10.14639/0392-100X-N1450
Objective. Oral lichen planus (OLP) is a chronic inflammatory disease. There are no markers that can be used to identify the risk of a malignant transformation of OLP to oral squamous cell carcinoma (OSCC). Methods. Immunohistochemical staining was performed among 56 patients with OLP and 66 patients with OSCC for p53, HSP90 and E-cadherin expression and presence of HPV16/18. Results. Significant differences in p53 and HSP90 expression between OLP and OSCC were found (p = 0.01 and p = 0.006, respectively). A positive correlation between HSP90 and p53 expression was seen in OLP (p = 0.017). Univariate analysis identified HSP90 expression and HPV16/18 presence as prognostic factors for overall survival time (OS) (p &lt; 0.05). In multivariate analysis, only HSP90 expression was an independent prediction factor for shorter OS of OSCC patients (p = 0.016). Conclusions. The present study suggests that cooperation between p53 and HSP90 as well as between HPV16/18 and HSP90 exists in OLP and may affect the biological behaviour of OLP. The observed expression of HSP90 and p53 in OLP and their increase in OSCC suggests that these proteins participate in the malignant transformation of OLP. HSP90 may be a potential independent prognostic biomarker that can predict poor prognosis in OSCC.
text/xml
1827-675X
1450
en
Małgorzata Radwan-Oczko
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
immunohistochemistry
Comparison of p53, HSP90, E-cadherin and HPV in oral lichen planus and oral squamous cell carcinoma
Text.Serial.Journal
https://www.actaitalica.it/
41
Head and neck
2021-12-21
2021-02-03
2021-12-22
2021-12-22
514-522
1
Małgorzata Radwan-Oczko
2021/12/21
10.14639/0392-100X-N1450
514
Department of Oral Pathology, Wroclaw Medical University, Poland
1827-675X
immunohistochemistry
522
Comparison of p53, HSP90, E-cadherin and HPV in oral lichen planus and oral squamous cell carcinoma
https://www.actaitalica.it/article/download/1450/712
41
ACTA OTORHINOLARYNGOLOGICA ITALICA 2021;41:514-522; doi: 10.14639/0392-100X-N1450




Head and neck


Comparison of p53, HSP90, E-cadherin and HPV
in oral lichen planus and oral squamous cell carcinoma
Confronto di p53, HSP90, E-cadherin, e HPV nel lichen planus e nel carcinoma
squamocellulare del cavo orale
Julia K. Bar1, Piotr Cierpikowski1, Anna Lis-Nawara1, Paula Duc2, Agnieszka Hałoń3, Małgorzata Radwan-Oczko2
1
 Department of Immunopathology and Molecular Biology, Wroclaw Medical University, Poland; 2 Department of Oral Pathology,
Wroclaw Medical University, Poland; 3 Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University,
Poland
*J.K. Bar and P. Cierpikowski contributed equally to this work.


