P53 Immunostaining Research Articles

Hepatocellular Carcinoma Score and Subclassification Into Aggressive Subtypes Using Immunohistochemical Expression of p53, β-Catenin, CD133, and Ki-67.

Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy in adults. Several studies have classified HCC into molecular subtypes aiming at detecting aggressive subtypes. The aim of the present study was to investigate the role of p53, β-catenin, CD133, and Ki-67 in subclassification of HCC into different aggressive subtypes and the correlation between those markers and the clinicopathologic characteristics of HCC patients. This retrospective study was conducted on paraffin-embedded blocks of 114 HCC specimens. Tissue microarray was constructed and immunostaining for p53, β-catenin, CD133, and Ki-67 was performed and HCC score was formulated. P53 expression was associated with old age (P=0.028), large tumor size (P=0.019), poorly differentiated HCC (P=0.012), hepatitis B virus (HBV) positivity (P=0.032), and hepatitis C virus (HCV) negativity (P =0.046). β-catenin expression was associated with small sized tumors (P=0.005), HBV negativity (P=0.027), early-staged tumors (P=0.029), and prolonged recurrence-free survival (P=0.045). High percentage of CD133 expression was associated with old patients (P=0.035) and HBV positivity (P= 0.045). Ki-67 expression was associated with large tumor size (P= 0.049), vascular invasion (P= 0.05), old age (P=0.035), and previous treatment of HCV by direct acting antiviral agents (P=0.005). Cases with high HCC score showed significant association with old patients (P=0.002), previous treatment of HCV by direct acting antiviral agents (P<0.001), large tumor size (P<0.001), and poorly differentiated tumors (P= 0.009). The proposed HCC score can divide HCC patients into subtypes necessitating tailoring of treatment strategy according to this proposed score to target and optimally treat the aggressive subtypes. This score needs to be further validated on large number of patients with longer follow-up period.

1p/19q co-deleted fibrillary astrocytomas: Not everything that is co-deleted is an oligodendroglioma

The presence of chromosome 1p/19q co-deletion is one of the hallmark required criteria for the diagnosis of oligodendroglioma, using the 2016 World Health Organization (WHO) Classification of Tumours of the Central Nervous System. Descriptions in the literature of astrocytomas, primarily glioblastomas, demonstrating partial losses on one or the other chromosome have been described. The significance of these small deletions is uncertain. Only rarely have cases of fibrillary astrocytoma been described as having co-deletion, which may potentially cause diagnostic confusion with oligodendroglioma. The goal of this study is to examine a large number of fibrillary astrocytomas to document how often 1p/19q co-deletions are present by Fluorescent In Situ Hybridization (FISH) testing (the testing method of choice in many institutions) and to evaluate what other markers may be helpful in avoiding misdiagnosis.This study is a retrospective evaluation of 359 fibrillary astrocytomas (55 grade II, 62 grade III and 242 grade IV) encountered between June 2016 and June 2019, we identified 11 tumors (3.1%) that had 1p/19q co-deletion by FISH testing. The clinical and pathologic features of these cases were reviewed. The 11 cases with co-deletion included 5 females who ranged in age from 37 to 86 years (median 63 years). Tumors arose in the temporal lobe in 5 patients, frontal lobe in 2, parietal lobe in 2, occipital lobe in 1, and cerebellum in 1. Final diagnoses included glioblastoma in 8 patients, anaplastic astrocytoma in 2, and diffuse astrocytoma in 1. Only 1 case (anaplastic astrocytoma) demonstrated evidence of IDH-1 immunoreactivity; none of the other 10 tumors showed evidence of an IDH1/2 mutation by PCR testing. Four tumors demonstrated p53 immunostaining of 30% or more. ATRX mutation as evidenced by loss of staining was observed in only 2 cases. Evidence of EGFR amplification by FISH testing was noted in 5 cases. Of particular note in the one case that demonstrated both 1p/19q co-deletion and an IDH-1 mutation, LOH testing was done and showed only partial losses on both chromosomes. Additionally, this tumor also demonstrated evidence of ATRX and p53 mutations by immunohistochemistry. In conclusion, co-deletions were noted in a minority of astrocytomas (3.1% of cases in the current study). Only 1 of 11 of these cases also demonstrated evidence of an IDH mutation, potentially raising differential diagnostic confusion with oligodendroglioma. Use of LOH 1p/19q testing, if available, or other markers such as ATRX, p53 and EGFR may be helpful in avoiding misclassification of such tumors as oligodendroglioma.

