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Ultrastructural Pathology

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Potential ultrastructure predicting factors forhepatocellular carcinoma in HCV infected patients

Soheir S. Mansy, Eman El-Ahwany, Soheir Mahmoud, Sara Hassan,Mohammed I. Seleem, Amr Abdelaal, Ahmed H. Helmy, Mona K. Zoheiry,Ahmed S. AbdelFattah & Moataz H Hassanein

To cite this article: Soheir S. Mansy, Eman El-Ahwany, Soheir Mahmoud, Sara Hassan,Mohammed I. Seleem, Amr Abdelaal, Ahmed H. Helmy, Mona K. Zoheiry, Ahmed S. AbdelFattah &Moataz H Hassanein (2017): Potential ultrastructure predicting factors for hepatocellular carcinomain HCV infected patients, Ultrastructural Pathology, DOI: 10.1080/01913123.2017.1316330

To link to this article: http://dx.doi.org/10.1080/01913123.2017.1316330

Published online: 11 May 2017.

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ORIGINAL ARTICLES

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCVinfected patientsSoheir S. Mansya, Eman El-Ahwanyb, Soheir Mahmoudc, Sara Hassana, Mohammed I. Seleemd, Amr Abdelaale,Ahmed H. Helmyf, Mona K. Zoheiryb, Ahmed S. AbdelFattahg, and Moataz H Hassaneing

aElectron Microscopy Research Department (Pathology), Theodor Bilharz Research Institute, Giza, Egypt; bImmunology Department, TheodorBilharz Research Institute, Giza, Egypt; cParasitology Department, Theodor Bilharz Research Institute, Giza, Egypt; dHepatobiliary surgery andliver Transplantation, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt; eSurgery Department, Faculty of Medicine,Ain Shams University, Cairo, Egypt; fSurgery Department, Theodor Bilharz Research Institute, Giza, Egypt; gHepatogastroenterologyDepartment, Theodor Bilharz Research Institute, Giza, Egypt

ABSTRACTHepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Althoughthe early diagnosis of HCC is vital for successful curative treatment, the majority of lesions arediagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study ofexperimentally gradually induced HCC, surgically resected HCC, and potential premalignantlesions from HCV-infected patients, with the prospect to detect cellular criteria denoting prema-lignant transformation. Among the main detected pathological changes which are postulated toprecede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmenta-tion, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of differentelectron density loaded with small sized rounded monotonous mitochondria, increase junctionalcomplexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellularproteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER),sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation oflipolysosomes, large autophagosomes, and micro-vesicular fat deposition.

In conclusion, the present work has visualized new hepatocytic division or regenerativeprocess that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, newobservations that may be of value or assist in predicting HCC and identifying the appropriatepatient for surveillance have been reported. Moreover, it has pointed to the possible malignantpotentiality of liver stem/progenitor cells.

For reliability, the results can be subjected to cohort longitudinal study.

ARTICLE HISTORYReceived 15 November 2016Accepted 3 April 2017Published online 12 May2017

KEYWORDSExperimental HCC; hepaticprogenitor cell; hepatocellu-lar carcinoma; liver regen-erative nodules; liver stem/progenitor cells; oval cell

Introduction

Hepatocellular carcinoma is an aggressive diseasewith poor outcome [1,2]. It is the fifth most diag-nosed cancer and the second cause of cancer deathin men all over the world [1,3]. Chronic hepatitisC virus (HCV) infection is considered a leadingrisk factor for hepatocellular carcinoma worldwide[4–7]. It accounts for 20% of all HCC [8]. Despitethe development of effective antiviral treatment, itis advised that treated patients with advancedfibrosis or cirrhosis undergo life-long screeningfor HCC [9–11]. Curative treatments are accessibleon condition that HCC is perceived early and aslong as the liver function is preserved [12,13].

Thus, population screening to identify patientswith cirrhosis and subsequently thorough surveil-lance for HCC are mandatory to decrease relatedmortality [3,14,15].

Nowadays, screening of hepatic nodules by radi-ologic imaging techniques reveals great progress.Meanwhile, the lesions that do not exhibit classicalimaging features still represent real challenge[16,17]. It was reported by Roncalli et al. [18]that imaging features of regenerative and dysplas-tic hepatocellular nodules between 0.5 and 1.5 cmare rarely diagnostic, and they are usually biopsied.

Many studies tackled the histopathological dif-ferentiation of hepatic nodules comprising largeregenerative nodule, low and high grade dysplastic

CONTACT Soheir Mansy soher.mansy@live.com, s.mansy@tbri.gov.eg Electron Microscopy Research Department, Theodor Bilharz Research Institute,ElNile St. Warrak-ElHadar Imbaba 12411, Giza, Egypt.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/iusp

ULTRASTRUCTURAL PATHOLOGYhttps://doi.org/10.1080/01913123.2017.1316330

© 2017 Taylor & Francis

lesions as well differentiated hepatocellular carci-noma [18–21]. Also, electron microscopic study ofHCC was undertaken by many authors in experi-mentally induced HCC [22,23] or in human biop-sies [24–27]. Meanwhile, no specifichistopathological criteria were postulated by thesestudies as a predictor of malignancy.

The aim of this work is to compare ultrastructuralpathological changes which occur in liver tissuesthroughout the development of experimentally gra-dually induced hepatocellular carcinoma in micewith the liver specimens of surgically resectedhuman hepatocellular carcinoma over-existingHCV; the corresponding surgical safety free tumorresection margin; chronic hepatitis C with nodularcirrhosis; and regenerative nodules. The emphasesare to identify at an ultrastructural level any mor-phological difference between the early stages ofexperimentally induced HCC and the pathologicalchanges occurring in human HCC and the potentialpremalignant studied lesions. In the attempt todepict criteria denoting or predicting malignanttransformation. Thus, we can identify the patientsat risk who should have a closer follow-up and wecan guaranty early diagnosis of malignancy with amore promising curative rate.

