorly differentiated hepatocellular
carcinoma (HCC), lymphocyte rich. In this case, programmed cell death 1
expression was observed. Immune checkpoint inhibitor treatment may be
effective in such cases, although there have been no reports of their
use for poorly differentiated HCC, lymphocyte-rich.%
\end{abstract}%



\sloppy


\emph{CASE REPORT}

A case of poorly differentiated hepatocellular carcinoma,
lymphocyte-rich.

Ginji Omori\textsuperscript{1}, Takahiro Osuga\textsuperscript{1}, Koji
Miyanishi\textsuperscript{1}*, Kota Hamaguchi\textsuperscript{1}, Shingo
Tanaka\textsuperscript{1,2}, Hiroyuki Ohnuma\textsuperscript{1},
Kazuyuki Murase\textsuperscript{1}, Kohichi Takada\textsuperscript{1},
Minoru Nagayama\textsuperscript{3}, Yasutoshi Kimura\textsuperscript{3},
Ichiro Takemasa\textsuperscript{3}, Yasuhiro Kikuchi\textsuperscript{4},
Toshihiko Torigoe\textsuperscript{4}, Junji Kato\textsuperscript{1}

\textsuperscript{1}Department of Medical Oncology, Sapporo Medical
University School of Medicine, Hokkaido, Japan

\textsuperscript{2}Department of Infection Control and Laboratory
Medicine, Sapporo Medical University School of Medicine, Hokkaido, Japan

\textsuperscript{3}Department of Surgery, Surgical Oncology and Science,
Sapporo Medical University School of Medicine, Hokkaido, Japan

\textsuperscript{4}Department of Pathology, Sapporo Medical University
School of Medicine, Hokkaido, Japan

\textsuperscript{*}To whom correspondence should be addressed.

E-mail: miyako@sapmed.ac.jp

\textbf{Contact Information}

Koji Miyanishi, M.D., Ph.D., Department of Medical Oncology, Sapporo
Medical University School of Medicine, South-1, West-16, Chuo-ku,
Sapporo, Hokkaido 060-8543, Japan.

TEL: (+81)-11-611-2111(ext.32540); FAX:(+81)-11-612-7987

e-mail: miyako@sapmed.ac.jp

\textbf{Abstract}

A 70-year-old woman was diagnosed poorly differentiated hepatocellular
carcinoma (HCC), lymphocyte rich. In this case, programmed cell death 1
expression was observed. Immune checkpoint inhibitor treatment may be
effective in such cases, although there have been no reports of their
use for poorly differentiated HCC, lymphocyte-rich.

\textbf{Key Clinical Message:} Pathological study in a case of poorly
differentiated hepatocellular carcinoma, lymphocyte-rich suggested that
immune checkpoint inhibitor may be effective. The histological type is
an important factor in determining treatment choices.

\textbf{Key words:} hepatocellular carcinoma, hepatocellular carcinoma,
lymphocyte rich, lymphoepithelial hepatocellular carcinoma, Immune
checkpoint inhibitors, programmed cell death ligand 1



\textbf{INTRODUCTION}

In the past, poorly/undifferentiated hepatocellular carcinoma (HCC) with
high lymphocytic infiltration was classified as
lymphoepithelioma-like-hepatocellular carcinoma
(LEL-HCC)\textsuperscript{1}. In the 2019 revision of the WHO
classification, it was incorporated into the lymphocyte-rich subtype,
regardless of differentiation\textsuperscript{2}. We experienced a case
of poorly differentiated hepatocellular carcinoma (HCC),
lymphocyte-rich, which corresponds to LEL-HCC in the previous
classification, and performed a pathological investigation.

\textbf{Case Report}

In 2019, a 70-year-old woman visited her local doctor with a complaint
of weight loss of 15 kg in a month. Abdominal ultrasound revealed a
liver tumor, and she was referred to our hospital. There was no history
of alcohol intake. Blood examination (Table 1) showed aspartate
aminotransferase elevation (AST; 66 U/L), alanine aminotransferase
elevation (ALT; 68 U/L), positive HBs antigen, HBV-DNA elevation (5.6
LogIU/mL), and negative hepatitis C virus antibody. Alfa-fetoprotein
(AFP) was markedly elevated (3946 ng/mL). Abdominal ultrasound revealed
a 22 x 20 mm mass with an internal mosaic pattern in segment 5 of the
liver (Figure 1). Angiographic computed tomography (AGCT) scan revealed
low absorption in CT during arterioportography (CTAP) and uniformly high
absorption in both early and delayed CT during hepatic arteriography
(CTHA) (Figure 2). EOB-MRI revealed a high signal in early phase, low
signal in hepatocellular phase, high signal in diffusion-weighted image,
and low signal in ADC (Figure 3). The lesion was solitary and there were
no findings suggestive of vascular invasion. Laparoscopic partial
hepatectomy was performed subsequent to the diagnosis of classical
hepatocellular carcinoma (HCC) UICC Stage Ib. Microscopically, there
were many infiltrating lymphocytes in the background, and tumor cells
with round to oval nuclei with prominent irregularities and abundant
eosinophilic sporophytes had grown in a plump, sheet-like pattern. Some
tubular structures corresponding to the moderately differentiated type
remained at the tumor margins; however, the majority of the cells were
the poorly differentiated type (Figure 4). Immunostaining of the tumor
cells was positive for glypian3 and negative for EBER. Programmed cell
death ligand 1 (PD-L1) was positive in about 40\% of tumor cells, and
inflammatory cells around the tumor were also partially positive,
showing scattered positive cells in the form of small clusters. The
infiltrating lymphocytes showed a mixture of CD3- and CD8- positive
cytotoxic T cells and CD4- and FOXP3-positive regulatory T cells (Figure
5-8). Twenty months have passed since hepatectomy and no recurrence has
been observed to date.