Assessing the nature of the association of human papillomavirus in oral cancer with and without known risk factors

BackgroundPublished studies assessing the association of human papillomavirus (HPV) and oral cancer, often include cases with additional confounders such as alcohol, tobacco, thus, precluding the assessment of HPV as a independent risk factor. Further apart from eliciting the mere presence of HPV, it is vital that the nature of the association is delineated for eliciting a causal inference. Thus, the present study assessed the presence and nature of the association of HPV in oral cancer cases with and without known risk factors.MethodsThe study compares the prevalence of HPV in oral cancer cases with known risk factors (group 1, n=15) to oral cancer (group 2, n=15) and oral epithelial dysplasia (group 3, n=15) cases with no known risk factors using PCR. Cases which are positive for HPV were subjected to p16 and p53 immunostaining to determine potential causal inference.ResultsHPV 16 was detected in only 1 case of group I, 3 cases of group II, and was absent in group III. HPV 18 was negative in all the 3 groups. All the HPV positive cases were negative for p16 and positive for p53 immunostaining.ConclusionsOnly a minor proportion of oral cancer cases without risk factors were positive for HPV 16. Even among these HPV 16 positive cases, the immunostaining profile (p53 positive and p16 negative) excludes the involvement of E6 and E7 mediated carcinogenesis. Thus even the weak HPV association noted in the present study cases may not be of causal nature.

MED12 exon 2 mutation is uncommon in intravenous leiomyomatosis: clinicopathologic features and molecular study

Intravenous leiomyomatosis (IVL) is a rare neoplasm that is characterized by smooth muscle cell proliferation within venous vessels. The aim of this study is to investigate the clinicopathological features, immunophenotypes, and MED12 gene mutations in IVL. Nine cases of IVL from the Affiliated Hospital of Qingdao University were collected, and the clinicopathological features were reviewed. The immunohistochemical expressions of p16, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alpha thalassemia/mental retardation syndrome X-linked (ATRX), retinoblastoma 1 (RB1), fumarate hydratase (FH), and p53, were evaluated. The mutation status of MED12 gene exon 2 was detected by Sanger sequencing. All the 9 patients were women ranging from 32 to 58 years, and uterine leiomyomas were identified in 5 patients. Immunohistochemical staining showed that all IVL and leiomyoma samples were positive for estrogen receptor and progesterone receptor, but negative for CD34. IVL displayed similar immunostaining patterns with their uterine counterparts with focal p16 immunostaining. FH, PTEN, ATRX, and RB1 were variably positive, and p53 and Ki-67 positive rates were less than 5% in all cases. Two novel genetic variations at MED12 exon 2, a synonymous mutation c.141C>T (p.Asn47=), and an in-frame deletion mutation c.133_147del15 (p.Phe45_Pro49del) were identified in two IVL cases. One missense mutation c.131G>A (p.Gly44Asp) was identified in one uterine leiomyoma. The remaining 11 tumor samples (7 IVL cases and 4 uterine leiomyomas) showed no mutations at MED12 exon 2. Our results showed two novel MED12 mutations in IVL. The MED12 mutations are different between IVL and uterine leiomyoma. These findings indicate that IVL is a unique entity and different from uterine leiomyoma.

Loss of p16 and high Ki67 labeling index is associated with poor outcome in esophageal carcinoma.

The p16 tumor suppressor is coded by CDKN2A (9p21) and plays an important role during carcinogenesis and tumor progression in numerous tumor entities. The aim of our study was to evaluate the prognostic role of p16 expression and CDKN2A deletion in esophageal cancer (EC). Therefore, we analyzed p16 and KI67 expression by immunohistochemistry and 9p21 deletion by fluorescence in-situ hybridization on a tissue microarray including 398 adenocarcinomas (AC) and 293 squamous cell carcinomas (SCC) with clinical follow up-data. p16 positivity was found in 30.2% of AC and 13.9% of SCC and CDKN2A deletion in 32.1% of AC and 33.5% of SCC. In SCC p16 immunostaining correlated with low tumor stage (P = 0.014). In AC Ki67 positivity was associated with high tumor stage (P = 0.001), presence of lymph node metastasis (P = 0.009), high UICC stage (P = 0.001) and poor grading (P = 0.005). Overall survival (OS) was shorter for patients with high Ki67 labeling index (Ki67LI; P = 0.009) and negative p16 immunostaining (P = 0.026). In both histological tumor types, CDKN2A deletion showed no association with phenotype or outcome. Proportional cox-regression modeling revealed patients’ age, tumor stage, lymph node metastasis and Ki67 labeling index as independent prognostic markers in AC. In SCC, only patients’ age and tumor stage proved to be independent prognosticators. In summary, our study shows that loss of p16 expression and high Ki67LI is linked to shortened OS in AC. CDKN2A deletion shows no relevant association with tumor phenotype and patient outcome.