SUMMARY
Objective. Oral lichen planus (OLP) is a chronic inflammatory disease. There are no mark-
ers that can be used to identify the risk of a malignant transformation of OLP to oral squa-
mous cell carcinoma (OSCC).
Methods. Immunohistochemical staining was performed among 56 patients with OLP
and 66 patients with OSCC for p53, HSP90 and E-cadherin expression and presence of
HPV16/18.
Results. Significant differences in p53 and HSP90 expression between OLP and OSCC
were found (p = 0.01 and p = 0.006, respectively). A positive correlation between HSP90           Received: February 3, 2021
and p53 expression was seen in OLP (p = 0.017). Univariate analysis identified HSP90              Accepted: June 26, 2021
expression and HPV16/18 presence as prognostic factors for overall survival time (OS)
(p < 0.05). In multivariate analysis, only HSP90 expression was an independent prediction         Correspondence
factor for shorter OS of OSCC patients (p = 0.016).                                               Julia K. Bar
Conclusions. The present study suggests that cooperation between p53 and HSP90 as well            Department of Immunopathology and Molecular
as between HPV16/18 and HSP90 exists in OLP and may affect the biological behaviour               Biology, Wroclaw Medical University, Bujwida 44,
                                                                                                  50-345 Wroclaw, Poland
of OLP. The observed expression of HSP90 and p53 in OLP and their increase in OSCC
                                                                                                  E-mail: julia.bar@umed.wroc.pl
suggests that these proteins participate in the malignant transformation of OLP. HSP90 may
be a potential independent prognostic biomarker that can predict poor prognosis in OSCC.          Funding
KEY WORDS: OLP, OSCC, biomarkers, HPV, immunohistochemistry                                       The research work was supported by grants No
                                                                                                  ST.B132.17.0007 and No ST.B132.16.045 from the
                                                                                                  Wroclaw Medical University.
RIASSUNTO
Obiettivo. Il lichen planus orale (OLP) è una malattia cronica infiammatoria. Non ci sono         Conflict of interest
marcatori predittivi del rischio di trasformazione di OLP nel carcinoma a cellule squamose        The Authors declare no conflict of interest.
del cavo orale (OSCC).
Metodi. Con il metodo immunoistochimico è stata valutata la presenza di p53, HSP90, E-              How to cite this article: Bar JK, Cierpikowski P,
caderina, HPV in 56 pazienti affetti da OLP e 66 da OSCC.                                           Lis-Nawara A, et al. Comparison of p53, HSP90,
Risultati. Sono state rilevate le differenze, statisticamente significative, nell’occorrenza di     E-cadherin and HPV in oral lichen planus and
p53 e HSP90 nell’OLP e OSCC (p = 0,01; p = 0,006; rispettivamente). La correlazione                 oral squamous cell carcinoma. Acta Otorhi-
positiva tra l’occorrenza di HSP90 e p53 è stata notata in OLP (p = 0,017). L’analisi mo-           nolaryngol Ital 2021;41:514-522. https://doi.
novariata ha evidenziato che HSP90 e HPV costituiscono i fattori prognostici per il tempo           org/10.14639/0392-100X-N1450
di sopravvivenza dei malati (OS) di OSCC (p < 0,05). L’analisi multivariata ha evidenziato
che HSP90 è un fattore prognostico indipendente correlato ad una OS più breve negli OSCC          © Società Italiana di Otorinolaringoiatria
                                                                                                  e Chirurgia Cervico-Facciale
(p = 0,016).
Conclusioni. Questo studio suggerisce che esiste una correlazione fra interazione di p53 e
                                                                                                                 OPEN ACCESS
HSP90 e di HSP90 ed HPV ed il comportamento biologico di OLP. L’accertamento della
presenza di p53 e HSP90 in OLP e l’aumento della loro espressione in OSCC indica che              This is an open access article distributed in accordance with
                                                                                                  the CC-BY-NC-ND (Creative Commons Attribution-Non-
queste proteine possono partecipare al processo di trasformazione di OLP. I rapporti evi-         Commercial-NoDerivatives 4.0 International) license. The
denziati tra la presenza di HSP90 e il tempo di OS di OSCC indica che HSP90 è un fattore          article can be used by giving appropriate credit and mentio-
prognostico indipendente in OSCC.                                                                 ning the license, but only for non-commercial purposes and
                                                                                                  only in the original version. For further information: https://
PAROLE CHIAVE: OLP, OSCC, biomarcatori, HPV, immunoistochimica                                    creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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                                                                                                 Biomarker expression in OLP and OSCC




