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Background: Oral squamous cell carcinoma (OSCC) is characterized by a broad spectrum of genomic imbalances, including gross chromosomal (polysomy/aneuploidy) ones as well as specific gene alterations. Aberrant expression of anaplastic lymphoma kinase (ALK) seems to be a useful molecular marker for discriminating patients based on genetic signatures in a variety of solid malignancies, such as lung carcinoma. Our aim was to analyze ALK protein expression patterns in a series of OSCCs., Materials and Methods: Fifty (n=50) OSCC tissue sections were analyzed by implementing an ALK-based immunohistochemistry protocol. Digital image analysis was performed for measuring the corresponding protein expression levels., Results: ALK overexpression was observed in 14/50 (28%) OSCC tissue sections, whereas the rest 36/50 (72%) demonstrated low expression levels. ALK expression was negatively associated with grade (p=0.027) and stage (p=0.0028) of the examined cases., Conclusion: Abnormal ALK expression in subsets of patients with OSCC seems to be related to an aggressive phenotype (advanced stage/progressive dedifferentiation). ALK protein overexpression may be used as a significant marker for applying targeted therapeutic regimens., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Oncogene up-regulation combined with suppressor gene down-regulation is a crucial genetic combination that promotes cell neoplastic phenotype and progressively malignant transformation in solid malignancies, including laryngeal squamous cell carcinoma (LSCC). Among oncogenes, the Kirsten ras oncogene homolog (K-Ras) is involved in LSCC onset and progression., Patients and Methods: Sixty (n=60) primary LSCC tissue sections were analyzed by immunohistochemistry (IHC). Digital image analysis (DIA) was also implemented for measuring K-Ras protein expression levels., Results: High K-Ras protein expression levels were observed in 20/60 (33.3%) LSCC tissue sections, whereas the rest of the cases (n=40; 66.7%) demonstrated low expression. Overall K-Ras expression was borderline significantly associated to the grade of the examined malignancies (p=0.048), whereas no other strong statistical correlations were identified. A progressive K-Ras overexpression was observed in all grades of the examined cases., Conclusion: K-Ras over expression is correlated to a progressive dedifferentiation in LSCC., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Oral squamous cell carcinoma (OSCC) demonstrates aggressive biological behavior in subgroups of patients with specific molecular characteristics. Concerning metastatic potential, disruption of cell to cell adhesion is a critical event in epithelial malignancies including OSCC. Our aim was to investigate the role of E-Cadherin expression in OSCC patients as a valuable protein marker., Materials and Methods: Fifty (n=50) tissue sections derived from primary OSCCs were analyzed by implementing an immunohistochemistry (IHC) assay based on a proper anti-E-cadherin antibody. Digital image analysis was also implemented for an objective evaluation of the corresponding protein expression levels., Results: E-cadherin altered expression (low to negative) was observed in 34/50 (68%) cases, whereas the rest (16/50-32%) demonstrated normal (high to moderate) expression. E-Cadherin abnormal expressionwas associated with the stage of the examined malignancies (p=0.023), whereas no significant correlations with grade, gender, smoking status or human papilloma virus (HPV) history were observed., Conclusion: E-Cadherin down regulation is frequently observed in OSCC and is correlated to a progressively aggressive phenotype of the malignancy in the corresponding patients (advanced stage), but it seems that the impact of HPV persistent infection on these patients is not a critical parameter., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy due to its increased ability for local metastases and distant lymph node metastases. Extensive cytogenetic analyses have detected chromosome instability (CI) patterns in OSCC including gross chromosome numerical alterations, such as polysomy and sporadically monosomy that negatively affect the biological behaviour of the malignancy. Our aim was to investigate the frequency and impact of chromosome 17 (Chr 17) numerical imbalances in OSCC., Materials and Methods: Fifty (n=50) formalin-fixed, paraffin-embedded primary OSCCs tissue sections were used. Chromogenic in situ hybridization (CISH) was implemented for detecting Chr 17 centromeric numerical imbalances. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented., Results: Chr 17 multiple copies were observed in 12/50 (24%) of the examined cases. Polysomy was observed in 10/50 (20%) tissue sections, monosomy in 2/50 (4%), whereas the rest of them demonstrated a normal, diploid pattern (38/50-76%). Chr 17 numerical differences were associated with the grade of differentiation of the examined tumors (p=0.001)., Conclusion: Chr 17 numerical imbalances (polysomy predominantly and monosomy) are observed in sub-groups of OSCCs correlating with a progressive dedifferentiation of malignant tissues. The proposed grid-based platform on CISH slides provides a novel, fast and accurate screening-mapping mechanism for detecting chromosome numerical aberrations., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Topoisomerase II alpha (TopoIIa) is a critical gene associated with response to chemo-therapeutic agents, such as anthracyclines, especially in breast adenocarcinoma. The aim of this study was to investigate the role of aberrant TopoIIa protein expression in laryngeal squamous cell carcinoma (LSCC)., Materials and Methods: Fifty (n=50) LSCC cases were enrolled in the study. Immunohistochemistry and a digital image analysis assay were implemented., Results: TopoIIa protein overexpression was observed in 32/50 (64%) cases, whereas low expression rates were detected in 18/50 (36%). TopoIIa overall expression presented strong association with the grade of the examined malignant tissues and borderline association with stage. TopoIIa overexpression correlated also with Human papillomavirus (HPV) positivity., Conclusion: TopoIIa overexpression was observed in significant subsets of LSCCs, and correlated predominantly with the grade of differentiation. HPV persistent infection seems to be associated with increased TopoIIa protein expression. TopoIIa expression analysis appears to be critical in identifying sub-groups of patients eligible for specific chemotherapy., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs., Material and Method: Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented., Results: Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001)., Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area., .

Background: P53 is a key regulator of genomic stability and function, acting as a tumor suppressor protein. Our aim was to correlate P53 expression with murine double minute 2 (MDM2), a proto-oncogene that interacts with P53 and forms an auto-regulatory pathway, in laryngeal squamous cell carcinoma (LSCC)., Materials and Methods: A total of 50 LSCC cases were included in the study. Immunohistochemistry was applied by using antibodies to P53 and MDM2 in the corresponding tissue sections. Protein expression levels for both molecules were measured by implementing a digital image analysis assay (immunostaining intensity levels, densitometric evaluation)., Results: Overexpression of P53 protein was observed in 16/50 (32%) LSCC cases, while 22/50 (44%) cases strongly expressed MDM2 protein. Interestingly, in 13/50 (26%) cases, combined overexpression of P53/MDM2 was detected. Overall P53 was strongly positively correlated with MDM2 expression (p=0.001). Both P53 and MDM2 overexpression were significantly correlated with advanced stage of LSCC (p=0.032 and p=0.001, respectively). Additionally, MDM2 was found to be associated with poorer survival of patients (p=0.046)., Conclusion: Aberrant co-expression of P53 and MDM2 is associated with advanced stage in LSCC. Furthermore, MDM2 overexpression is a frequent and critical genetic event in LSCC and seems to negatively affect survival., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Epidermal growth factor receptor (EGFR) acts as an oncogene in malignancies. Our aim was to examine the role of combined EGFR/ anaplastic lymphoma kinase (ALK) expression as molecular markers in laryngeal squamous cell carcinoma (LSCC) patients., Materials and Methods: Fifty (n=50) tissue sections derived from twenty-five (n=25) primary LSCCs were analyzed by immunohistochemistry (IHC)., Results: EGFR overexpression was observed in 17/25 (68%) cases. Concerning ALK, 23/25 (92%) demonstrated low expression. EGFR expression was associated with grade (p=0.049), whereas ALK expression was correlated with stage (p=0.048). ALK overexpression was detected at advanced-stage EGFR-positive cases. A biphasic EGFR protein expression pattern was observed in five (n=5) LSCC cases, whereas ALK expression was stable in all cases., Conclusion: EGFR overexpression is frequently observed in LSCC combined with