Materials and methods

The materials of this study consisted of 63 liver biop-sies processed for electron microscopic examination.The source of these liver biopsies was as follow:

1. Twenty ultrasound guided liver core biopsies

They were collected from patients withchronic hepatitis positive for serum HCVRNA. They were of stage F4 compensatinghepatic cirrhosis according to the METAVIRscoring system [28]. They were selected fromthe patients consecutively admitted to thehepato-gastroenterology department ofTheodor Bilharz Research Institute hospitalbetween the periods of December 2013 andJanuary 2015. The exclusion criteria of theinvolved patients were the presence of anyadditional cause of chronic liver diseases;following any regimen for anti-HCV, antifi-brotic or immunosuppressive therapy; thepresence of any concomitant general diseases

as rheumatoid arthritis, autoimmune diseaseor hepatocellular carcinoma. Liver cirrhosiswas confirmed on the basis of clinical exam-ination, computerized tomography (CT) scanand histopathological examination of theliver biopsies processed for light microscopicexamination. All the cases revealed cirrhoticnodules except that five cases showed criteriaof regenerative nodules. This differentiationwas done according to the InternationalWorking Party (IWP) criteria [29].

2. Twenty-eight specimens from the surgicallyresected HCC and the corresponding tumorfree surgical margin.They were harvested from 14 patients having

well circumscribed solitary tumor mass, under-going partial surgical liver resection. Thepatients had pre-existing HCV infection anddid not have any associating causes of chronicliver diseases, or general diseases. They were notexposed previously to antiviral, immunosup-pressive, or any HCC treatment.The liver specimens were harvested from the

tumor mass and the corresponding free tumorresection margin. The preoperative diagnosis ofHCC was based on triphasic multislice CT.While postoperative diagnosis of primary HCCand histopathological changes of free tumorresectionmargin depended on lightmicroscopicexamination of paraffin embedded resected liverspecimens. The included samples for electronmicroscopic examination were nine early welldifferentiated HCC < 2 cm in maximum dia-meter and five progressed HCC. They have tra-becular patterns with occasional pseudo acinarconfiguration. The specimens from the safetyfree tumor resection margin revealed criteria ofliver cirrhosis with often high grade dysplasticnodular changes (HGDN). Histopathologicaldiagnosis and classification of the tumor wereperformed according to the international con-sensus group for hepatocellular neoplasia [30](Figure 1A).

3. Twelve liver samples collected from threegroups of male albino mice Balb C injectedwith the hepatocarcinogenic reagent dimethyl-nitrosamine (DEN) [23,31].

Also, three liver samples from a normal controlgroup of three mice were included in this study.

2 S. S. MANSY ET AL.

A new experimental mice model of hepatocar-cinogenesis was accomplished by weekly intra-peritoneal injection of dimethylnitrosamine(DEN) at a dose of 50 mg/kg for 16 weeks. Agroup of four mice each was sacrificed at theinterval of 4, 8, and 16 weeks. Lesion progress upto the onset of HCC was inspected grossly andmicroscopically. The mice breeding and feedingwere maintained under standard conditions atthe Schistosome Biological Material SupplyCentre (SBSC) of the Theodor Bilharz ResearchInstitute (TBRI), Giza, Egypt. All manipulations

performed with the animals were in the frame ofthe ethical guidelines for care of laboratoryanimals.

Grossly the mice liver revealed congestion andenlargement at the fourth week from the begin-ning of DEN injection. The mice liver showeddiffuse nodular pattern at the 8th week of injec-tion. The nodules were about 1mm in diameter.Protruded gross was seen at the 16th week. It wasmore than 4 mm in diameter (Figure 1B). Lightmicroscopic examination of liver samples of 4weeks exposed mice revealed cellular hyperplasia

Figure 1A. a: Haematoxylin and eosin stained liver section 20× of HCV cirrhotic micronodule (head of arrow). Note the wellcircumscribed nodule by thick fibrous tissue of portal tract (arrow). −b: Haematoxylin and eosin stained liver section 20× of HCVregenerative nodule. Note, the peripheral compressed hepatocyte (arrow) and the fat infiltrated hepatocytes. −c and d:Haematoxylin and eosin stained liver section 20× of free tumour resection margin. Figure (c) Shows two large nodules. Theirborder are demarcated with bands of connective tissue. They reveal high grade dysplastic changes with autonomous small cellsproliferation, high cell density, compressed peripheral parenchyma (arrow), two to three cell thick plates and structural atypia. Figure(d) reveals small cells high grade dysplastic changes with irregularly increase in cells density and atypia. – e and f: Haematoxylin andeosin stained liver section of hepatocellular carcinoma trabecular pattern 20×. Figure (e) shows well differentiated HCC with thicktrabecular growth in which the cells pertaining the shape of hepatocytes with sinusoidal nuclear alignment. Figure (f) revealsprogressive HCC with cell of irregular size and shape, increased nuclear cytoplasmic ratio and mitotic activities.

ULTRASTRUCTURAL PATHOLOGY 3

with low grade dysplastic cellular foci. The 8 weekstreated mice showed high grade dysplasticnodules. Sixteen weeks exposed mice to DENrevealed HCC nodules (Figure 1C).

Specimens for light microscopic examinationwere fixed in 10% buffered formalin and processeduntil embedded in the paraffin block. Four μmthick sections were stained with hematoxylin andeosin and Masson trichrome stain. Specimens forelectron microscopic examination were dividedinto tiny pieces of 1 mm3 and fixed in 4% bufferedglutaraldehyde in 0.2 M cacodylate then washed inan equal volume of 0.3 M cacodylate with sucrose0.4 M. Samples were post fixed in 2% osmiumtetroxide in distilled water, washed in water thendehydrated in ascending ethyl alcohol concentra-tion, infiltrated, and embedded in Epoxy resinthen polymerized at 60 C°. Semithin sectionswere prepared from the polymerized gelatin cap-sule using Ultracut R ultramicrotome (Leica,Vienna, Austria). The midzonal liver area was thesite of choice for performing ultrathin sections.Semithin sections were stained with methylene

blue and Azur II. Ultrathin sections were con-trasted with uranyl acetate and lead citrate. Thesections were examined using Philips EM 208S atan accelerating voltage of 80 kW.