Introduction                                                        son to the normal epithelium tissue, suggests their poten-
                                                                    tial as prognostic biomarkers 8,14. Some researchers have
Oral lichen planus (OLP) is a chronic disorder that affects
                                                                    found a strong correlation between HPV16/18 infection
the oral mucosa of 1-2% of adult population 1,2. The le-            and oral premalignant lesions, particularly in OLP 15. How-
sions appear symmetrically and bilaterally, and are white           ever, epidemiologic studies on HPV infection in OSCC
or red-white in colour. There are five subtypes of OLP le-          and OLP have shown some discrepancies 5,16. The preva-
sions, i.e., reticular, plaque, atrophic, erosive, and bullous 3.   lence of HPV in OLP has been reported to range from 0.5
The aetiology and pathogenesis of OLP is still unclear and          to 2.5%, depending on the geographic location 16. The risk
has not been fully explained 3,4. There are conflicting data        of progression to malignancy was approximately 0.5% for
concerning the rate of OLP malignant transformation into            non-erosive OLP, compared to at least 3.5-4.0% for erosive
oral squamous cell carcinoma (OSCC), which is estimated             OLP over similar follow-up periods 16. Recent data suggest
at 0.4-5.0% over a four-to-five-year period 3,5,6. Several hy-      that HPV is a risk factor for OSCC development, especially
potheses have been proposed to explain the mechanism of             OSCC occurring in the oropharynx, which includes the base
malignant transformation of OLP 3,6. A potential mecha-             of the tongue and tonsils 17,18. To understand the biological
nism leading to the neoplastic transformation of OLP is as-         behaviour of OLP and its transformation into a malignant
sociated with the genetic mutation or overexpression of the         lesion, a comparative analysis of molecular changes in both
p53 protein, heat shock protein 90 (HSP90) and E-cadher-            OLP and OSCC is needed. As of today, there are no stud-
in 1,4,6-8. It was found that human papillomavirus 16 and 18        ies analysing the relationship between the presence of p53,
(HPV16/18) infection in OLP may increase the risk of the            E-cadherin, HSP90 and HPV in OLP and OSCC lesions to
malignant transformation to OSCC 5. There are no prog-              determine their impact on the possibility of an OLP malig-
nostic biomarkers that can identify OLP lesions with a high         nant transformation. We assumed that a comparison of p53,
risk of developing into malignant lesions 4. Some biomark-          HSP90 and E-cadherin expression and HPV16/18 presence
ers expressed in OSCC can also be observed in OLP 1,2,4,6,7.        in precancerous and cancerous lesions would allow us to
One such biomarker is the p53 protein, which plays an im-           discover their role in the risk of OSCC development.
portant role in cell cycle control and apoptosis 1,2,4. The loss    The study aimed to assess p53 protein, HSP90 and E-cad-
of the suppressive function of the p53 protein due to a mu-         herin expression and presence of HPV16/18 in two sub-
tation in the TP53 gene leads to abnormal cell proliferation,       types of OLP in comparison to their expression in OSCC to
resulting in the development of cancer 2,4,9. It was found that     reveal the role of these proteins in the progressive growth
p53 overexpression was associated with an increased risk            of OLP and OSCC as part of an analysis of their clinical
of OLP malignancy 1,3,9,10. E-cadherin and HSP90, which             behaviour.
are frequently observed in oral carcinoma, are also present
in the OLP tissue, but their role is unknown 11,12. E-cad-
herin is a transmembrane glycoprotein primarily found in            Materials and methods
human and animal epithelial cells; the mediated junctions           Patients
between cells play an important role in tumorigenesis and           Fifty-six patients with OLP and 66 patients with OSCC took
the invasion of tumor cells 10,12. Studies revealed that E-         part in the study between 2012 and 2017. Biopsy specimens
cadherin may be associated with the pathological features           of OLP and surgical specimens of OSCC were obtained
and biological behaviour of tumours 12. The downregula-             from the Department of Pathomorphology and Oncological
tion of mRNA E-cadherin levels in OSCC suggests that it             Cytology of the Wroclaw Medical University, Poland. Tis-
is likely to be an early factor in tumour growth 13. Only a         sue specimens were formalin-fixed and paraffin-embedded.
few studies concerning E-cadherin expression in OLP have            The OLP group consisted of 21 men and 35 women, whereas
been published 6,12. The focal loss of E-cadherin expression        the OSCC group consisted of 44 men and 22 women. OLP
in OLP lesions may increase their growth and cause malig-           biopsy specimens were verified histopathologically. The
nancy 8,12. HSP90 is a molecular chaperone that is crucial          fifty-six samples of two subtypes of OLP included 43 cases
for cell growth, communication, protein degradation and             of reticular OLP and 13 cases of erosive OLP. Five-micron
signal transduction 11. Some studies note that HSP90 may            sections from paraffin blocks were prepared and stained us-
be involved in the pathogenesis of the inflammatory pro-            ing haematoxylin-eosin. Microscopic slides were examined
cess, which can be observed in OLP 8,11. It was suggested           to verify the subtype of oral OLP (reticular and erosive). The
that HSP90 overexpression in OLP may be associated with             tissue specimens were classified as the reticular subtype, with
the persistence or chronicity of the disease 11. Significantly      clinical characteristics of Wickham striae without atrophic-
higher HSP90 expression in OSCC samples, in compari-                erosive areas and with specific histological features, e.g., the



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J.K. Bar et al.