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2020.007, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Prevalence and Detection of Sexually Transmitted Cases of Laryngeal Carcinoma.

Human papillomavirus (HPV) is among the reported etiologies of laryngeal squamous cell carcinoma (LSCC). Understanding the impact of HPV on LSCC may help reduce its incidence. This study investigates the association between HPV and LSCC as well as the roles for different immunohistochemical stains in HPV detection. A total of fifty-two formalin-fixed, paraffin-embedded tissue samples of LSCC, diagnosed between 2005 and 2015, were obtained from the archives of the Pathology Department. The samples were stained and processed to evaluate the relationship of HPV to LSCC. Patients had a mean age of 65.02 ± 14.341 years. By polymerase chain reaction (PCR), high-risk strains of HPV were detected in 15.4% of tissue samples. HPV-16 was found in 75% of the positive samples for an overall prevalence of 13.5%. P16 immunostaining was positive in 15.4% of cases while cyclin B and cyclin E were positive in 65.4% and 76.9%, respectively. Over half of the cases were histologically graded as moderately-differentiated, 28.8% as well-differentiated, and 15.4% as poorly-differentiated. The prevalence of HPV-positive LSCC was lower than previously stated in the literature. HPV-16 was the most commonly detected subtype, in concordance with the findings of multiple other studies. HPV-positive LSCC trended with higher histologic grade. P16 and cyclin E immunohistochemical stains were of limited use in identifying HPV in LSCC. In contrast, cyclin B had a high sensitivity which could be used to rule out HPV in LSCC.

Determination of high-risk HPV status of head and neck squamous cell carcinoma using the Roche cobas HPV test on cytologic specimens and acellular supernatant fluid.

High-risk human papillomavirus-positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine-needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy-based techniques, the ability of liquid-based assays was examined in the current study. The performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell-free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization. A total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty-three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty-two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell-free supernatant fluid. Two cell-free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas-positive cell pellet was deemed to be a false-positive result. The sensitivity of the cobas assay was 100% for pellet material and cell-free supernatant fluid, with specificities of 94.7% and 100%, respectively. cobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell-free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses.

Prognostic evaluation of human papillomavirus and p16 in esophageal squamous cell carcinoma.