The experimental work and human samplescollection were conducted after approval ofTheodor Bilharz Research Institute Review Boardfor ethics (TBRI RB). Enrolled patients in thisstudy have been asked to write an informed con-sent before being subjected to any medical inter-vention. For animal manipulations, TBRI RBfollows the ethics approved by the EthicalCommittee of the Federal Legislation andNational Institutes of Health Guidelines inthe USA.

Results

The main histopathological characteristic of thedifferent studied experimental and human lesionsat the level of light microscope was summarized inthe form of the figure legend.

Ultrastructural morphological characteristics ofexperimentally induced HCC

The ultrathin liver sections prepared from miceexposed to DEN for 4 weeks revealed hepatocyteswith different electron density; hepatic stellate cellsloaded with fat droplets located intercellularly andin the sinusoidal area; hypertrophied rough endo-plasmic reticulum (RER) sequestrating mitochon-dria with fragmented and disoriented cristae.Some of these mitochondria underwent degenera-tion and liberated their cristae in the cytoplasm inthe form of small vesicles (Figure 2). The electrondense hepatocytes showed an increase in uniformsmall sized round mitochondria, enrichment inRER arranged in parallel arrays, an increase incytoplasmic proteinaceous material and free ribo-somes. The cells disclosing a decrease in electrondensity revealed excess fat deposition and few cel-lular organelles or signs of degenerative changes.Also, it was depicted in the examined sectionshepatocytic regenerative changes with the forma-tion of a cellular rosette, loss of hepatocytes poly-gonal configuration, nuclei with irregularlyindented membrane and prominent nucleoli.

Figure 1B. Photos of the normal and exposed liver to DEN. – A:control Normal liver. – B: Liver of the mouse treated with DENfor four weeks appears congested and enlarged. – C: Liver ofthe mouse treated with DEN for 8weeks shows diffuse hepaticnodules of about 1–2 mm in diameter. – D: Liver of the mousetreated with DEN for 16 weeks reveals multiple protrudingtumour mass.

4 S. S. MANSY ET AL.

The liver sections of mice exposed 8 weeks toDEN were characterized by frequent detection ofHering canals and inter hepatocytic bile ductuleswith undeveloped luminal microvilli. The bileduct-like cells forming the bile ductules or sharingin the formation of Hering canals with hepatocyteswere rich in cytoplasmic organelles. They hadelongated oval nuclei with irregular nuclear mem-brane and dispersed chromatin condensation incomparison with typical bile duct cells or cholan-giocytes. Oval cells were seen in close vicinity tobile ductules and Hering canals. They appeared

small oval cells of about 5 microns with poorlydeveloped cytoplasmic organelles. Their large ovalnuclei showed clumped heterochromatin. Bilecanaliculi, not occasionally as normal, werebound with more than three or more hepatocytes.Some revealed amorphous inclusion or cellularorganelles in their lumens. Desmosomes boundingthe bile canaliculi were increased. Intermediatehepatic cells were often seen within hepatocytescords or clusters. They appeared larger than theoval cell but smaller than the mature hepatocytes.Their nuclei were rich in euchromatin and their

Figure 1C. a and b: Liver sections of 4 weeks exposed mice to DEN injection show different degree of cellular hyperplasia. Note infigure b the irregularity in cellular cytoplasmic density and microacinar formation (arrow) (Haematoxylin and eosin 20×). –c and d:Liver sections of 8 weeks exposed mice to DEN injection reveal high grade dysplastic changes. Note the irregular hyperchromaticnuclei in figure c, and the clear nuclei with prominent nucleoli as well as intraparenchymal bile ductules (arrow) in figure d(Haematoxylin and eosin 20×). Both sections show crowded hypercellularity of irregular size small cell changes, with alternatingdifferent cellular density. –e and f: Liver sections of 16 weeks exposed mice to DEN injection reveal HCC of thick trabecular cell plateswith hyperchromatic irregular sized nuclei and increased vascularity (Haematoxylin and eosin 20×). With the progress of the lesion,note the turnover of haematoxylin and eosin stained hepatocytes from acidophilic hepatocytic cytoplasm seen in figure (a) tobasophilic cytoplasm which reaches its maximum density in figure (f).

ULTRASTRUCTURAL PATHOLOGY 5

cytoplasm disclosed more developed cell orga-nelles. They appeared less` electron dense in com-parison with mature hepatocytes. They mayconstitute one or more cells in bile canaliculiboundary. Lipolysosomes or liposomes were oftenseen in hepatocytes and occasional extravasatedRBCs were detected in the examined fields(Figure 3). Beside these previously mentionedhepatocytic morphological changes which charac-terized this stage of DEN exposure, it was depictedfoci of small sized hepatocytes or trabeculae ofmore than two cells thick. These cells disclosedan increase in nearly uniform sized mitochondria,intracytoplasmic proteinaceous material, and largebizarre shape nuclei with prominent nuclear pores.The nuclei had an irregular nuclear membranewith prominent marginated nucleoli or pseudo-nuclear inclusions.

The liver sections of mice sacrificed after beeninjected intraperitoneally weekly by 50 mg/kg ofDEN for 16 weeks were characterized by the detec-tion of newly formed unpaired arteries with theirpeculiar protruding endothelial lining, abortive

elastic lamina and smooth muscle layer; absenceof Hering canals; occasional disclosure of bile duc-tules in between hepatocyte clusters or hepatictrabeculae; active division of intermediate hepato-cytes and the visualization of star shape enucleatedcytoplasmic cellular masses having dendritic pro-jections. Moreover, nuclear changes were promi-nent with an increase in the nuclear-cytoplasmicratio. The nuclei showed indentation, occasionalpseudo-cytoplasmic inclusion, multiple margi-nated nucleoli, and prominent nuclear pores.Some pores had an unclear diaphragm. Thenuclear chromatin was formed mainly of euchro-matin with condensation of heterochromatin inthe form of clumps at a regular interval along theinner side of the nuclear membrane. The circum-ference of many nuclei was encircled by condensedfine granular proteinaceous material, free ribo-somes, microtubules, and microfilaments. Thehepatocytes showed hyperplastic hypertrophiedrough endoplasmic reticulum arranged parallel inclose contact with the increased uniform, nearlyrounded of equal size mitochondria. Sequestrated

Figure 2. Liver sections of four weeks exposed mouse to DEN. –A: The liver section shows Intercellular hepatic stellate cells (S)loaded with fat droplets and hepatocytes (H) with different electron density. –B and C: Electron micrographs reveal hepatocytes withincrease electron density more than twice that in the vicinity. The electron dense cells (asterisks) illustrate an increase in freeribosomes, proteinaceous material, small size mitochondria and rough endoplasmic reticulum. The electron lucent cells (H) ofmicrograph B shows degenerative changes While in C reveals fluffy diffuse fat deposition and few cell organelles. –D: Sequestratedmitochondria with hypertrophied rough endoplasmic reticulum (double arrow). Note the degenerated mitochondrion with theliberation of its content in the form of microvesicles (arrow).