hydropic degeneration of basal cells. The tissue samples were     Table I. Clinicopathological parameters of patients with oral lichen planus
classified as the erosive subtype, with the clinical character-   (OLP) and oral squamous cell carcinoma (OSCC).
istic of Wickham striae at the periphery with the presence of      Parameter                              OLP                    OSCC
                                                                                                       n = 56 (%)             n = 66 (%)
atrophic-erosive areas and apparent hydropic degeneration of
basal cells. Sixty-six tissue samples of primary OSCC col-         Age
lected from the tongue, the floor of the mouth, and buccal            < 60 years                        17 (30.4)             24 (36.4)
mucosa were included in the study. Tissue specimens from              ≥ 60 years                        39 (69.6)             42 (63.6)
the primary OSCC were histologically verified to confirm the       Gender
diagnosis, histological type and tumour grade. The clinico-           Female                            35 (62.5)             22 (33.3)
pathological features of patients with OLP and patients with          Male                              21 (37.5)             44 (66.7)
OSCC are presented in Table I. Approval of the Ethics Com-         Duration
mittee of Wroclaw Medical University to perform this study            ≤ 37 months                       37 (66.0)                  -
was obtained (decision number KB230/2016).                            > 37 months                       19 (34.0)                  -
                                                                   Clinical type
Antibodies                                                            Reticular                         43 (76.8)                  -
Immunohistochemical staining was performed using the                  Erosive                           13 (23.2)                  -
following antibodies: mouse monoclonal antibody DO-7               Tumour grade
(clone DO-7), which reacts with both wild- and mutant-                G1                                    -                 23 (34.9)
type of the unphosphorylated human p53 protein (Novo-                 G2                                    -                 35 (53.0)
castra, Newcastle, UK), anti-HSP90 protein which rec-                 G3                                    -                 8 (12.1)
ognises the protein corresponding to the 306 amino acids
                                                                   Tumour site
of the C-terminus of the HSP90 molecule (clone JPB24,
                                                                      Tongue                                -                 30 (45.5)
Novocastra), anti-human cadherin (clone NCH-38) which
                                                                      Floor of the mouth                    -                 18 (27.3)
recognises the 120 kD mature form of E-cadherin (Dako,
                                                                      Buccal mucosa                     27 (48.2)             18 (27.3)
Copenhagen, Denmark) and anti-HPV16 E6 + HVP18 E6
                                                                      General involvement               25 (44.6)                 -
[C1P5] which reacts with the E6 protein (Abcam, USA).
                                                                      Other                              4 (7.2)                  -
Immunohistochemical staining (IHC)                                 T classification
Proteins were stained immunohistochemically on paraf-                 T1/T2                                 -                 29 (43.9)
fin-embedded OLP and OSCC tissue specimens using the                  T3/T4                                 -                 37 (56.1)
Universal Dako REAL EnVision Detection System, Per-                N classification
oxidase/DAB+, Rabbit/Mouse (Dako, Copenhagen, Den-                    N0                                    -                 37 (56.1)
mark) according to the manufacturer’s instructions. OLP               N1/N2/N3                              -                 29 (43.9)
and OSCC specimens were incubated with primary anti-               Clinical stage
bodies overnight at 4°C. After washing of specimens with              I/II                                  -                 21 (32.0)
0.1 M Tris buffer, pH = 7.4 (TBS), they were incubated                III/IV                                -                 45 (68.0)
with Dako REAL EnVision/HRP, Rabbit/Mouse (Dako)                   Survival
for 30 minutes at room temperature. The antigen-antibody              Alive                                 -                 20 (30.0)
reaction was visualised with DAB (3,3-diaminobenzidine)               Dead                                  -                 46 (70.0)
(Dako) as a chromogen. The sections were counterstained
with haematoxylin and mounted. The incubation buffer
(TBS) without the primary antibodies was used as a nega-          sessed by counting 1000 cells in 10-15 randomly selected
tive control. Positive controls for each antibody were per-
                                                                  high-power fields. The proportion of p53 and HPV16/18
formed according to the manufacturer’s recommendations.
                                                                  positive cells in tissue was assessed as follows: 0 = (0-
Interpretation of immunohistochemical staining                    10%), 1 = (> 10-30%), 2 = (31-50%), 3 = (> 50%).
Protein expression in OLP and OSCC tissues was assessed           E-cadherin and HSP90 expression was analysed based on
semiquantitatively, taking into account the intensity of im-      the intensity of membrane or cytoplasmic immunostaining
munostaining and the number of cells showing immuno-              and the percentage of stained tumour cells. The frequency
reactivity for the analysed proteins. The number of cells         of E-cadherin and HSP90 positive cells in tissue areas was
exhibiting staining for p53 protein and HPV16/18 was as-          scored as follows: 0% (no reaction-10%), 1 = (> 10-25%),



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2 = (26-60%), 3 = (> 61%). The colour of the IHC stain                                             surgery until death of patients with OSCC. Cox-regression
was used to evaluate its intensity as follows: negative (-, no                                     analyses were used to identify prognostic factors for OS-
colour), weak (+, light brown colour), moderate (++, dark                                          CC patients. Differences were considered significant for
brown colour), and strong (+++, very dark brown colour).                                           p < 0.05. Statistical analysis was performed using the STA-
In cases where no immunostaining or variable weak posi-                                            TISTICA v13.0 software (Statsoft, Krakow, Poland).
tivity occurred, negative scores were given for all protein
expressions (< 10% of cells).                                                                      Results
Statistical analysis                                                                               HSP90, p53 protein and E-cadherin expression and HPV
In all statistical analyses, p53, HPV16/18, E-cadherin and                                         16/18 infection in OLP and OSCC
HSP90 immunoreactivity was divided into two groups:                                                As shown in Table II, in OLP specimens HSP90, p53 pro-
negative or limited to 10% of tumour cells versus > 10%                                            tein and E-cadherin expression and HPV type 16/18 were
of positive cells. Associations between p53, HSP90, E-cad-                                         observed in 28/56 (50.0%), 18/56 (32.1%), 35/56 (62.5%)
herin, and HPV16/18 expression and clinicopathological                                             and 4/56 (7.1%) cases, respectively. A higher number of
parameters were evaluated using the Chi2, Mann-Whitney,                                            cells stained for p53 and HSP90 was observed in erosive
and Kruskal-Wallis tests. Correlations between proteins                                            OLP (expression of p53, 20-50%, and HSP90, 20-70%,
were analysed using Spearman’s rank correlation. Overall                                           of the positive tissue) compared to non-erosive OLP (ex-
survival (OS) was defined as the number of months from                                             pression of p53, 10-15%, HSP90, 10-50%, of the posi-