\textbf{Discussion}

Among undifferentiated cancers with abundant lymphocyte infiltration,
those derived from the head and neck region are called
lymphoepithelioma\textsuperscript{3}. Undifferentiated cancers with
abundant lymphocyte infiltration in organs other than the head and neck
are called lymphoepithelioma-like carcinomas (LELC), and in the WHO
classification 4th edition, LELC in the liver were classified as
lymphoepithelioma-like hepatocellular carcinomas
(LEL-HCC)\textsuperscript{1}. However, in the 2019 revision of the WHO
classification, HCC with abundant lymphocyte infiltration was included
in the classification of ''hepatocellular carcinoma, lymphocyte-rich''
regardless of the degree of differentiation\textsuperscript{2}. The
frequency of lymphocyte-rich HCC among all HCC cases is less than 1\%,
and it is reported that its prognosis is better than that of classical
HCC\textsuperscript{2,4}. Most cases of LELC in other organs are
associated with Epstein-Barr virus (EBV)\textsuperscript{5} Infection,
but most cases of HCC, lymphocyte-rich are negative for
EBV\textsuperscript{6}. We searched PubMed for articles from 1995 to
2019 using the keywords ''lymphoepithelioma-like hepatocellular
carcinoma'' or ''hepatocellular carcinoma lymphocyte rich''. As a
result, there were 58 cases corresponding to poorly differentiated HCC,
lymphocyte-rich (Table 2\textsuperscript{7-24}). We examined a total of
59 cases including the current case. The median age was 60 years; 27
cases were hepatitis B virus (HBV) positive and 18 were hepatitis C
virus (HCV) positive. EBV was negative in all but 1 case. Although there
are few reports on the proportion of infiltrating lymphocyte types,
there are many cases in which both CD4-positive cells, including
regulatory T cells, and CD8-positive cells, considered to be cytotoxic T
lymphocytes (CTLs), were present to the same extent. In this case as
well, CD4 and CD8 were equally positive. FoxP3 was positive in some of
the CD4-positive cells, indicating the presence of regulatory T cells.

In the IMbrave150 trial, the combination of the immune checkpoint
inhibitor (ICI) atezolizumab and the angiogenesis inhibitor bevacizumab
for unresectable HCC significantly prolonged overall survival compared
with sorafenib. As a result, this combination therapy has come to be
used clinically for unresectable HCC. The response rate for this
treatment in unresectable HCC is 27.3\%\textsuperscript{25}, which is a
good result, but it is still necessary to identify biomarkers for
predicting the effect of ICIs in terms of pharmacoeconomics and
personalized medicine. In addition, Chan et al. reported that there are
many mutations in the Wnt pathway, including CTNNB1, in normal HCC, as
well many mutations in CCND1 in LEL-HCC. These findings suggest that
LEL-HCC may have different tumor properties compared with normal
HCC\textsuperscript{26}. Therefore, we decided to histopathologically
assess the ICI treatment strategy for poorly differentiated HCC,
lymphocyte-rich. So far, there have been no reports of the use of ICIs
in poorly differentiated HCC, lymphocyte-rich, but there have been
multiple reports of cases in which ICIs were effective for LELC in other
organs\textsuperscript{27, 28}. In a study of 217 cases of HCC by
Calderaro et al., the PD-L1 positive rate (\textgreater{} 1\% of tumor
cell-positive cases were defined as PD-L1 positive) of LEL-HCC tumor
cells was 3 of 13 cases, which was not different from the positive rate
of 17\% for all HCC. However, the PD-L1 positive rate of surrounding
inflammatory cells was significantly high (\selectlanguage{english}PD-L1 expression by
inflammatory cells was classified as high if the number of PD-L1
positive clusters around the tumor was equal or superior to 6 {[}median
of the full series{]}) in 12 of 13 cases (92\%)\textsuperscript{29}. In
this case as well, multiple PD-L1-positive immune cell clumps were found
around the tumor (Fig. 8). It is known that ICIs induces an immune
response by inhibiting PD-L1 on tumor-related
macrophages\textsuperscript{30}, and ICIs may be effective in poorly
differentiated HCC, lymphocyte-rich. In the study by Calderaro et al.,
the analysis of the PD-L1 positive rate of LEL-HCC and tumor cells was
limited to only 13 cases. In the present case, 40\% of the tumor cells
were also PD-L1 positive. There are other reports\textsuperscript{7}that
have shown PD-L1 positivity in tumor cells and infiltrating lymphocytes
of poorly differentiated HCC, lymphocyte-rich, which is consistent with
our case. To better understand the tendency for PD-L1 expression in
tumor cells in poorly differentiated HCC, lymphocyte-rich cases, it is
necessary to accumulate more cases. Considering the aforementioned
details, it is likely that poorly differentiated HCC lymphocyte-rich,
which was once designated as LEL-HCC, can be expected to have a good
therapeutic response to ICIs, and these findings may contribute to the
stratification of HCC treatment in the future.