To the Editor: Esophageal cancer is the sixth most common cancer globally according to the World Health Organization.[1] Due to rapid clinical progression and extremely poor prognosis, the 5-year survival rate of esophageal squamous cell carcinomas (ESCC) remains less than 20%;[2] therefore, further developments in this field are needed. Human papillomavirus (HPV) is a small circular non-enveloped double-stranded DNA virus that primarily infects mucosa and cutaneous keratinocytes[3] and its infection rates in ESCC range from 11.7% to 38.9% worldwide.[4] P16 which is encoded by the CDKN2A gene and known as cyclin-dependent kinase inhibitor 2A, is a widely used immunohistochemical marker in squamous cell carcinomas and associated with high-risk HPV.[5] The present study included 228 cases from Henan Cancer Hospital, immunostaining for p53 and retinoblastoma protein (Rb) were previously performed for 147 samples, and then 73 ESCC cases were randomly selected for detecting HPV infection and p16 expression. A total of 20 cases were selected through healthy gastroscopy physical examination. This study was approved by the Institutional Ethics Committee of Henan Cancer Hospital (No. 201401017). Sections were incubated with HPV (ZM-0217, CAMVIR-1), p16 (ZM-0205, 1C1), p53 (ZM-0405, BP53.12), or Rb (ZM-0223, 13A10) antibodies from Beijing Zhongshan Jinqiao Biotechnology Co. Ltd, China as the primary antibodies and biotinylated goat anti-rabbit IgG as the secondary antibody. The RNA of HPV 16/18 was detected by the RNAscope 2.5 Assay Reagent Kit (Advanced Cell Diagnostics, Inc., Hayward, CA, USA). All statistical analysis were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Fisher exact test was used to identify predictors for HPV-positive tumors. The analysis of the 5-year survival rate between different groups was performed using the Kaplan-Meier method and log-rank test. Differences with a value of P < 0.05 were considered statistically significant. All statistical tests were two-sided. The characteristics of 73 ESCC patients were analyzed and revealed that there were no significant differences between the groups with respect to age (P = 0.708), stage (P = 0.788), T category (P = 0.417), N category (P = 0.788), differentiation grade (P = 0.141), smoking (P = 0.445), and alcohol consumption (P = 0.940). The positive cases of HPV, p53, p16, and Rb were 33 (45.2%), 40 (54.8%), 5 (6.8%), and 32 (43.8%), respectively. The mRNA expression of HPV 16/18 detected through RNAscope 2.5 Assay was negative. The HPV infection in the esophageal mucosa of 20 normal people was all negative. The cases of co-positive HPV and p16, p53, and Rb were 3 (4.1%), 22 (30.1%), and 12 (16.4%), respectively. Among the 73 ESCC patients (for five cases, survival information could not be confirmed), the total number of deaths was 24, and the 5-year survival was evaluated to compare the prognosis from the different groups. The number of deaths of patients with positive HPV, p16, p53, and Rb were 9, 1, 14, and 12; and the number of deaths of patients with negative HPV, p16, p53, and Rb were 15, 23, 10, and 12, respectively. As shown in Figure 1, there was no significant difference in 5-year survival rate between patients with positive or negative HPV, p16, p53, and Rb (χ2 = 0.664, 0.500, 0.008, and 0.262, P = 0.415, 0.479, 0.929, and 0.609, respectively, all P > 0.05).Figure 1: The Kaplan-Meier curve analysis of 5-year survival rate between patients with positive or negative HPV, p16, p53, and Rb, respectively. Number 1 and 2 indicates positive and negative HPV, p16, p53, or Rb, respectively. HPV: Human papilloma virus; Rb: Retinoblastoma protein.It is well-known that the p53 and p16 tumor suppressor genes are closely related to the cell cycle. Mutations in the p53 and p16 genes can disrupt the cell cycle regulatory mechanism and induce cell hyperplasia and tumors growth. The rate of HPV infection in 73 ESCC patients was 45.3%; however, statistical analysis showed that the HPV, p16, p53, and Rb were not correlated with the 5-year survival rate of ESCC (all P > 0.05). HPV infection may be related to the occurrence of ESCC, but it is not related to the 5-year survival rate of ESCC patients. The relationship between HPV and ESCC should to be further explored based on a large sample size. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Funding This work was supported by grants from the Henan Medical Science and Technology Project (general) (No. 201401017) and the National Natural Science Foundation of China (No. 81602637). Conflicts of interest None.

P16 Expression in Primary Vulvar Extramammary Paget Disease.

P16 immunohistochemistry has been widely used in facilitating the diagnosis of human papillomavirus (HPV)-related usual type vulvar intraepithelial neoplasm. However, studies of p16 expression in primary vulvar extramammary Paget disease (EMPD) are limited. We assessed the p16 expression by immunohistochemistry in 40 cases of primary vulvar EMPD, including 34 cases of intraepithelial vulvar EMPD and 6 cases of invasive vulvar EMPD and correlated p16 expression patterns with disease progression. Overall, p16 expression was present in 36 cases (90%), including 20 cases (50%) with focal staining pattern and 16 cases (40%) with diffuse staining pattern. All 20 cases with focal p16 staining pattern were intraepithelial vulvar EMPD. Diffuse p16 staining pattern was present in 10/30 cases (33.3%) of intraepithelial EMPD and in 6/6 cases (100%) with invasive vulvar EMPD. Negative p16 staining was present in four intraepithelial EMPD cases. Using a highly sensitive RNA in situ hybridization method, we did not detect high-risk HPV in the selected 10 cases with diffuse p16 staining pattern, including 6 cases of intraepithelial EMPD and 4 cases of invasive EMPD. We also observed that intraepithelial EMPD had predominantly cytoplasmic p16 immunoreactivity, whereas nuclear p16 immunoreactivity was mainly seen in invasive EMPD components. Our study demonstrated that the p16 positive immunostaining was seen in the majority of primary vulvar EMPD which is not related to HPV infection. Therefore, knowing the overlapping p16 immunostaining patterns in vulvar EMPD and usual type vulvar intraepithelial neoplasm is important to render the correct diagnosis.

Diagnostic significance of dual immunocytochemical staining of p53/cytokeratin20 on liquid-based urine cytology to detect urothelial carcinoma.