Table II. Correlation between clinicopathological parameters and immunoreactivity for HSP90, p53, E-cadherin and HPV in oral lichen planus.
                           HSP90, n (%)                         p53, n (%)                    E-cadherin, n (%)                   HPV16/18, n (%)
      Parameter          Negative       Positive      p-value Negative            Positive      p-value Negative           Positive     p-value Negative      Positive   p-value
    Age
      < 60 years             9              8          0.771a          13             4         0.362a           6               11     0.822 a      17           0      0.171a
                           (52.9)         (47.1)                     (76.5)        (23.5)                     (35.3)           (64.7)             (100.0)       (0.0)
       ≥ 60 years            19             20                         25            14                         15               24                  35           4
                           (48.7)         (51.3)                     (64.1)        (35.9)                     (38.5)           (61.5)              (89.7)      (10.3)
    Gender
      Female                 15             20         0.084b          21            14         0.104a          16               19     0.036b       31           4      0.108a
                           (42.9)         (57.1)                     (60.0)        (40.0)                     (45.7)           (54.2)              (88.6)      (11.4)
       Male                  13              8                         17            4                          5                16                  21           0
                           (61.9)         (38.9)                     (81.0)        (19.0)                     (23.8)           (76.2)             (100.0)       (0.0)
    Duration
      ≤ 37 months            23             14         0.011a          26            11         0.589a          18               19     0.016a       36           1      0.072a
                           (62.2)         (37.8)                     (70.3)        (29.7)                     (48.6)           (51.4)              (97.3)       (2.7)
       > 37 months            5             14                         12            7                          3                16                  16           3
                           (26.3)         (73.7)                     (63.2)        (36.8)                     (15.8)           (84.2)              (84.2)      (15.8)
    Tumour site
      Buccal                 12             15         0.503a          22             5         0.025c          13               14     0.282a       26           1      0.432a
      mucosa               (44.4)         (55.6)                     (81.5)        (18.5)                     (48.1)           (51.9)              (96.3)       (3.7)
      General                13             12                         13            12                         7                18                  22           3
      involvement          (52.0)         (48.0)                     (52.0)        (48.0)                     (28.0)           (72.0)              (88.0)      (12.0)
      Other                   3              1                         3             1                          1                3                   4            0
                           (75.0)         (25.0)                     (75.0)        (25.0)                     (25.0)           (75.0)             (100.0)       (0.0)
    Clinical type
       White striae         21             22          0.752a         27            16          0.140a         18             25        0.220a       40          3       0.930a
                          (48.8)         (51.2)                     (62.8)        (37.2)                     (41.9)         (58.1)                 (93.0)      (7.0)
       Erosive               7              6                         11             2                          3             10                     12          1
                          (53.8)         (46.2)                     (84.6)        (15.4)                     (23.1)         (76.9)                 (92.3)      (7.7)
    Total OLP            28 (50.0)      28 (50.0)                  38 (67.9)     18 (32.1)                  21 (37.5)      35 (62.5)              52 (92.9)   4 (7.1)
a
    Chi-square test; b Mann-Whitney U-test; c Kruskal-Wallis test. Bold values indicate statistical significance (p < 0.05).




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J.K. Bar et al.




 A                                       B




                                                                                   Figure 2. Comparison of HSP90 and p53 protein expression in OLP and
                                                                                   OSCC. Significant differences in HSP90 and p53 expression between OLP and
                                                                                   OSCC were found (*p = 0.006 and **p = 0.01, respectively).
C                                        D

                                                                                   was stronger (10-90% of positive tissue) than in OLP
                                                                                   specimens (10-60% of the positive tissue) (Fig. 1A-D).
                                                                                   In the majority of OSCC samples, HPV16/18 was re-
                                                                                   stricted to 10-20% of tumour cells, but in some individ-
                                                                                   ual cases, HPV16/18 was found in 30-40% of the OSCC
                                                                                   tissue (Fig. 1G-H). A comparative analysis of OLP and
 E                                        F
                                                                                   OSCC for the expression of p53 and HSP90 proteins and
                                                                                   HPV16/18 presence between OLP and OSCC specimens
                                                                                   revealed differences. A significant increase in HSP90
                                                                                   and p53 expression was observed in OSCC compared to
                                                                                   OLP lesions (p = 0.006; p = 0.01, respectively) (Fig. 2).
                                                                                   A positive correlation between HSP90 and p53 expres-
                                                                                   sion (p = 0.017) and a low positive correlation tendency
G                                        H                                         between HSP90 and HPV16/18 expression was found in
Figure 1. Representative immunostaining of HSP90, p53, E-cadherin and              OLP (p = 0.088, Tab. IV). No significant association be-
presence of HPV16/18 in OLP and OSCC cases. (A) HSP90 cytoplasmic immu-            tween the biomarkers analysed and HPV16/18 infection
nostaining observed in basal and superbasal epithelial cells of OLP; (B) HSP90     was found in OSCC samples (p > 0.05, Tab. IV).
membrane/cytoplasmic strong immunostaining observed in a high percentage
of the OSCC tissue; (C) p53 protein expression limited to small OLP tissue ar-
eas; (D) a high number of cells showed strong p53 expression in the OSCC tis-      Clinicopathological features correlated to HSP90, p53, E-
sue; (E) membrane/diffuse E-cadherin expression in superficial and deep lay-       cadherin and HPV16/18 expression in OLP and OSCC
ers of the epithelial tissue of OLP; (F) low E-cadherin expression restricted to   The relation between clinical parameters and expression of
a small tissue area of OSCC; (G) HPV16/18 infection detected in a low number
of cell nuclei in OLP; (H) HPV16/18 presence in malignant cells forming gland      biomarkers revealed correlations between HSP90 expres-
in OSCC tissue (EnVision technique). Figure 1A,C,D,E,F scale bar = 50 μm,          sion and longer duration of OLP (p = 0.011), p53 protein
Figure 1B,G,H scale bar = 100 μm.                                                  and general involvement of oral mucosa (p = 0.025), E-cad-
                                                                                   herin and male gender (p = 0.036) and longer disease dura-
                                                                                   tion (p = 0. 016). The differences between HPV16/18 in-
tive tissue). Percentage of E-cadherin staining was similar                        fection and duration were borderline significant (p = 0.072,
(10-50% of the positive tissue) in both OLP types. Con-                            Tab. II).
versely, HPV16/18 was limited to small (10-15%) tissue                             Analysis between the clinicopathological parameters of
areas (Fig. 1A-H). OSCC specimens revealed HSP90, p53                              OSCCs and biomarker expression revealed that HSP90
protein and E-cadherin expression and HPV type 16/18 in                            expression correlated with an advanced tumour stage and
49/66 (74.2%), 30/66 (45.5%), 42/66 (63.6%) and 11/66                              short survival time (p = 0.045 and p = 0.018, respective-
(16.7%) cases, respectively (Tab. III). A marked hetero-                           ly), while p53 protein correlated with a high tumour grade
geneity of protein expression was observed in OLP and                              (p = 0.032). E-cadherin expression correlated with lymph
OSCC. HSP90, p53 and E-cadherin expression in OSCC                                 node metastasis (p = 0.043). HPV16/18 infection correlat-



