Carcinoma Specimens Research Articles

The utility of claudin-4 versus MOC-31 and Ber-EP4 in the diagnosis of metastatic carcinoma in cytology specimens.

Claudin-4 is a sensitive and specific marker for carcinoma in effusion cytology. The authors examined the diagnostic use of claudin-4 versus MOC-31 and Ber-EP4 by comparing their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in differentiating carcinoma from mesothelioma and benign/mesothelial hyperplasia in effusion specimens. This retrospective study comprised a cohort of 229 cytology specimens, including 211 effusion fluid and 18 fine-needle aspiration specimens. Cytologic categories included 134 carcinoma, 28 mesothelioma, 46 indefinite (suspicious and atypical), and 21 benign. Cell block sections were stained for claudin-4 and compared with those previously stained for MOC-31 and Ber-EP4. Indefinite cases were further reclassified based on clinical and pathologic findings into benign (26 cases), mesothelioma (11 cases), and carcinoma (nine cases). None of the mesotheliomas (0/39) or benign effusions (0/47) were positive for claudin-4, whereas 134 of the 143 carcinoma specimens were positive. Compared to MOC-31 and Ber-EP4, claudin-4 had the highest specificity and PPV (100% for each), followed by Ber-EP4. Claudin-4 showed high sensitivity (93.7%), albeit lower than MOC-31. MOC-31 had the lowest specificity and PPV but the highest sensitivity and NPV. Ber-EP4 had the lowest sensitivity (91.6%). Claudin-4 can be used as a single marker for carcinoma with high sensitivity and superior specificity compared with MOC-31 and Ber-EP4. Mesothelial lineage can be ruled out when claudin-4 is positive. In equivocal cytology samples with few scattered cells of interest, a panel of claudin-4 and Ber-EP4 results in the highest combined sensitivity and specificity.

Correlation of P53 Expression with Clinicopathological Parameters, Hormone Receptors and HER 2 Neu Status in Breast Carcinoma

Background: Progesterone receptor (PR), estrogen receptor (ER) expression, human epidermal growth factor 2 (HER 2 neu) amplification/ overexpression, and p53 nuclear protein accumulation are all significant prognostic biomarkers in breast cancer (BC). This work aims to assess the co-presence of p53 overexpression with ER, PR, and HER 2 neu expression in invasive BC specimens by IHC and compare their expression with various clinicopathological features. Objectives: To evaluate the expression of ER, PR, HER-2neu and p53 in invasive breast carcinoma specimens and to correlate the expression of p53 with ER, PR, HER-2 neu and the clinicopathological parameters. Methods: Cases of Modified Radical Mastectomy received from June 2015 to May 2017 were studied. In addition to hematoxylin and eosin sections, immunohistochemical staining was done for ER, PR, HER 2 neu and p53. The expression of ER, PR, HER 2 neu and p53 was noted and the findings were correlated with other clinicopathological parameters. Results: Among 86 cases studied most of them belonged to the age group 41-60 years (54, 62.8%). A significant positive correlation of p53 over expression with ER and PR positive tumours and Triple Negative tumours was noted. Also p53 overexpression showed a positive significant correlation with tumour grade. Conclusion: P53 overexpression in breast carcinoma indicates an aggressive clinical course and can be used as a marker of poor prognosis.

Molecular portraits of clear cell ovarian and endometrial carcinoma with comparison to clear cell renal cell carcinoma

ObjectiveAdvanced clear cell gynecologic malignancies remain among the most challenging diseases to manage. We evaluated ovarian and endometrial clear cell carcinoma (OCCC and ECCC) specimens using comprehensive sequencing technology to identify mutational targets and compared their molecular profiles to histologically similar clear cell renal cell carcinoma (ccRCC). MethodsUsing next-generation sequencing (NGS), fragment analysis (FA), and in situ hybridization (ISH), 164 OCCC, 75 ECCC and 234 ccRCC specimens from 2015 to 2018 were evaluated and compared. ResultsThe highest mutation rates in ECCC and OCCC were noted in: ARID1A (75.0%, 87.5%), TP53 (34.8%, 11.1%), PIK3CA (25.0%, 46.8%), PPP2R1A (8.7%, 16.7%), MSI-high (8.8%, 6.4%) and PTEN (8.3%, 7.1%). Among these mutations, there was no significant difference between OCCC and ECCC mutation prevalence except in TP53, with higher mutation rates in ECCC versus OCCC (34.8 vs. 11.1%, respectively, p < 0.05). ccRCC demonstrated different mutation profiles with higher mutation rates in VHL (80.3%), PBRM1 (43.9%), SETD2 (31.1%), and KDM5C (29.2%). By contrast, VHL, PBRM1, and SETD2 mutations were not found in ECCC and OCCC (0.0%). Compared to ccRCC and ECCC, OCCC was found to have a significantly higher tumor mutation burden (TMB) (19.1%). ConclusionGynecologic and renal CCC demonstrate separate and disparate somatic profiles. However, OCCC and ECCC are diseases with similar profiles. TMB and MSI analyses indicate that a subset of OCCC may benefit from immunotherapy. Prospective clinical trials are needed and are underway to examine targeted therapies in these gynecologic disease subtypes.