6 S. S. MANSY ET AL.

mitochondria with RER were a common finding.Also, distended RER cisternae loaded with a pro-teinaceous material were seen liberated fromdegenerated cells. The hepatocytes did not main-tain their polygonal configuration and theyrevealed an increase in electron density. Theyshowed well-developed Golgi apparatus, anincrease in cytoplasmic proteinaceous material,autophagosomes, lipolysosomes, and fat deposi-tion mainly in the form of microvesicular droplets.Moreover, fat droplets infiltrated with microfila-ments or dissected with electron dense condensedcytoplasmic microfilaments were perceived in theexamined sections. The intercellular spacesbetween tumor cells were often filled with afibrin-like material. The detected sinusoidal spaceswere capillarized and showed hypertrophiedKupffer cells loaded with residual lysosomes(Figures 4 and 5).

Characteristic of surgically resected HCC

The examined ultrathin sections of surgicallyresected HCC revealed comparable ultrastructuralcellular criteria to what seen in the 16 weeksDEN-exposed mice. Small irregular sized hepato-cytes were arranged in trabeculae of more thantwo cells thick or forming pseudo acini. Thehepatocytes appeared either as clear electronlucent cells displaying minimal cellular organellesand diffuse fat deposition; or electron dense cellsshowing proliferated rounded small size mito-chondria with electron dense matrix, depositionof microvesicular fat droplets, increase cytoplas-mic proteinaceous material and hypertrophiedRER. Also, hepatocytes were configured as bizar-rely shaped cells revealing few cytoplasmic cellu-lar organelles and fat droplets traversed by afibrillary material. This later tumor cell config-uration was seen in progressive HCC. The tumor

Figure 3. Liver sections of eight weeks exposed mouse to DEN. –A: Bile ductule (B) seen in between hepatocytes (H), undergoingformation by the assembly of newly formed bile duct like cells rich in cytoplasmic organelles. Oval cell (O) is seen in the vicinity of itsbasement membrane. B: Canalicular ductular junction or Hering canal formed by the junction of hepatocytes (H) with the biliaryepithelial cell (B). The formed lumen (L) shows rudimentary microvilli. Note the oval cell (O) in close contact with the canal. –C: Bilecanaliculi bordered by hepatocytes (H) and intermediate hepatocyte of progenitor cell origin (IHP) showing oval nucleus perpendi-cularly oriented to the lumen (L) with increase in cell junctions at the margin of the lumen. –D: Hepatocyte (H) shows small sizemitochondria with sparse cristae, Hyperplastic disrupted rough endoplasmic reticulum studded with ribosomes, increase cytoplasmicproteinaceous material, liposomes or lipolyosomes (arrow), free ribosomes, nucleus (N) with prominent pores (curved arrow) inbetween there are heterochromatin clumps.

ULTRASTRUCTURAL PATHOLOGY 7

Figure 4. Liver sections of 16 weeks DEN exposed mice. –A: Active intermediate hepatocyte of progenitor cell origin (IHP) inbetween hepatocytes showing replicated centrosomes (arrow) for the beginning of mitotic cell cycle. –B: Intermediate hepaticprogenitor cells (IHP) with increase nuclear cytoplasmic ratio forming trabeculae of more than two cell thick. The nuclear chromatinis formed mainly of euchromatin. –C: The circumference of the nucleus is sequestrated with an area of fine granular proteinaceousmaterial, free ribosomes and microfilaments. The nuclear chromatin is formed mainly of euchromatin with regular heterochromatincondensation along the nuclear membrane demarcating nuclear pore. –D: Star shape cytoplasmic masses (M) with excess stuntedprocesses.

Figure 5. Electron micrograph of Liver sections of 16 weeks DEN exposed mice. –A: Newly formed small vessels (arrow). Theendothelial lining (E) protruding in the lumen mimic that of small arterioles. Meanwhile, the smooth muscle and elastic tissue arepoorly developed. –B:Interhepatocytic small arteriole in a formation phase (arrow). Note the oval progenitor cell (O) constituting theendothelial lining which protrudes intraluminal; the elastic tissue and smooth muscle cell are not clearly determined. –C: Fat droplets(L) infiltrated with microfilaments and dissected with electron dense condensed cytoplasmic microfilaments. –D: Distendedvesiculated rough endoplasmic reticulum loaded with proteinaceous material and microtubules (arrow).

8 S. S. MANSY ET AL.

cells revealed either monotonous, regular roundednuclei formed of euchromatin with or withoutsmall size nucleoli or bizarre shape large irregu-larly indented nuclei showing dispersed clumps ofheterochromatin with segregated and marginatednucleoli. The nuclei of binucleated cells lackedsimilarity. They were of different size and shape.Hepatocyte progenitor cells were often seen inbetween hepatocytes clusters or trabeculae. Aswell as constituent of the newly produced bileductules in the form of cholangiocyte like cells.Sinusoidal like space, extravasated RBCs,Unpaired arteries with abortive elastic internaand smooth muscle, cannibalistic cells weredetected in the examined sections (Figure 6).Apart from the previously mentioned morpholo-gical changes observed in HCC sections, whichwere as well distinguished at the level of lightmicroscopy, it was depicted in the examined sec-tions hepatocytes showing mouse mammarytumor virus-like in their cytoplasm. The laterappeared as sequestrated mitochondria by thecisternae of RER. The mitochondria showed

nearly homogenous electron dense matrix with-out cristae or revealed very few undeveloped cris-tae. The cisternae of RER showed depositedproteinaceous material at regular intervals alongthe outer membrane of the cisternae. Also, it wasdisclosed increase in junction complex adjoiningcell membrane along the canalicular and lateralsurfaces of adjacent hepatocytes and evenbetween cellular microvilli, pseudo nuclear inclu-sions, autophagosomes showing RER or mito-chondria, ribosomes studded vesicles, vesiclessequestering polyribosomes, degranulated RERvesicles, membrane-bound lipidic bodies or lipo-somes, detached cellular parts bounded by intactmembrane, star shape cytoplasmic masses withextended processes seen detached from hepaticprogenitor cell or malignant cells (Figure 7).