Background:Urine cytology is a noninvasive and inexpensive method; however, it is limited in low sensitivity for detecting and monitoring urothelial carcinoma (UC). To overcome limitation of cytology, several tests using urine samples have been attempted that immunocytochemical staining is an inexpensive and easy to perform ancillary technique. Dual immunocytochemical staining for p53 and cytokeratin 20 (CK20) is assessed in liquid-based urine cytology slides.Materials and Methods:Liquid-based urine cytology samples collected between 2008 and 2013 and matched follow-up biopsy samples of high-grade UC (HGUC) (n = 44) and low-grade UC (LGUC) (n = 14) were analyzed.Results:Urine cytology showing atypical cells was subjected to dual-color immunostaining for p53 and CK20. The sensitivity of urine cytology combined with p53 and CK20 immunostaining was 77.3% in HGUC and 52.9% in LGUC. Of 20 cases diagnosed with atypia by urine cytology, 13 (65%) were positive for p53 or CK20. Dual immunocytochemical staining for p53/CK20 improved the diagnostic accuracy of urine cytology.Conclusions:The present results indicate that cytomorphology combined with p53/CK20 immunostaining is useful for the detection of HGUC and LGUC.

Étude de la prévalence du papillomavirus (HPV) dans les cancers des voies aéro-digestives sur une cohorte unicentrique française de 372 patients

French data about HPV role in head and neck carcinomas are sparse, although French patients are mostly heavy smokers. In this series of oropharyngeal et non-oropharyngeal tumors, we aimed to determine what were the clinicopathological features associated with HPV and evaluate survival of patients according to HPV status. Three hundred and seventy-two cases of head and neck squamous cell carcinomas were reviewed and clinicopathological data were detailed. For each case, we performed a HPV PCR and an immunostaining against p16 protein (paraffin embedded tissues). The series contained 90% of heavy smokers and 36% of tumors were located in oropharynx. HPV DNA was detected in 46% of oropharyngeal carcinomas and 16% of non-oropharyngeal carcinomas. Genotype 16 was the most frequently detected (84%). Clinicopathological features significantly associated with HPV DNA were: oropharyngeal location; absence of tobacco smoking; nodal involvement; poorly-differentiated non-keratinizing histology; positive p16 immunostaining. HPV infection was significantly associated with a longer survival for oropharyngeal carcinomas. It was not the case for non-oropharyngeal carcinomas. In this French series with lot of heavy smokers, under half of carcinomas are HPV induced. Clinicopathological features and survival data associated with HPV infection are the same as those classically described in literature.

Identification of A Novel Papillomavirus Associated with Squamous Cell Carcinoma in A Domestic Cat.

Papillomaviruses infect the skin and mucosal surfaces of diverse animal hosts with consequences ranging from asymptomatic colonization to highly malignant epithelial cancers. Increasing evidence suggests a role for papillomaviruses in the most common cutaneous malignancy of domestic cats, squamous cell carcinoma (SCC). Using total DNA sequencing we identified a novel feline papillomavirus in a nasal biopsy taken from a cat presenting with both nasal cavity lymphoma and recurrent squamous cell carcinoma affecting the nasal planum. We designate this novel virus as Felis catus papillomavirus 6 (FcaPV6). The complete FcaPV6 7453 bp genome was similar to those of other feline papillomaviruses and phylogenetic analysis revealed that it was most closely related to FcaPV3, although was distinct enough to represent a new viral type. Classification of FcaPV6 in a new genus alongside FcaPVs 3, 4 and 5 is supported. Archived excisional biopsy of the SCC, taken 20 months prior to presentation, was intensely positive on p16 immunostaining. FcaPV6, amplified using virus-specific, but not consensus, PCR, was the only papillomavirus detected in DNA extracted from the SCC. Conversely, renal lymphoma, sampled at necropsy two months after presentation, tested negative on FcaPV6-specific PCR. In sum, using metagenomics we demonstrate the presence of a novel feline papillomavirus in association with cutaneous squamous cell carcinoma.

Diagnostic Utility of Immunohistochemical Expressions of IMP3 Versus DOG1 and p63 in Salivary Gland Tumors.