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples.

Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics. Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex© Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]. The combination of FusionPlex© Solid tumor with ArcherDX® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens. Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

The Contribution of Matrix Metalloproteinase-7 Promoter Genotypes to Hepatocellular Carcinoma Susceptibility.

Metalloproteinase-7 (MMP-7) has been previously found to be up-regulated in hepatocellular carcinoma (HCC) specimens and cells, favoring epithelial-mesenchymal transition. However, the contribution of MMP-7 genotypes to HCC has not been revealed to date. The study aimed to evaluate the contribution of MMP-7 promoter A-181G (rs11568818) and C-153T (rs11568819) genotypes on the risk of HCC in Taiwan, where HCC incidence is extremely high compared to worldwide data. In this case-control study, MMP-7 genotypes and their association with cigarette smoking and alcohol drinking habits were determined in 298 HCC patients and 889 healthy subjects by a typical polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Ever smokers and alcohol drinkers were represented with higher percentages in the case group compared to the control group. MMP-7 rs11568818 genotypes were not found differentially distributed in case and control groups (p for trend=0.5246). People of the analyzed cohort of the present study were all of CC genotypes at their rs11568819 polymorphic sites, without any CT or TT genotypes. As for gene-lifestyle interactions, people with variant genotypes at MMP-7 rs11568818 had the same odds for HCC development compared to the wild-type AA genotype, no matter whether the subjects belonged to the smoker, non-smoker alcohol drinker, or non-drinker groups. MMP-7 variant genotypes did not present any significance towards being a marker for HCC risk in Taiwanese.

Programmed cell death 1 and programmed cell death ligand 1 expression in invasive breast carcinoma using CAL10 and NAT105 immunostaining

ABSTRACT Increased incidence of breast cancer has stimulated development of new diagnostic and therapeutic methods. The programmed cell death 1 (PD1) pathway and its inhibitors are promising avenues for investigation. PD1 includes PD ligands 1 (PDL1) and 2 (PDL2). We investigated the expression of PD1 and PDL1 in invasive breast carcinomas using immunohistochemical staining. We used 171 invasive breast carcinoma specimens from which tissue microarray blocks were created. Immunohistochemical staining of PD1 using NAT105, and PDL1 using CAL10 was performed on tissue microarray sections. NAT105 and CAL10 are useful clones for detecting expression of PD1 and PDL1. PD1 and PDL1 immunostaining was significantly stronger in carcinomas with basal-like phenotype compared to other molecular breast cancer types. PD1 and PDL1 expression also was associated with a high histologic grade and a high Ki-67 index. PD1 expression also was associated with lymphovascular invasion and axillary metastasis. PD1 and PDL1 expression is associated with aggressive tumor behavior and a basal-like phenotype in breast cancer. We suggest that inhibition of the PD1/PDL1 pathway, particularly in triple negative breast carcinomas with basal-like phenotype, might be useful for targeted immunotherapy.

Fructose-1,6-bisphosphatase 1 functions as a protein phosphatase to dephosphorylate histone H3 and suppresses PPARα-regulated gene transcription and tumour growth.