Characteristic of surgical safety free tumorresection margin

Prepared ultrathin sections from free tumor resectionmargin revealed foci of cellular changes comparable

Figure 6. Electron micrographs of liver sections demonstrating different morphological configuration of HCC. –A: group of small sizehepatocytes of early well differentiated HCC showing increase nuclear cytoplasmic ratio arranged around vascular space (arrow)giving the picture of pseudo acinar pattern. –B: Malignant cells of early well differentiated HCC showing increased intercellularjunction (arrow), nuclear chromatin consisted of euchromatin. –C: Progressive HCC showing enucleated cytoplasmic masses (M) withextended microvilli, mitochondria with electron dense matrix, many liposomes (arrow). –D: Group of small sized cells of progressiveHCC showing bizarre shape nuclei with condensed heterochromatin along nuclear membrane seems to be the result of anuncontrollable division of intermediate hepatocytes of progenitor cell origin.

ULTRASTRUCTURAL PATHOLOGY 9

to the cells of HCC sections but without the revelationof small bizarre shaped cells. They displayed abnormalarchitectural configuration with pseudoacinar forma-tion, thick liver cell plate more than three cells wide,and increase in the nuclear-cytoplasmic ratio. Heringcanal, bile ductules, and bile canaliculi were oftendetected in this category of studied liver samples.This is comparable to the findings of eight weeksDEN-exposed mice. Hepatic progenitor cells wereoften depicted between hepatocytes, or as a constitu-ent of bile canaliculi in the form of intermediatehepatocytes. Cholangiocyte-like cells formed intercel-lular bile ductules. Moreover, the examined sectionsrevealed an increase in desmosomes like structurealong the biliary pole of hepatocytes, frequent detec-tion of kupffer cells and cellular debris in capillarizedblood sinusoids, intracytoplasmic lipofuscin granules,residual lysosomes, and small RER vesicles notstudded with ribosomes, and neovascularizationseen in between the hepatocytic cords. Star shapecytoplasmic masses similar to that distinguished inthe ultrathin sections of surgically resected HCC and

experimentally induced HCC were distinguished inthe examined sections (Figure 8).

Electron microscopic examination of cirrhoticand regenerative nodules

Cirrhotic nodules revealed abnormal architecturalconfiguration. Hepatocytes were dissected byfibrous tissue or forming a rosette around vascularchannel or bile canaliculus. Cellular regenerationshowed autonomous cell proliferation. Bi-nucleated cell disclosed nearly similar nuclei inshape and size. Nuclei with prominent nucleoliwere depicted in most the examined hepatocytes.The mitochondria in cirrhotic nodules were ofirregular size and shape with electron densematrix. The RER showed hypertrophy withincreased proteinaceous material in its cisternae.It showed occasional condensation around thenuclei or in close relation to mitochondria. Thecytoplasm was occupied with diffuse fat depositionor large fat droplets. Tight junction is preserved

Figure 7. Electron micrographs of liver sections demonstrating some changes associating HCC. –A: Detached star shape cytoplasmic mass(M) with protruding microvilli from intermediate hepatocyte of progenitor cell (IHP) and Hepatic progenitor cell (HP). –B: Sequestratedmitochondria by rough endoplasmic reticulum. Themitochondria show very sparse cristae or devoid of cristae and the outer surface of RERreveals regular electron dense deposits (arrow) different from ribosomes, mimic the core and capsid of mouse mammary tumor virus. –C:Well demarcated Cytoplasmic cellular mass (arrow) shows multiple autophagic vacuoles containing electron dense filamentous andproteinaceous material. –D: Well-formed unpaired small artery showing peculiarly shaped endothelial cells.

10 S. S. MANSY ET AL.

between adjacent hepatocytes at the edge of bilecanaliculi. No Hering canal or intercellular bileductules were disclosed in the examined sections.(Figure 9).

The five cases diagnosed at the level of lightmicroscopy as regenerative nodules revealed apartfrom the previously reported changes seen in cirrho-tic nodules, they displayed star shape enucleatedcytoplasmic masses with processes seen mainlyalong the hepatocytes sinusoidal pole. They appeareddetached from viable hepatocytes with intact active

nuclei rich in euchromatin. These cells seem toundergo specific kind of primitive cell division orsplitting. These hepatocytes did not disclose anycriteria of apoptotic changes or necrosis. Smallsized regenerating cells arranged concentrically andlook like cholangiocytes were disclosed in betweenmature hepatocytes. Some of these cells were devoidof a nucleus. Also, hepatic stellate cells were fre-quently detected intercellularly or along blood sinu-soids meanwhile hepatic progenitor cells wereoccasionally disclosed (Figure 10).

Figure 8. Electron micrographs of liver sections demonstrating pathological changes of free tumor resection margin. –A: Bilecanaliculi bordered by more than two hepatocytes (arrow) showing an increase in junctional complexes at the lumen margin andalong the cell membranes interface (arrow head). –B: Canalicular-ductular junction or Hering canal lined by two intermediatehepatocytes (IHP) and two ductular epithelial like cells (DEC). –C: Small unpaired arteriole (arrow) with characteristic endothelial celland wall. –D: inter-hepatocytic hepatic progenitor cell (HP) constituting part of undergoing newly vessel formation. Note thecytoplasmic cellular projection (arrow) which encircle the progenitor cell (H: hepatocyte). –E: Star shape cytoplasmic masses (M) withstunted microvilli mimic the picture seen in experimental work. –F: Hepatocyte shows liposomes (arrow), sequestrated mitochondriawith RER and condensed rim of proteinaceous material around the nucleus.