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Table III. Correlation between clinicopathological parameters and immunoreactivity for HSP90, p53, E-cadherin and HPV in oral squamous cell carcinoma.
                           HSP90, n (%)                         p53, n (%)                    E-cadherin, n (%)                  HPV16/18, n (%)
      Parameter         Negative       Positive      p-value Negative           Positive      p-value Negative   Positive   p-value Negative   Positive   p-value
    Age
      < 60 years            6              18         0.915a         15              9        0.327a      8         16      0.699a     19         5       0.492a
                          (25.0)         (75.0)                    (62.5)         (37.5)               (33.3)     (66.7)             (79.2)     (20.8)
       ≥ 60 years           11             31                        21             21                   16         26                 36         6
                          (26.2)         (73.8)                    (50.0)         (50.0)               (38.1)     (61.9)             (85.7)     (14.3)
    Gender
      Female                 7             15         0.426a         14              8        0.294a      8         14      1.000a     19         3       0.640a
                           (31.8)        (68.2)                    (63.6)         (36.4)               (36.4)     (63.6)             (86.4)     (13.6)
       Male                  10            34                        22             22                   16         28                 36         8
                           (22.7)        (77.3)                    (50.0)         (50.0)               (36.4)     (63.6)             (81.8)     (18.2)
    Tumour grade
      G1                     6             17                        15             8                    8          15                 21          2
                          (26.1)         (73.9)                    (65.2)         (34.8)               (34.8)     (65.2)             (91.3)      (8.7)
       G2                   11             24         0.186a         20             15        0.032a     15         20      0.268a     27          8      0.347a
                          (31.4)         (68.6)                    (57.1)         (42.9)               (42.9)     (57.1)             (77.1)     (22.9)
       G3                    0              8                         1             7                    1           7                  7          1
                           (0.0)        (100.0)                    (12.5)         (87.5)               (12.5)     (87.5)             (87.5)     (12.5)
    Tumour site
      Tongue                8              22                        13             17                   10         20                 26         4
                          (26.7)         (73.3)                    (43.3)         (56.7)               (33.3)     (66.7)             (86.7)     (13.3)
       Floor of the         4              14         0.919a         12              6        0.234a      7         11      0.897a     14         4       0.726a
       mouth              (22.2)         (77.8)                    (66.7)         (33.3)               (38.9)     (61.1)             (77.8)     (22.2)
       Buccal               5              13                        11              7                    7         11                 15         3
       mucosa             (27.8)         (72.2)                    (61.1)         (38.9)               (38.9)     (61.1)             (83.3)     (16.7)
    T classification
       T1/T2                 11            18         0.045a         17             12        0.556a     10         19      0.779a     27          2      0.059a
                           (37.9)        (62.1)                    (58.6)         (41.4)               (34.5)     (65.5)             (93.1)      (6.9)
       T3/T4                  6            31                        19             18                   14         23                 28          9
                           (16.2)        (83.8)                    (51.4)         (48.6)               (37.8)     (62.2)             (75.7)     (24.3)
    N classification
      N0                     7             30         0.151a         20             17        0.928a     10         27      0.043b     34          3      0.035a
                           (18.9)        (81.1)                    (54.1)         (45.9)               (27.0)     (73.0)             (91.9)      (8.1)
       N1/N2/N3              10            19                        16             13                   14         15                 21          8
                           (34.5)        (65.5)                    (55.2)         (44.8)               (48.2)     (51.7)             (72.4)     (27.6)
    Clinical stage
       I/II                  7             14         0.067b         13              8        0.412a      6         15      0.369a     19          2      0.288a
                           (33.3)        (66.7)                    (61.9)         (38.1)               (28.6)     (71.4)             (90.5)      (9.5)
       III/IV                10            35                        23             22                   18         27                 36          9
                           (22.2)        (77.8)                    (51.1)         (48.9)               (40.0)     (60.0)             (80.0)     (20.0)
    Survival
      Alive                  9             11         0.018a         12              8        0.558a      6         14      0.479a     19          1      0.089b
                           (45.0)        (55.0)                    (60.0)         (40.0)               (30.0)     (70.0)             (95.0)      (5.0)
       Dead                   8            38                        24             22                   18         28                 36         10
                           (17.4)        (82.6)                    (52.2)         (47.8)               (39.1)     (60.9)             (78.3)     (21.7)
    Total OSCC               17            49                        36             30                   24         42                 55         11
                           (25.8)        (74.2)                    (54.5)         (45.5)               (36.4)     (63.6)             (83.3)     (16.7)
a
    Chi-square test; bMann-Whitney U-test; Bold values indicate statistical significance (p < 0.05).