Tumour cells exhibit greater metabolic plasticity than normal cells and possess selective advantages for survival and proliferation with unclearly defined mechanisms. Here we demonstrate that glucose deprivation in normal hepatocytes induces PERK-mediated fructose-1,6-bisphosphatase 1 (FBP1) S170 phosphorylation, which converts the FBP1 tetramer to monomers and exposes its nuclear localization signal for nuclear translocation. Importantly, nuclear FBP1 binds PPARα and functions as a protein phosphatase that dephosphorylates histone H3T11 and suppresses PPARα-mediated β-oxidation gene expression. In contrast, FBP1 S124 is O-GlcNAcylated by overexpressed O-linked N-acetylglucosamine transferase in hepatocellular carcinoma cells, leading to inhibition of FBP1 S170 phosphorylation and enhancement of β-oxidation for tumour growth. In addition, FBP1 S170 phosphorylation inversely correlates with β-oxidation gene expression in hepatocellular carcinoma specimens and patient survival duration. These findings highlight the differential role of FBP1 in gene regulation in normal and tumour cells through direct chromatin modulation and underscore the inactivation of its protein phosphatase function in tumour growth.

Spatial maps of Tcell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response.

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic Tcell receptors (TCRs). Determining the spatial distributions of Tcell clonotypes in tissues is essential to understanding Tcell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire. Here, we developed Slide-TCR-seq, a 10-μm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. We confirmed the ability of Slide-TCR-seq to map the characteristic locations of Tcells and their receptors in mouse spleen. In human lymphoid germinal centers, we identified spatially distinct TCR repertoires. Profiling Tcells in renal cell carcinoma and melanoma specimens revealed heterogeneous immune responses: Tcell states and infiltration differed intra- and inter-clonally, and adjacent tumor and immune cells exhibited distinct gene expression. Altogether, our method yields insights into the spatial relationships between clonality, neighboring cell types, and gene expression that drive Tcell responses.

MiR-146a participates in the regulation of cancer stemness of oral carcinoma cells

Background/purposeIncreasing evidence regarded the existence of cancer stem cells (CSCs) as a leading cause of therapy failure and tumor relapse due to their self-renewal and differentiation abilities. Although ectopic overexpression of micro-RNAs (miRNAs) can modulate the cancer stemness and tumor development in oral cancer, their molecular mechanism is still unclear. Therefore, in the present study, we attempt to uncover the role of miR-146a in the maintenance of oral CSCs. Materials and methodsThe expression of miR-146a was determined using qRT-PCR analysis. Aldehyde dehydrogenase (ALDH) enzymic activity and sphere formation assays were used to evaluate the cancer stemness and self-renewal, respectively. Functional assays, including migration/invasion Transwell and colony formation assay, were used to evaluate the aggressive abilities. Luciferase reporter assay was performed to validate the relationship between miR-146a and Numb. ResultsIn the present study, we reported an increased expression of miR-146a in the oral squamous cell carcinoma (OSCC) specimen, primary OSCC cells sphere, and high ALDH1 activity population within OSCC cells. Inhibition of miR-146a significantly suppressed the ALDH1 activity, self-renewal capacity, and aggressive abilities, including migration, invasion, and colony formation. Moreover, we demonstrated that Numb is a functional target of miR-146a in OSCC-CSCs. Notably, silencing of Numb could retrieve the self-renewal and migration impaired by knockdown of miR-146a. ConclusionOur results indicate that miR-146a can regulate the cancer stemness in OSCC by modulating Numb, and hence miR-146a/Numb axis can serve as a potential target for oral cancer therapy.

Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH.

Although molecular profiling of DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor specimens has become more common in recent years, it remains unclear how discrete FFPE processing variables may affect detection of copy number variation (CNV). To better understand such effects, array comparative genomic hybridization (aCGH) profiles of FFPE renal cell carcinoma specimens that experienced different delays to fixation (DTFs; 1, 2, 3, and 12 hours) and times in fixative (TIFs; 6, 12, 23, and 72 hours) were compared to snap-frozen tumor and blood specimens from the same patients. A greater number of regions containing CNVs relative to commercial reference DNA were detected in DNA from FFPE tumor specimens than snap-frozen tumor specimens even though they originated from the same tumor blocks. Extended DTF and TIF affected the number of DNA segments with a copy number status that differed between FFPE and frozen tumor specimens; a DTF ≥3 hours led to more segments, while a TIF of 72 hours led to fewer segments. Importantly, effects were not random as a higher guanine-cytosine (GC) content and/or a higher percentage of repeats were observed among stable regions. While limiting aCGH analysis to FFPE specimens with a DTF <3 hours and a TIF <72 hours may circumvent some effects, results from FFPE specimens should be validated against fresh or frozen specimens whenever possible.