ULTRASTRUCTURAL PATHOLOGY 11

Figure 9. A and B: Electron micrographs illustrating ultrastructural morphological changes in the cirrhotic liver. –A: Hepatocyteshows irregular sized mitochondria with electron dense matrix. The chromatin of the nucleus is formed of euchromatin. –B: Group ofhepatocytes, separated by fibrillary and proteinaceous deposit, show fat droplet deposition, the characteristic pyknotic mitochondriaof hepatitis. –C and D: Electron micrographs illustrating ultrastructural morphological changes of the regenerative nodule in cirrhoticliver. C: Hepatic stellate cell loaded with fat droplets. –D: Hepatocyte shows sequestrated nucleus with rough endoplasmic reticulum,small rounded mitochondria and hypertrophied rough endoplasmic reticulum studded with ribosomes.

Figure 10. Electron micrographs of liver sections of the regenerative nodule demonstrating the star shape cytoplasmic masses withextending processes. –A: cytoplasmic mass nearly taking star configuration andmany are seen in between hepatocytes. –B: Detached part(M) from a hepatocyte (H) forming bile canaliculus with the mother cell (curved arrow). Note the site of cleavage (arrow) (TEM: ×5600). –C:Liberated cytoplasmic star shape masses (M) with extended processes showing electron dense small round and rod shape inclusion mimicmitochondria (TEM: ×5600). –D: The hepatocyte split up into small pieces (arrow) showing electron dense mitochondria and have theability to produce small processes for mobility. Note the nucleus (double arrows) rich in euchromatin with intact nuclear membrane andprominent nucleolus, the mitochondria appear small in size and electron dense (TEM: ×4400).

12 S. S. MANSY ET AL.

The main histomorphological differentiatingfeatures of the studied lesions at the level of lightmicroscope and electron microscopy was summar-ized in Tables 1 and 2. Also, the main proposedcriteria for predicting malignant transformation orassisting in the diagnosis of inconclusive liverbiopsy examined at the level of the light micro-scope by the use of electron microscopy was illu-strated in Tables 2 and 3. The absent and thepresent morphological feature symbolized by −and + reported in the tables assist in the diagnosisof the corresponding lesion and its differentiationfrom the other studied lesions. The uncommonmorphologic feature symbolized by ± in the tablepoints to the criteria when present predicts malig-nant transformation, if it is equally mentioned ascriteria of malignant lesion.

Discussion

To our knowledge, this is the first ultrastructuralwork which tackles a comparative study betweengradually induced HCC in experimental micemodel versus human surgically resected HCCand potential premalignant nodular liver with thecoexistence of HCV infection. This was done withthe prospect, to deduce the changes that precedethe development of frank HCC and which canassist in its prediction. This in turn, can be of

Table 1. Main histomorphological differentiating features of thestudied lesions at the level of light microscope.

Parameter CN RN HGDN HCC

Light microscopicexamination

Trabecular cell platesmore than 3 cells thick

− − − +

Increase nuclearcytoplasmic ratio

− − ± +

Increase cell densitymore than twice thenormal surrounding

− − ± +

Mitosis ((1–5/10 HPF) − − ± +Cellular atypia − − + +Unpaired arteries − − ± +Ductural reaction + + + ±Absence of portal tract − − ± +Stromal invasion − − − +Peripheral compressedhepatocytes

− + + +

CN = cirrhotic nodule; RN = regenerative nodule; HGDN = high gradedysplastic nodule; HCC = Hepatocellular carcinoma.

±: may be present; +: commonly present; −: absent

Table 2. Main histomorphological differentiating features of thestudied lesions at the level of Electron microscopy.

Parameter CN RN HGDN HCC

Electronmicroscopicexamination

Trabecular cell platesmore than 3 cells thick

− − − +

Increase nuclearcytoplasmic ratio

− − ± +

Nuclear indentationand nuclearcytoplasmic inclusion

− − ± +

Cellular atypia − − + +Condensed RER aroundthe nucleus

− + + −

Alternating electronlucent and electrondense hepatocytes

− − + +

Increase cytoplasmicproteinaceous material

− − ± +

Hepatocyte packedwith small roundmonotonousmitochondria

− − − +

Hepatocytes showmitochondria ofdifferent size andshape

+ + + −

Hepatocyte progenitorcells

− ± + +

Actively dividingintermediatehepatocytes

− − − +

Bile canaliculi boundedwith more than threehepatocytes withincreased junctioncomplex

− − + +

Interhepatocytic bileductules

− − + ±

Diffuse cytoplasmic fatdeposition

+ + ± −

Microvesicular fatdroplets infiltrated bymicrofilaments

− − − +

Lipolysosomes(membrane boundlipidic material withcentrally locatedelectron dense deposit)

− − ± +

Hering canals formedwith cholangiocyte likecells

− − + −

Kupffer cells ± + + −Hepatic stellate cells ± + + −Star shape enucleatedcytoplasmic masses

− + + +

Frequentautophagosomes

− − ± +

Unpaired arteries − − ± +mouse mammarytumor virus-like

− − ± +

CN = cirrhotic nodule; RN = regenerative nodule; HGDN = high gradedysplastic nodule; HCC = Hepatocellular carcinoma.

±: may be present; +: commonly present; −: absent

ULTRASTRUCTURAL PATHOLOGY 13

value in the distinction between early HCC andHGDN which still represents an important histo-pathological challenge especially in liver biopsyrather than resected tissue [18].

In the present work, the reported main ultra-structural pathological changes characterizingHCC (Figures 6A, B, D, and 7D) and cirrhoticliver on top of HCV (Figures 9A and B) were inagreement with IWP guidelines [29] and the datareported by many other authors[18,19,22,24,25,32–39]. Also, the ultrastructurefindings of this study have substantiated that theprogress of liver toxicity induced byDimethylnitrosamine (DEN) to the developmentof HCC [31,40] elicits pathological changes com-parable to changes seen in human hepatocellularcarcinoma (Figures 4B and D; 5A, B, and D with6C and D; 7A, C and D) and liver tissue takenfrom the safety free tumor margin after resection(Figures 3A–D; 4D; 5A and B with 8A–C, E and

F). In addition, the present article highlightedsome new ultrastructural observations which mayrepresent pathological potential of HCCdevelopment.