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Table IV. Spearman’s rank correlation between immunoreactivity for HSP90, p53, E-cadherin and HPV in oral lichen planus (OLP) and oral squamous cell car-
cinomas (OSCC).
                                         HSP90                           p53                       E-cadherin                       HPV16/18
 OLP                                           r              p-value                r             p-value      r             p-value     r              p-value
 HSP90                                                                            0.318             0.017     0.184            0.175    0.230              0.088
 p53                                        0.318              0.017                                          0.068            0.618    0.197              0.146
 E-cadherin                                 0.184              0.175              0.068             0.618                               0.156              0.254
 HPV16/18                                   0.230              0.088              0.197             0.146     0.156            0.254
 OSCC
 HSP90                                                                            0.095             0.449     0.181            0.146    -0.116             0.352
 p53                                        0.095              0.449                                          0.100            0.423     0.117             0.350
 E-cadherin                                 0.181              0.146              0.100             0.423                               -0.120             0.337
 HPV16/18                                  -0.116              0.352              0.117             0.350     -0.120           0.337
Bold values indicate statistical significance (p < 0.05). r: Spearman’s rank correlation coefficient.



ed with lymph node metastasis (p = 0.035) and showed a                                           tients. As part of multivariate analysis, it was determined
correlation trend with survival time (p = 0.088, Tab. III).                                      that tumour grade (HR 2.30, p = 0.016), T classification
Univariate and multivariate analyses (Tab. V) were con-                                          (HR 1.77, p = 0.001), lymph node involvement (HR 2.14,
ducted based on the clinicopathological and immunohis-                                           p = 0.021) and HSP90 expression (HR 2.78, p = 0.016)
tochemical characteristics of the OSCC patient cohorts to                                        were independent factors associated with the shorter OS of
determine prognostic factors. Univariate analysis identified                                     OSCC patients.
tumour grade (p = 0.036), T classification (p < 0.001), clin-
ical stage (p = 0.005), lymph node involvement (p = 0.004),
HSP90 expression (p = 0.021), and HPV16/18 presence
                                                                                                 Discussion
(p = 0.032) as prognostic factors for the OS of OSCC pa-                                         Several attempts have been made to identify biomarkers that


Table V. Univariate and multivariate Cox regression analysis of overall survival in patients with oral squamous carcinoma.
          Parameter                                         Univariate                                                     Multivariate
                                                     HR (95% CI)                            p-value                  HR (95% CI)                 p-value
 Gender
    Female/male                                    0.50 (0.25-0.98)                          0.045
 Tumour grade
    G1/G2+G3                                       2.00 (1.05-3.81)                          0.036                  2.30 (1.17-4.52)             0.016
 T classification
    T1+T2/T3+T4                                    1.97 (1.42-2.74)                         < 0.001                 1.77 (1.25-2.51)             0.001
 N classification
    N0/N1+N2+N3                                    2.38 (1.32-4.29)                          0.004                  2.14 (1.12-4.08)             0.021
 Clinical stage
    I+II/III+IV                                    1.66 (1.17-2.37)                          0.005
 HSP90 expression
    Positive/negative                              2.47 (1.14-5.33)                          0.021                  2.78 (1.21-6.40)             0.016
 p53 expression
    Positive/negative                              1.00 (0.96-1.04)                          0.918
 E-cadherin expression
    Positive/negative                              0.74 (0.41-1.33)                          0.311
 HPV16/18 expression
    Positive/negative                              2.18 (1.07-4.44)                          0.032
HR: hazard ratio; CI: confidence interval. Bold values indicate statistical significance.