372. ASSESSMENT OF TUMOR REGRESSION GRADE IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA AFTER CHEMORADIOTHERAPY: COMPARISON OF 3 COMMONLY USED SCORING SYSTEMS

Abstract Tumor regression grade (TRG) is a measure of histopathological response of cancer to neoadjuvant chemoradiotherapy (nCRT) and is associated with outcomes. Several TRG system are used in esophageal cancer: Becker (0%,1 ~ 10%,10 ~ 50%, &amp;gt;50% vital residual tumor cell [VRTC]), Chirieac (0%,1 ~ 50%, &amp;gt;50% VRTC) and Japan TRG (0%,1 ~ 33%,33 ~ 66%, &amp;gt;66% VRTC). Although these methods are generally accepted, currently there is no common standard. We compared the application of three systems for assessment of TRG. Hematoxylin and eosin–stained slides from 482 resection specimens of esophageal squamous cell carcinoma following nCRT were independently reviewed by three pathologists. Interobserver agreement was analyzed by kappa statistics. The prognostic strength of each TRG system was assessed using the concordance index. Interobserver agreement for the Chirieac system showed the best kappa value (0.93) followed by the Becker (0.90) and JTRG (0.87) system. All three models predicted survival (P &amp;lt; 0.0001) independent of yp-stage in multivariate analysis. Concordance indices of the Chirieac, Becker and JTRG were: 0.627, 0.627 and 0.633, respectively (higher number = better prediction). When comparing with Chirieac system, the JTRG system more accurately predicted survival (p = 0.03) while the Becker system did not (p = 0.12). Chirieac system is most reproducible for the evaluation of esophageal cancer response to nCRT. However, the JTRG system seemed to be more accurate in predicting survival.

435. A DIAGNOSTIC PITFALL; SMALL CELL CARCINOMA-LIKE FEATURES IN BASALOID SQUAMOUS CELL CARCINOMA OF THE ESOPHAGUS

Abstract Esophageal basaloid squamous cell carcinoma may resemble small cell carcinoma biopsy specimens and cause difficulties in pathology diagnosis. We aimed to explore the pathological features of basaloid squamous cell carcinomas in biopsy specimens that mimic small cell carcinoma and to clarify their diagnostic and prognostic features. This study will contribute to draw attention to an important pitfall in pathology diagnosis in endoscopic biopsy specimens of the esophagus. Thirty biopsy specimens of esophageal basaloid squamous cell carcinoma were reviewed and compared with 13 matched surgical specimens. A small cell carcinoma-like feature was defined as, when at least one of the three following findings was observed: 1) diffuse growth pattern, 2) nuclear molding and 3) nuclear crush artifact. Immunohistochemistry of synaptophysin, chromogranin A, CD56 (neuroendocrine markers) and p16 were performed in all cases. Small cell carcinoma-like features, such as diffuse growth, nuclear molding, or nuclear crush artifact, were identified in 80% (24/30) of the biopsies and in 77% (10/13) of the surgery specimens, but in a proportionally much smaller area in the surgical specimens than in the biopsy samples. The presence of a small cell carcinoma-like feature had no impact on patients´ outcome. Immunohistochemically, synaptophysin and chromogranin A were consistently negative, while CD56 was expressed in 42% (10/24) basaloid squamous cell carcinomas with small cell carcinoma-like features. p16, a highly sensitive marker for small cell carcinoma, was also expressed in 8% (2/24). In conclusion, small cell carcinoma-like features are frequent and conspicuous in biopsies, which may probably be caused by exogenous factors such as friction and external pressure that occur in biopsy procedure and in the tumor environment. Small cell carcinoma-like features may lead to a misinterpretation of a true small cell carcinoma, if CD56 is an only neuroendocrine marker expressed. p16 expression may be also detected in basaloid squamous cell carcinoma.

Differential DNA Methylation of THOR and hTAPAS in the Regulation of hTERT and the Diagnosis of Cancer.

Simple SummaryBecause of its high prevalence of >45% in 9 out of 11 (82%) cancer types screened, THOR hypermethylation has been suggested to be a frequent telomerase-activating mechanism in hTERT-expressing tumor types, e.g., in cancers of the prostate, breast, blood, colon, lung, bladder, and brain. In this prime example, we present detailed DNA methylation profiles in urothelial cancer that reveal the exact positions of the most differentially methylated CpG dinucleotides within the THOR region in order to design an efficient Methylation-Specific PCR (MSPCR) approach for diagnostic and prognostic purposes. Furthermore, our data suggest an epigenetic mechanism regulating hTERT expression through the methylation status of THOR and lncRNA hTAPAS.Background: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5′ of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). Methods: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. Results: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. Conclusions: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided.