The diverse site of origin and classification complexity of salivary glands tumors increase difficulties in their diagnosis. This study aimed to evaluate the specificity and diagnostic ability of immunohistochemical expressions of IMP3 versus DOG1 and p63 in cases of such tumors. Thirty paraffin-embedded salivary gland tumors were obtained from the Pathology Department Archive. Their diagnosis was confirmed. The specimens were then re-classified and evaluated using the IMP3, DOG1 and p63 immunohistochemical markers. There were 8 pleomorphic adenoma (PA), 12 mucoepidermoid carcinoma (MEC) and 10 adenoid cystic carcinoma (ADC) cases. All 12 MECs (100%) were IMP3 positive, while 30% of ADCs and only 25% of PAs were positive for IMP3. There was a statistically significant relationship between salivary gland tumors and IMP3 immunostaining (P =0.03). As regards to DOG1 results, 12.5% of PAs showed variable luminal positive immunostaining and 40% of ADCs showed weak luminal and abluminal immunostaining while 16.7% of MEC showed cytoplasmic staining. On the other hand, all ADCs (100%) showed moderate p63 reactivity in the nuclei of abluminal cells. All MEC cases (100%) were also p63-positive, showing a strong diffuse nuclear reactivity. A statistically significant relationship was noticed between salivary gland tumors and p63 immunostaining (P < 0.05). IMP3 is more sensitive for diagnosis of MEC than ADC. p63 is statistically significant in diagnosing salivary gland tumors (MEC and ADC). On the other hand, DOG1 staining is not sensitive in diagnosis of studied malignant salivary gland tumors, limiting its diagnostic utility.

Clinicopathological and Molecular Differences Between Gastric-type Mucinous Carcinoma and Usual-type Endocervical Adenocarcinoma of the Uterine Cervix.

We investigated differences in the clinicopathological and molecular characteristics between gastric-type mucinous carcinoma (GMC) and usual-type endocervical adenocarcinoma (UEA). We collected the clinicopathological information and performed targeted genomic sequencing analysis. GMCs exhibited significantly deeper invasion depth, larger horizontal spread, more advanced stage, more frequent distant metastasis, and more frequent parametrial and vaginal extension. Disease-free survival time of GMC patients was significantly shorter than that of UEA patients. GMCs displayed mutant p53 immunostaining pattern, whereas UEAs exhibited p16 block positivity. GMCs harbored mutations in KRAS, TP53, NF1, CDKN2A, STK11, and ARID1A. One GMC exhibited MDM2 amplification. In contrast, UEAs harbored mutations in HRAS, PIK3CA, and BRCA2. Two UEAs were found to have novel TP53 mutations. GMC is associated with more aggressive behavior than UEA. Distinctive p53 and p16 immunostaining patterns enable differential diagnosis. GMC and UEA exhibit genetic heterogeneity with potentially actionable molecular alterations.

Predictors of survival and recurrence after primary surgery for cervical metastasis of unknown primary.

Cervical metastasis from unknown primary (CUP) is commonly classified as an advanced overall stage. P16 or human papillomavirus (HPV) positivity in metastatic lymph nodes (LN) might be associated with a favorable survival outcome of CUP. Therefore, we evaluated the prognostic values of p16 immuno-positivity in LN and other clinicopathological factors in patients with squamous cell carcinoma CUP (SCCUP). This study involved 83 patients who underwent therapeutic neck dissection and panendoscopic examination and biopsy for suspected CUP. P16 immunostaining and HPV typing in LN were performed in 56 patients. Cox proportional hazard regression analyses were used to identify factors associated with overall survival (OS) and disease-free survival (DFS). Postoperatively, primary tumors (PT) were found in 32 (38.6%) patients, mainly (90.6%) in the oropharynx, and not found in 51 (61.4%) patients. The clinicopathological data (except for histological grade) and 5-year OS and DFS rates did not significantly differ between patients with and without PT identification (all P > 0.05). P16 positivity was associated with favorable OS and DFS outcomes in the patients with PT (P < 0.05) but not in those without PT (P > 0.1). Multivariate analyses showed that age (> 60years) and LN ratio (≥ 0.1) were the independent predictors of OS and DFS outcomes (all P < 0.05). P16 positivity or other factors were not independent factors. Age and LN ratio are significant risk factors of survival and recurrence after primary surgery for SCCUP. Prognostic significance of LN p16 positivity should be further studied.

Low prevalence of human papillomavirus infection in sinonasal inverted papilloma and oncocytic papilloma.