520
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may be useful in examining the biological characteristics of      healthy oral tissue to malignancy. The lack of differences
OLP and make it possible to predict clinical outcomes for         in E-cadherin expression between OLP and OSCC found in
OLP patients 5,8,17. Several reports analysing the role of p53,   this study is in line with the previous results, which showed
HSP90, E-cadherin and HPV16/18 in malignant transfor-             downregulation of E-cadherin in the early stage of OSCC
mation of OLP towards OSCC have been published 1,5-7,11.          development that did not significantly change during tu-
However, the role of these biomarkers in this process is still    mour progression 13. The topic of the oncogenic potential
a controversial topic 5,10,18,19. Nonetheless, many biomarkers    of HPV infection in OLP and OSCC is controversial 5,16. As
expressed in OLP are also overexpressed in OSCC 8,20. The         with the earlier reports, in our study HPV16/18 infection
correlation between HSP90 expression, low E-cadherin ex-          was detected in a small percentage of OLP, but the number
pression and disease duration for OLP patients observed in        of HPV16/18 positive cases increased in OSCC and was
this study are partly consistent with previous reports, which     associated with worse clinical outcomes of patients with
suggests that in cases of long disease duration, the risk of      HPV positive tumours 16,18. According to published data,
genetic changes may be increased 6,7,20. In our study, HSP90      our results indicate that the HPV16/18 infection may con-
expression was more frequently observed in non-erosive            tribute to the malignant transformation of OLP and gener-
OLP, but it was limited to small tissue areas, and this type      ate the HPV16/18 positive subtype of OSCC with aggres-
of OLP is associated with longer disease duration. There          sive growth 5,15,18. To our knowledge, this is the first study
are reports which define the HSP90 protein as a marker as-        that had revealed a positive correlation between HSP90
sociated with the malignant transformation of OLP 11. As a        and p53 protein, and between HSP90 and HPV16/18 infec-
chaperone protein, HSP90 is involved in the stabilisation         tion in OLP, but not in OSCC. These findings suggest that
and maturation of a large number of proteins, including the       as a chaperone protein HSP90 is crucial for the stability
p53 protein, which contributes to the aggressive behaviour        of the p53 protein; and although the interaction between
of tumours 11,21. We found that low E-cadherin expression         HSP90 and the E6/E7 proteins expressed by HPV16/18
is associated with shorter OLP duration. These results in-        is not fully described, it was found that HSP90β induces
dicate that cell-cell contact is distorted at an early stage of   the stability of E6/E7 proteins 24,25. Nevertheless, the ob-
OLP development 20. The association between the p53 pro-          served interaction between HSP90 and p53 and HPV16/18
tein and the areas of oral mucosa observed in non-erosive         E6 and E7 proteins indicates that HSP90 may prolong the
OLP in this study is in line with the observations of Shiva et    presence of p53 and E6/E7 proteins in cells. On the other
al. 22, who observed p53 protein expression in non-erosive        hand, we may consider that the constant expression of E6/
OLP. The results of this study, which show increased p53          E7 oncoproteins may interfere with the p53 signaling path-
and HSP90 expression and lower E-cadherin expression in           way, leading to the p53 protein degradation and causing
OSCC in comparison to OLP are in line with other data             loss of suppressive function and increase the possibility of
indicating that dysfunctions of these proteins may lead to        malignant transformation 18,25. The progressive character of
malignant lesions in some cases 6,7,10,11. The impact of these    these proteins in OLP was confirmed in this study through
proteins on the aggressive biological behaviour of OLP le-        univariate analysis of HSP90, p53 and E-cadherin expres-
sions is reflected in our data, which shows their increased       sion and presence of HPV16/18 as independent factors in
expression in OSCC, as well as a correlation with worse           OSCC, but only HSP90 expression in multivariate analysis
clinical and pathological features of tumours. Similar data       showed a prognostic value in OSCC. The results reveal that
were presented by other authors, who found a correlation          the progressive growth of OSCC requires complex changes
between p53, E-cadherin and HSP90 protein expression              in various molecules, which may occur in premalignant le-
and worse OSCC clinical parameters, and suggests that             sions as indicators of OLP malignancy 10.
these proteins are involved in the progressive growth of
tumours 8,9,13,18,23. Moreover, our observation of significant
differences in p53 and HSP90 expression between OLP and
                                                                  Conclusions
OSCC may reflect genetic alterations or protein structural        In conclusion, our data suggest that a positive correlation
changes occurring as part of the malignancy process 3,7,10.       between HSP90 and p53 protein expression, as well as be-
In this study, we were unable to reveal the prognostic value      tween HSP90 and HPV16/18 in OLP, indicates that coop-
of p53 and HSP90 expression due to the short observation          eration between these proteins exists in OLP. Moreover, we
time of OLP patients. Nevertheless, the results obtained by       can propose that the network between the host cell proteins
Giuliani et al. 2, who described the overall malignant trans-     and E6/E7 proteins indicates that this interaction can mod-
formation in 1.4% of cases, including 1.37% for OLP, con-         ulate the differential profile of the expression of host cell
cluded that p53 expression may cause the progression from         proteins, affecting the biological behaviour of OLP. The



                                                                                                                             521
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