Real-World Comprehensive Genomic Profiling Success Rates in Tissue and Liquid Prostate Carcinoma Specimens.

Challenges with sequencing tissue samples from patients with prostate cancer have been reported in clinical trials. To assess the success rate of comprehensive genomic profiling (CGP) for prostate cancer patients, we analyzed a real-world cohort who underwent sequencing of their prostate tissue sample as well as a subset of patients with a reflex liquid biopsy. Overall, a significant majority (82%) of tissue prostate carcinoma samples yielded reportable CGP results. Of those samples that were unsuccessful, most (75%) were inadequate samples that did not meet pre-established criteria to advance into sequencing. For cases where liquid CGP was performed if tissue CGP was unsuccessful, mutations that were likely attributable to prostate carcinoma were observed in most cases and all cases were successful in generating a report. These results suggest that, for CGP testing, prostate cancer tissue is a reasonable matrix type and that liquid samples can be effectively used as an alternative to tissue.

Exploring APOBEC3A and APOBEC3B substrate specificity and their role in HPV positive head and neck cancer

SummaryAPOBEC3 family members are cytidine deaminases catalyzing conversion of cytidine to uracil. Many studies have established a link between APOBEC3 expression and cancer development and progression, especially APOBEC3A (A3A) and APOBEC3B (A3B). Preclinical studies with human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) and clinical trial specimens revealed induction of A3B, but not A3A expression after demethylation. We examined the kinetic features of the cytidine deaminase activity for full length A3B and found that longer substrates and a purine at −2 position favored by A3B, whereas A3A prefers shorter substrates and an adenine or thymine at −2 position. The importance and biological significance of A3B catalytic activity rather than A3A and a preference for purine at the −2 position was also established in HPV+ HNSCCs. Our study explored factors influencing formation of A3A and A3B-related cancer mutations that are essential for understanding APOBEC3-related carcinogenesis and facilitating drug discovery.

Analysis of TERT Rs2736100 genotype distribution in laryngeal squamous cell carcinoma patients.

The applicability of the telomerase reverse transcriptase (TERT) gene Rs2736100 polymorphism in cancer research has been well documented for various malignancies except for head and neck cancers, where data is sparse. This study aimed to analyze this polymorphism with the pathological characterizers of laryngeal squamous cell carcinoma (LSCC) patients. Genetic testing was performed using the Real-time PCR technique on 56 paired samples of biological material (blood and tissue). Data were analyzed using Epi info 7 software. The subjects were predominantly male (95% vs. 5%), with a median age of 62 years, and smokers (89%). The primary tumor origin site was the glottic region (34%), and the advanced clinical stages III-IV were more common (46% vs. 18%). Results show high frequencies for the mutated variants of Rs2736100 (CC 36%>AC 34%>AA 30%), while distribution according to tumor classification criteria leaned towards moderately differentiated carcinoma specimens in T3-T4 stages for the AC/CC variants (P-value without statistical significance) but positively favored the relationship between the AA variant and lack of lymph node metastasis (P=0.0106). The genotypes tend to associate themselves with a better histological presentation regarding the pattern of tumor invasion and, thus, better prognostic values for LSCC. Results suggest that the wild-type genotype of TERT Rs2736100 may be a protective factor for lymph node metastasis and histological pattern of tumor invasion in LSCC. Results regarding the synergistic relationship between cancer and smoking corroborate literature data for moderate to severe smokers regardless of the genetic variant.

Detection of High-Risk Human Papillomavirus DNA in Invasive Ductal Carcinoma Specimens.