The aim of this study was to investigate the role of human papillomavirus (HPV) in sinonasal inverted papilloma (SIP) and sinonasal oncocytic papilloma (SOP) from a single institution and whether p16 can serve as a surrogate marker for HPV infection. This study included 49 subjects with SIP and 36 subjects with SOP. Formalin-fixed paraffin-embedded tissues were used to extract genomic DNA, and HPV detection was performed by utilizing a valid nested polymerase chain reaction approach that can detect all known HPV subtypes. Immunohistochemistry was used to evaluate the expression of p16 in all tumor sections. The presence of HPV DNA was found in 6.1% (3/49) of the SIP patients and 11.1% (4/36) of the SOP patients. All identified HPV subtypes in SIP were high-risk HPV, including HPV-16 (two patients) and HPV-58 (one patient). Regarding SOP, there were three patients positive for HPV-16 and one with low-risk HPV (type 6). In total, 11/49 (22.4%) SIP lesions and 10/36 (27.8%) SOP lesions were considered p16 positive, with p16 staining in more than 70% of tumor cells. There was only one SIP and one SOP that were positive for both HPV (high-risk HPV type 16) and p16 staining. HPV does not play an etiologic role in inverted papilloma or oncocytic papilloma of the sinonasal region. p16 immunostaining should not be used as a surrogate marker to evaluate the HPV infection status in these lesions.

Histo- and immunohistochemistry-based estimation of the TCGA and ACRG molecular subtypes for gastric carcinoma and their prognostic significance: A single-institution study.

Gastric cancers comprise molecularly heterogeneous diseases; four molecular subtypes were identified in the cancer genome atlas (TCGA) study, with implications in patient management. In our efforts to devise a clinically feasible means of subtyping, we devised an algorithm based on histology and five stains available in most academic pathology laboratories. This algorithm was used to subtype our cohort of 107 gastric cancer patients from a single institution (St. Michael’s Hospital, Toronto, Canada), which was divided into 3 cases of EBV-positive, 23 of MSI, 27 of GS and 54 of CIN tumours. 87% of the tumours with diffuse histology were classified as GS subtype, which was notable for younger age. Examining for characteristic molecular features, aberrant p53 immunostaining was seen most frequently in the CIN subtype (43% in CIN vs. 6% in others), whereas ARID1A loss was rarely seen (6% vs. 35% in others). HER2 overexpression was seen exclusively in CIN tumours (17% of CIN tumours). PD-L1 positivity was seen predominantly in the EBV and MSI tumours. As with the TCGA study, no survival differences were seen between the subtypes. A similar strategy was employed to approximate the Asian Cancer Research Group (ACRG) molecular subtyping, with the addition of p53 IHC to the algorithm. We observed rates of ARID1A loss and HER2 overexpression that were comparable to the ACRG study. In summary, our algorithm allowed for clinically feasible means of subtyping gastric carcinoma that recapitulated the key molecular features reported in the large scale studies.

Estimation of genomic instability and mutation induction by graphene oxide nanoparticles in mice liver and brain tissues.

The rapidly growing interest in using graphene-based nanoparticles in a wide range of applications increases human exposure and risk. However, very few studies have investigated the genotoxicity and mutagenicity of the widely used graphene oxide (GO) nanoparticles in vivo. Consequently, this study estimated the possible genotoxicity and mutagenicity of GO nanoparticles as well as possible oxidative stress induction in the mice liver and brain tissues. Nano-GO particles administration at the dose levels of 10, 20, or 40mg/kg for one or five consecutive days significantly increased the DNA breakages in a dose-dependent manner that disrupts the genetic material and causes genomic instability. GO nanoparticles also induced mutations in the p53 (exons 6&7) and presenilin (exon 5) genes as well as increasing the expression of p53 protein. Positive p53 reaction in the liver (hepatic parenchyma) and brain (cerebrum, cerebellum, and hippocampus) sections showed significant increase of p53 immunostaining. Additionally, induction of oxidative stress was proven by the significant dose-dependent increases in the malondialdehyde level and reductions in both the level of reduced glutathione and activity of glutathione peroxidase observed in GO nanoparticles administered groups. Acute and subacute oral administration of GO nanoparticles induced genomic instability and mutagenicity by induction of oxidative stress in the mice liver and brain tissues.