The first remarked change in our experimentalmodel of mice exposed for 4 weeks to DEN wasthe alternating electron dense and electron lucentarea of liver parenchyma (Figures 2A–C) with thefrequent detection of hepatic stellate cells intercel-lularly (Figure 2A). This different electron densityof hepatocytes was reported as the morphologicalmalignant sign of liver nodules denoted equally atthe level of light microscope [18,19,20,25,40]. Inthe present work, the frequent detection of hepaticstellate cells in this early stage of experimentalHCC induction and in the examined regenerativenodules of HCV (Figure 9C) infected patients sup-ports the previously reported postulation of Yinet al. [41] that hepatic stellate cells have a pro-found impact in liver regeneration and cancer.Also, it was reported that mature hepatic stellatecells result in differentiation of liver stem/progeni-tor cells or oval cells into cholangiocytes[33,42,43]. Through either paracrine signalingpathways or cell–cell interaction [42–44]. Thiscan justify the frequent detection of newly formedHering canals and bile ductules in which cholan-giocytes like cells formed part of their constituentsin 8 weeks DEN-treated mice (Figures 3A and B)and the specimens of the free tumor surgical mar-gin (Figure 8B). Conversely, Human HCC and16weeks DEN-exposed mice revealed occasionalintercellular bile ductules but did not showHering canal in the examined sections. Thus, thispathological transition may represent a pathologi-cal potential for HCC development.

It is worth noting, that our results regarding thelocation of oval cells or hepatic progenitor cells inthe vicinity of Hering canals and bile ductules areconsistent with the observations made by [45–47]and support the statement that oval cells or hepaticprogenitor cells are the progeny of periductularstem cells [1,31,47,48].

By the 16th week of DEN exposure neovascu-larization and formation of unpaired or isolatedarteries were disclosed (Figures 5A and B). This isconsistent with the fact that malignant lesionsupon growth become hypervascular and unpairedarteries within liver nodule are a specific feature of

Table 3. Histopathological features which are considered bythis study transitional stage or predictor for malignant transfor-mation and recommend close patient follow up in inconclusivediagnostic biopsy at light microscopic examination.Relevant morphological criteria with high diagnostic value1 – Clonal fragmentation of hepatocytes or star shape enucleatedcytoplasmic mass

2 – Frequent detection of hepatic progenitor cell or dividingintermediate hepatocyte.

3 – Biliary tree changes:Hering canal showing cholangiocyte like cells.Bile ductule in between hepatocyte trabeculae.Bile canaliculi bordered with more than three hepatocytes orintermediate hepatocytes.

Increase short intercellular not well formed junctional complex.4 – Alternating electron light and electron dense hepatocytesshowing abundant proteinaceous material.

Subsidiary morphological criteria with moderate diagnosticvalue

1 – Packed hepatocytes with uniform monotonous round small sizemitochondria.

2 – Small round vesiculated degranulated RER filling the cellcytoplasm.

3 – Presence of cytoplasmic lipolysosomes, large autophagosomesand liberated large vesicles of RER distended with proteinaceousmaterial.

4 – loss of the diffuse appearance of fat deposition or demarcatedmicrovesicular fat deposit.

5 – sequestration of the nuclei by RER or proteinaceous materials.6 – Lack resemblance of nuclei of binucleated cells.7 – Increase tight junction between hepatocytic membranes andcellular microvilli.

8 – Presence of cytoplasmic structure that mimics mouse mammarytumor virus.

NeovascularizationUndeveloped parenchymal vessels.Unpaired arteries.

14 S. S. MANSY ET AL.

HCC [17,19]. Even, in the present work, theunpaired artery was displayed in three liver sam-ples of surgical free tumor margin of progressiveHCC (Figure 8C). Also, all liver specimen of freetumor surgical margin displayed foci showing cel-lular changes (Figure 8E) as in HCC specimens(Figures 6C and 7A) and the 16 weeks exposedmice to DEN (Figure 4D). This is in agreementwith Okusaka et al. [49], Sasaki et al. [50] andkudo [40] who reported the presence of micro-scopic foci of malignant cells at the edge of hepatictumor resection. Also, these findings reinforce thereported high recurrence rate of HCC which reach40–75% with 5-year survival rate of less than 10%[51,52]. Thus, patients with resected tumor mustpursue restricted follow-up regimen.

Oval cells or Hepatic progenitor cells weredepicted in the groups of mice treated with DENfor the induction of HCC, in liver specimens ofhuman HCC, surgically free tumor margin, andliver regenerative nodule. Moreover, activelydividing intermediate hepatic cells were depictedin 16 weeks experimentally exposed mice to DEN(Figure 4A). In the present work, hepatic progeni-tor cells or intermediate hepatocytes were notdepicted in the cirrhotic nodule of HCV infectedliver at the level of electron microscopy.Meanwhile, malignant intermediate hepatocytelike cells was seen in progressive human HCCspecimens (Figure 6D). Based on this, we canassume that the visualization of hepatic progenitorcells or the actively dividing intermediate hepaticcells into liver sections at the level of electronmicroscopy is considered predicting factors forthe progression to malignant transformation. Itwas reported that small cells in dysplastic fociwhich constitute the earliest premalignant lesionsin hepatocellular carcinoma are formed mainly ofprogenitor cells and intermediate hepatocytes [53].Moreover, many studies have drawn attention tothe possible involvement of Hepatic progenitorcells in the process of hepatic tumorigenesis.[42,51,53,54]. It is generally accepted that cancerarises from the malignant transformation of stemcells [55], and, tumors revealing hepatic progeni-tor cell structures have a poorer prognosis and ahigher recurrence rate versus tumors lacking thesefeatures [56,57]. In consequence, the frequentdetection of hepatic progenitor cells in liver

sections at the level of electron microscopy isconsidered by this study a transitional stagebetween benign and malignant microenvironmentinitiation predicting the swing to malignant trans-formation. Thus, Attention should be paid to thetumorigenic ability of hepatic stem progenitor cellsin the liver therapeutic field.