\textbf{Conclusion}

We experienced a rare case of poorly differentiated HCC,
lymphocyte-rich, which may be present in HCCs showing typical imaging
findings. For treatment of advanced cases, ICIs may be effective, and it
was considered necessary to confirm pathological findings when selecting
treatment.

\textbf{ACKNOWLEDGEMENTS}

All authors would like to thank the patient and his family for allowing
this case study.

\textbf{CONFLICT OF INTEREST}

The authors have no conflict of interests.

\textbf{AUTHOR CONTRIBUTIONS}

GO, TO and KM: collected and analyzed data and wrote and edited the
manuscript. KH, ST, HO, KT, KM and JK: involved in the patient's care.
MN, YK and IT: were abdominal surgeons providing hepatectomy. YK and TT:
were pathologists providing diagnosis.

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\textbf{Figure legends}

\textbf{Figure 1.} Abdominal ultrasonography images. The tumor was
present on segment 5 and showed a mosaic pattern (white arrow).

\textbf{Figure 2.} CT during angiography. The tumor showed low
absorption on CT during arterial portography (a) and high absorption on
CT during hepatic arteriography both in the early phase (b) and late
phase (c).

\textbf{Figure 3.} EOB-MRI images. The tumor showed a high signal on
early phase (a), low signal on T1-weighted hepatocyte phase (b), high
signal on diffusion-weighted imaging (c), and low signal on apparent
diffusion coefficient (d).

\textbf{Figure 4.} Histological findings in resected lymphocyte rich
hepatocellular carcinoma (loupe image, H-E stain). The enlarged image is
shown in the black frame. White line shows the border of the tumor and
liver background. H-E staining showed poorly differentiated tumor cells
with prominent nuclear irregularities and high degree of lymphocytic
infiltration in the background.

\textbf{Figure 5.} H-E staining and immunostaining images of tumor. H-E
(a), Glypican3 (b), Hepatocyte (c), EBV-encoded small RNA (EBER) (d).
Many infiltrating lymphocytes were found around tumor cells stained with
Glypican3 and Hepatocyte. EBER was negative.

\textbf{Figure 6.} Immunostaining images of infiltrating lymphocytes.
CD8 (a), CD4 (b), CD3 (c), CD20 (d). Both CD8 and CD4 were positive in
50\%. CD3 was positive in almost 100\% and CD20 was negative.

\textbf{Figure 7.} Immunostaining images. FOXP3 (a), Programmed cell
death 1 (PD-1) (b), Programmed cell death ligand 1 (PD-L1) (c). FOXP3
was weakly positive in CD4 positive cells. PD-1 was positive in almost
all lymphocytes. PD-L1 was positive in about 40\% of tumor cells.

\textbf{Figure 8.} Low-power field showing immunostaining of PD-L1 (a).
High-power field showing Glypican 3 immunostaining (b) and PD-L1 (c).
Multiple small foci of PD-L1 positive inflammatory cell clusters were
founded around Glypican 3-positive tumor cells (circles).

\textbf{Tables}

\textbf{Table 1.} Laboratory data on admission.

\textbf{Table 2.} The 59 cases of poorly differentiated HCC,
lymphocyte-rich\textbf{.}

We searched PubMed for articles from 1995 to 2019 using the keywords
''lymphoepithelioma-like hepatocellular carcinoma'' or ''hepatocellular
carcinoma lymphocyte rich''. As a result, 58 cases of poorly
differentiated HCC, lymphocyte-rich were identified. The table shows a
total of 59 cases, including the current case reported.

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