CD5 Expression Correlates with Inferior Survival and Enhances the Negative Effect of p53 Overexpression in Diffuse Large B Cell Lymphoma

Purpose De novo CD5+ DLBCL is increasingly recognized as a distinct pathologic phenomenon with a specific clinical picture. However, De novo CD5+ DLBCL has not been studied on a large scale in China. In this study, we evaluated the frequency, clinicopathological characteristics of de novo CD5+ DLBCL and prognostic impact of CD5 expression, and patient survival in our center. Methods In this study, we retrospectively investigated 745 DLBCL cases treated at Tianjin medical university cancer institute and hospital between 2000 and 2017, sub-classifying them as germinal center B cell-like (GCB) and non-GCB type by immunohistochemical staining with CD10, BCL6 and MUM-1, and then comparing the prognosis. We used a cutof ≥50% tumor cells for CD5 to be considered positive. Results In the enrolled DLBCL patients, 64 (9.2%) were CD5+ and 631 (90.8%) were CD5-. There was no significant difference in age, sex, extranodal involvement, serum LDH, C-myc overexpression and CNS relapse between these two groups. In the CD5+group, the cell of origin was non-GCB type in 46 cases (71.9%); the ratio of non-GCB type in the CD5+ group was higher than that in the CD5-group (P=0.033). Comparison of the clinical characteristics of CD5+ vs CD5-DLBCL patients showed that CD5+ DLBCL patients were more frequently elderly (&gt;60 years), and had B-symptoms, high performance status, stage III-IV, an IPI score &gt;2, and BM involvement. 46.9% of the CD5+ patients, compared to 9.4% CD5-DLBCL patients, showed BM involvement at diagnosis. Almost 84.4% of CD5+DLBCL patients had concurrent overexpression (≥50% of the tumor cells) of antiapoptotic Bcl-2, an unfavorable biomarker. This frequency was significantly higher than that in CD5-DLBCL patients (67.2%, P=0.032). Similarly, the P53 positive rate (≥50% of the tumor cells) of CD5+DLBCL (46.9%) is significantly higher than that of CD5-DLBCL (17.6%, P=0.011). Univariate Cox analysis identified the following prognostic factors: CD5 positive, age &gt;60 years, IPI≥3, BM/PB involvement, performance status and stage (III or IV). Intensive chemotherapy was not identified as significantly prognostic by univariate analysis. The CD5+GCB group showed no significant differences compared to CD5-GCB group for both PFS and OS, whereas the CD5+ non-GCB DLBCL and CD5- non-GCB DLBCL showed significantly worse prognosis compared to other groups. (P &lt; 0.001, PFS and OS, respectively) (Fig.1A; 1B; 1C; 1D) In CD5+ DLBCL, PFS and OS in patients treated with rituximab were significantly better than those without rituximab. Three-year PFS was 41.1% for the former and 15.4% for the latter (P=0.036, Fig. 1E), and three-year OS were 60.7 and 46.2% (P=0.047, Fig. 1F). Next, we evaluated the therapeutic responses of different chemotherapy regiments. A total of 20 patients received treatment with R-CHOP and 24 patients received DA-EPOCH-R. Patients treated with R-CHOP showed similar PFS and OS compared with intensive treatment group (Fig. 1G,1H). Of the 631 cases of CD5- DLBCL, only 111 cases (17.6%) showed p53 overexpression. In contrast, p53 was overexpressed in 30 (46.9%) of 64 CD5+ DLBCL. As shown in Fig. 1, PFS and OS in patients with overexpression of either p53 or CD5 alone were significantly different from those in patients with p53- /CD5- DLBCL. However, patients with p53 and CD5 co-overexpression had the worst PFS and OS (P &lt; 0.001, PFS and OS, respectively) (Fig. 1I, 1J). These data suggest that the negative prognostic impact of p53 and CD5 overexpression was augmented when both variables existed. In fact, all 30 patients with p53 and CD5 co-overexpression died within 40 months of diagnosis. Conclusion In summary, in this study we show that de novo CD5+ DLBCL, which occurs at a frequency (9.2%), was associated with unfavorable clinicopathologic variables and with inferior survival following R-CHOP and DA-EPOCH-R treatment. CD5+ DLBCL has a high frequency of p53 overexpression and CD5 augments the negative effect of p53 overexpression in DLBCL. Fig. 1 PFS (A) and OS (B) of patients with DLBCL according to the presence or absence of CD5. PFS (C) and OS (D) for the four DLBCL groups: CD5+ GCB DLBCL, CD5+ non-GCB DLBCL, CD5- GCB DLBCL and CD5- non-GCB DLBCL. PFS (E) and OS (F) of CD5+ DLBCL in the chemotherapy group and in the R-chemotherapy group. PFS (G) and OS (H) in CD5+ DLBCL treated with RCHOP and DA-EPOCH-R regimens. PFS (I) and OS (J) in patients with de novo DLBCL stratified according to p53 and CD5 immunostaining status. Figure 1 Disclosures No relevant conflicts of interest to declare.