low ALK expression. LSCC patients with EGFR/ALK protein overexpression should be eligible for targeted therapeutic strategies., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Deregulation of apoptosis is critical regarding the development and progression of malignancies, including laryngeal squamous cell carcinoma (LSCC). B-Cell lymphoma 2 (BCL2) (gene locus:18q21.33), located on the outer mitochondrial membrane, acts mainly as an anti-apoptotic factor suppressing and blocking apoptotic signal transduction., Materials and Methods: Fifty (n=50) primary LSCC tissue sections were used. Immunohistochemistry and digital image analysis were implemented for evaluating BCL2 protein expression levels., Results: High BCL2 protein expression levels were observed in 21/50 (42%) LSCC tissue sections, whereas the remaining cases (n=29) demonstrated a low expression. Overall, BCL2 expression was associated with grade (p=0.046) and anatomical region of the examined malignancies (transglottic, p=0.047). Interestingly, high BCL2 expression levels were strongly associated with radiotherapy-based only regimens (p=0.01) in corresponding patients., Conclusion: BCL2 overexpression was found to be correlated with an aggressive phenotype (advanced grade of differentiation) in LSCC, also demonstrating a potential selective anatomic localization (transglotic region). Additionally, BCL2 overexpression appears to be a negative regulator for successful radiotherapy implementation by reducing the apoptotic process in patients., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: p16 (gene locus: 9p21) tumor suppressor gene is considered an important biomarker for the progression and prognosis in a variety of malignancies and pre-cancerous lesions, including high-risk (HR-) human papilloma virus (HPV)-mediated squamous intraepithelial lesions (SILs), based on cytological and the corresponding cervical intraepithelial neoplasia (CIN) histopathological categorization. p16 acts as a cyclin-dependent kinase-4 inhibitor negatively regulating the cell cycle. In persistent HPV infection, E7 oncogenic protein binds retinoblastoma protein leading to its proteolytic transformation, also triggering E2F dissociation, which increases DNA transcription and progression to S phase. This mechanism promotes aberrant p16 over-expression. Our aim was to comparatively analyze p16 protein expression patterns in laryngeal squamous cell carcinomas (LSCC) and also in SILs., Materials and Methods: Fifty (n=50) primary LSCCs tissues all non-HPV-dependent, and a set of 50 liquid-based SILs, were analyzed by immunohistochemistry and immunocytochemistry, respectively. Concerning the screening process in cytological slides, a novel real-time reference and calibration grid platform was implemented and employed., Results: Decreased protein expression was observed in 34/50 (68%) tissues regarding LSCCs. Overall p16 expression was associated to smoking status of the patients (p=0.001), and also with the p-stage of the examined malignancies (p=0.033). A strong statistical significance was assessed correlating LSIL/HSIL cases with a progressive p16 over expression (p=0.001), also reflecting a higher CIN diagnosis (p=0.001)., Conclusion: p16 down-regulation is a frequent genetic event in LSCCs, which is associated with advanced disease. In contrast to this, p16 over-expression triggered by a specific molecular mechanism shows a strong relationship with a progressively aggressive phenotype due to upgraded SIL/CIN cervical categorization. The first described application of the grid platform demonstrated a considerable improvement in the immunocytochemistry slide screening process enhancing the diagnostic reliability., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Oral squamous cell carcinoma (OSCC) is an aggressive neoplasm. Many chromosomal and gene alterations have been identified in OSCC, including structural and numerical changes. In this study, we implemented a molecular assay of chromosome 7 (Chr7) in order to investigate the level of its numerical instability in OSCC., Materials and Methods: Using tissue microarray technology, 30 primary OSCCs were cored and re-embedded into one recipient block. Chromogenic in situ hybridization assay was performed based on Chr7 centromeric probedetection., Results: Chr 7 numerical analysis detected polysomy (trisomy/ tetrasomy) in 4/30 (13.3%) of the examined tissue OSCC cores. Statistical significance was assessed correlating Chr7 numerical aberrations with stage (p=0.015), especially detected in cases not related to human papillomavirus (HPV) (p=0.01)., Conclusion: Although Chr7 polysomy is a relatively rare gross genetic event in OSSC, it affects their biological behavior leading toa progressively aggressive phenotype (advanced stage). Furthermore, Chr7 polysomy is observed more frequently in non-viral (HPV) cases., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Phosphatase and tensin homolog (PTEN) (gene locus: 10q23.3) -a tumor suppressor gene- is deleted, mutated or epigenetically hyper-methylated in a variety of malignancies. PTEN acts as a negative regulator in PI3K/AKT/mTOR signaling transduction pathway. Our aim was to investigate PTEN protein expression patterns in laryngeal squamous cell carcinomas (LSCC)., Materials and Methods: Using tissue microarray technology, fifty (n=50) primary LSCCs were cored and re-embedded into one recipient block. Immunohistochemistry and digital image analysis were implemented for evaluating protein expression levels., Results: Abnormal protein expression (low to negative staining intensity values) was observed in 28/50 (56%) tissue cores. Overall PTEN expression was associated with the anatomical region of the malignancies (p=0.039), whereas a borderline correlation with the differentiation grade was also assessed (p=0.05)., Conclusion: Aberrant expression of PTEN tumor-suppressor gene in LSCCs seems to affect their biological behavior. Well-differentiated tumors express moderate to high protein levels, an evidence of normal gene function, whereas loss of its expression correlates with a progressive tumor dedifferentiation. Additionally, loss of its expression is detected more frequently in specific anatomical regions of the larynx (glottis, predominantly, and partially supraglottis)., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

A study of laryngeal carcinomas was performed in order to analyze (a) the expression of p53/p21, cyclin D1/cyclin E, p21/p27 (b) the relation of normal and abnormal protein expression, with the proliferation status, as determined by the expression of Ki67 and PCNA and (c) the correlation of our findings with prognosis. We performed a retrospective analysis of 57 cases of squamous cell carcinomas of the larynx. We applied monoclonal antibodies against p53, p21, p27, cyclin D1, cyclin E, Ki67 and PCNA, using streptavidin-biotin method. Analysis of the p53/p21 proteins, revealed abnormalities in 25/37 cases (67.57%), while 12/37 (32.43%) cases displayed normal phenotype (p53-/p21-). Analysis of cyclins revealed overexpression in 17/48 cases (35.42), while the majority 31/48(64.58%) displayed normal phenotype (cyclin D1-/cyclin E-). Concerning CDKIs expression, the majority 30/50(60%) presented high levels of both inhibitors (p21+/p27+). Cases with simultaneous overexpression of CDKIs demonstrated significantly higher levels of Ki67 protein (p = 0.05). Analysis of p53/p21, cyclin D/cyclin E, p21/p27 patterns showed no association between the presence of one or two alterations and prognosis. In conclusion, we demonstrated that p53 tumor suppressor pathway is frequently disrupted in laryngeal cancer. Furthermore, levels of CDKIs, although they act as cell cycle activity blockers, are not reliable markers for the estimation of laryngeal neoplastic cells growth fraction.

Background: Cell cycle progression and transition of cells from the first gap phase (G1) to the DNA replication phase (S) depend on a finely tuned balance between the levels of cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs)., Materials and Methods: We analyzed 57 squamous cell invasive carcinomas of the larynx, 10 in situ carcinomas, 56 cases of dysplasia, 11 papillomas and 26 keratosis. We investigated: a) the immunohistochemical expression of CDKIs, p21 and p27, b) any possible relation between normal and abnormal immunoprofiles of these proteins and p53 protein and proliferation status as determined by the expression of Ki67 and PCNA, and c) their presence in pre-malignant and malignant laryngeal lesions., Results: Expression of p21 and p27 was observed in 58.9% and 89.5% of the laryngeal carcinomas, respectively. High levels of p21 were significantly correlated with increased cyclin D (p=0.001), cyclin E (p<0.001) and Ki67 (p<0.001), while increased expression levels of p27 were associated with p53 accumulation (p=0.02) and with increased proliferation status as expressed by Ki67 (p=0.05)., Conclusion: Due to the increased expression levels of CDKIs in laryngeal carcinomas, we suggest the existence of a mechanism by which tumor cells tolerate the inhibitory effect of these proteins on cell cycle progression.