In the present work, the studied specimens ofregenerative nodules elucidated splitting or frag-mentation of some hepatocytes into cytoplasmicmasses (Figure 10D) taking nearly star shapeappearance with extended processes. These hepa-tocytes did not show signs of apoptotic changes ornecrosis. This may denote failure of hepatocyteregeneration as it is associated with the occasionaldetection of hepatic progenitor cells. It is reportedthat impairment of hepatocytes regeneration maytrigger the expansion of stem/progenitor cellscounterbalancing the inhibited regenerative abilityof mature hepatocytes [58,59]. This hepatocytefragmentation or splitting can be considered clonalfragmentation or kind of regeneration. This kindof primitive division or fragmentation looks likethe division which occur in starfish. In anotherword, each fragment can develop to produce anew cellular structure or live as in autotomy, liveindependently. Especially, the separated part hasthe ability to move freely and seems contain elec-tron dense mitochondria with replicated DNA.

Hence, based on the previously mentioned dis-cussion and reported cellular morphologicalchanges in this work there is increasing evidencethat some electron microscopic hepatocytic criteriacan be postulated by this study as predictor factorsfor malignant transformation in HCV-infectedpatients with cirrhosis. They can be summarizedinto four relevant and ten auxiliary or subsidiarycriteria that can be considered as factors whichmay postulate carcinogenic potential. The fourmore relevant criteria proposed by this study arethe detection of star shape cytoplasmic massesdetached from progenitor cells or hepatocyteswhich may designate the impairment of normalcell regeneration or division and the resort toalternate cell replication (Figures 10D and 8E);the frequent elucidation of hepatic progenitorcells and dividing intermediate hepatocytes likecell in the examined sections (Figure 8D); biliarytree changes in the form of the frequent detection

ULTRASTRUCTURAL PATHOLOGY 15

of Hering canal (Figure 8B), bile ductules inbetween hepatocyte clusters or trabeculae, andbordered bile canaliculi with more than threehepatocytes or intermediate hepatocyte-like cells,with increase intercellular junctions (Figure 8A);and different electron density of hepatocytes. Theten subsidiary morphological criteria were equallyobserved in the early stages of experimentallyinduced HCC, in human HCC and specimens offree tumor surgical margin. Meanwhile, they wereabsent in the specimens of cirrhotic nodules. Theycomprise packed hepatocytes with uniform mono-tonous round small size mitochondria with med-ium or deep electron dense matrix (Figures 2C and8A); small round vesiculated degranulated RERfilling the cell cytoplasm; formation of lipolyso-somes (Figures 3D and 8E); liberated cellularRER vesicles distended with proteinaceous mate-rial (Figure 5D); increase in autophagosomes(Figure 7C); dissected microvesicular demarcatedfat droplets showing or not microfilament(Figure 5c); sequestration of the nuclei by RER orproteinaceous material (Figure 4C and 9D); lacksimilarity between newly formed nuclei; increaseintercellular junctions between hepatocytic mem-branes or cellular projections (Figure 3C and 8A).It is worth noting, that these previously mentionedchanges were reported by Ghadially [60] as criteriaseen in tumor cells. The tenth postulated subsidi-ary criterion is the sequestration of mitochondriawith RER cisternae (Figure 7B). The mitochondriashowed electron dense homogenous matrix withabsent or abortive cristae. The RER revealed reg-ular proteinaceous deposit along the outer cister-nal membrane, mimic mouse mammary tumorvirus-like. This pathological figure was previouslyreported by Mansy et al. [61] in cases of HCV.They postulated its possible implication in theprocess of cellular transition to malignancy.

We believe that these assumed premalignantmorphological criteria should not be consideredapart from each other and be better evaluatedusing an initiated scoring system. This scoreincludes two groups: the four relevant and theten subsidiary observations reported in thisstudy. Each criterion in the first group is pre-sented by the figures 0 absent or 3 as presentand each item of the second group is presented

by the figures 0 absent or 1 if present. Theevaluation of these criteria grades 0–22.

In conclusion, the present study has shed lighton peculiar morphological hepatocytic alterationsas a potential predictor of HCC. These morpholo-gical changes would help understand mechanisti-cally what is happening in the process ofhepatocarcinogenesis, which seems for reliabilitybe subjected to cohort longitudinal studies. Also,the study has supported the previously reportedspeculation of the malignant potentiality of liverstem/progenitor cell and the impact of HSC onthis process. Additionally, this article has drawnattention to a new hepatocyte fate or division, thehepatocytes fragmentation which needs furtherdetailed study.

It is recommended that patients underwent sur-gical resected HCC or having a cirrhotic liver withregenerative nodules must be subjected to anappropriate surveillance. Importantly, if a biopsyis required, it must be subjected to light andadjunct electron microscopic examination withthe application of molecular and immunohisto-chemical tumor markers analysis. These collabora-tive methods may offer an important contributionto predicting malignant transformation and realizeaccurate diagnosis.

Author’s contribution

Soheir Mansy obtained funding of the project 8k,put the idea of the article and the design of thehuman part of the work, performed microscopicexamination and interpretation of the human andexperimental enrolled samples, analyzed, and dis-cussed the obtained results, wrote the manuscript.Eman El-Ahwany obtained funding of the project111T, put the idea and the design for the experi-mental animal part of the work. Sarah Hassanharvested and screened the specimens of humanHCC. Soheir Mahmoud injected and supervisedmice breeding. Mona Zoheiry contributed to thesupervision of the mice injection and breeding.Mohammed I Seleem, Amr Abdelaa, and AhmedH Helmy provided the work with resected HCCand patient‘s clinical data. Ahmed S. AbdelFattahand Moataz H Hassanein provided the work with

16 S. S. MANSY ET AL.

the core liver biopsies and patient’s clinical data.All authors approved the manuscript.

Declaration of interest

The authors who have taken part in this work declared thatthey do not have any conflict of interest in regarding thismanuscript.

Funding

The authors are grateful to the Theodor Bilharz ResearchInstitute for supporting the realization of this work by fund-ing the two projects 8k and 111T.

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