According to several studies, there is an association between human papillomavirus (HPV) and breast cancer. Therefore, detection and genotyping of HPV seem important. The present study aimed to investigate the presence of HPV DNA in breast tissues by analyzing the L1 gene. This case-control study was conducted on 63 formalin-fixed paraffin-embedded (FFPE) tissues of invasive ductal carcinoma (IDC) as the case group and 32 FFPE tissues of fibroadenoma as the control group. HPV DNA was detected using the polymerase chain reaction assay. Positive samples were then subjected to genotyping. All statistical analyses were performed in SPSS version 22.0. The patients' age ranged from 15 to 92 years, with a mean age of 43.54±16.36 years. HPV DNA was detected in 17/95 (17.89%) samples, including 9/32 (28.12%) fibroadenoma samples and 8/63 (12.69%) IDC samples. No significant difference was observed regarding the presence of HPV DNA between the IDC and fibroadenoma tissues (P=0.08). However, a significant difference was found in the detection of high-risk HPV (HR-HPV) between the case and control groups (P=0.03). In the case group, 87.5% of the detected viruses (7/8 samples) were HR-HPV, while in the control group, 22.22% of positive samples (2/9 samples) were HR-HPV (P=0.03). Based on the results, HR-HPV and low-risk HPV genotypes were detected in 53% (9/17) and 47% (8/17) of positive samples, respectively. In this study, 12.69% of IDC samples were positive for HPV genomes, and HR-HPV was detected in 87.5% of these samples. The present results suggest the important role of HR-HPV in the development of breast cancer.

1235P Analytical performance of PD-L1 IHC 28-8 pharmDx in gastric, gastroesophageal junction (GEJ), and esophageal carcinoma evaluated using combined positive score (CPS)

PD-L1 IHC 28-8 pharmDx was used in conjunction with Combined Positive Score (CPS) in the phase III CheckMate -649 clinical trial sponsored by Bristol Myers Squibb to assess gastric, gastroesophageal junction (GEJ), and esophageal adenocarcinoma. The assay demonstrated clinical utility in identifying patients who have the greatest overall survival benefit and progression-free survival benefit from first-line treatment with nivolumab (OPDIVO®) in combination with chemotherapy. The precision, robustness, and external reproducibility of PD-L1 IHC 28-8 pharmDx was assessed in gastric, GEJ, and esophageal carcinoma. Analytical specimens represented multiple carcinoma subtypes, including adenocarcinoma. Antigen retrieval was conducted on PT Link and staining was performed with Autostainer Link 48 platform using PD-L1 IHC 28-8 pharmDx. PD-L1 staining was assessed using CPS. Assay performance was validated at the CPS ≥ 5 expression cutoff. PD-L1 expression was measured on 1226 unique commercially procured cases of human FFPE specimens (439 gastric, 375 GEJ, and 412 esophageal carcinoma). Combined precision was measured in a single study which included the following conditions: inter-day, inter-run, inter-instrument, inter-operator, and reagent inter-lot. Assay precision demonstrated an overall agreement of 95.6% with a corresponding two-sided 95% confidence interval of (92.7%, 98.3%). Assay robustness was measured for: PT Link Incubation Time, PT Link Incubation Temperature, Target Retrieval Solution pH, Target Retrieval Solution Reuse, and Tissue Section Thickness. Assay robustness agreements were estimated above 90% with the lower bound of two-sided 95 percent confidence intervals at 85.5% or higher. When tested at three external sites, intra- and inter-site reproducibility, and intra- and inter-observer agreements were estimated above 91% with the lower bound of two-sided 95 percent confidence intervals at 86.0% or higher. PD-L1 IHC 28-8 pharmDx has shown to be precise, robust, and reproducible in detecting PD-L1 expression in FFPE human gastric, GEJ, and esophageal carcinoma specimens using the CPS scoring algorithm.

Combined evaluation of both WEE1 and phosphorylated cyclin dependent kinase 1 expressions in oral squamous cell carcinomas predicts cancer recurrence and progression

Background/purposeWEE1 is a mitotic inhibitor at G2 checkpoint of the cell cycle that negatively regulates cyclin-dependent kinase 1 (CDK1) through inhibitory phosphorylation. This study assessed whether the expressions of both WEE1 and phosphorylated CDK1 in specimens of oral squamous cell carcinoma (OSCC) might predict the OSCC recurrence and progression. Materials and methodsThis study used immunohistochemistry to examine the expressions of WEE1 and phosphorylated CDK1 proteins in 75 specimens of OSCC and 30 specimens of normal oral mucosa (NOM). ResultsThe mean WEE1 labeling index (LI) was significantly lower in 75 OSCC samples than in 30 NOM samples (P < 0.001), whereas the mean phosphorylated CDK1 LI was significantly higher in 75 OSCC samples than in 30 NOM samples (P < 0.001). We found a significant association of low WEE1 LI (<21%) with OSCC recurrence (P = 0.047) and a significant association of low phosphorylated CDK1 LI (<10%) with larger tumor size (P = 0.011) and more advanced clinical stages (P = 0.021) of OSCC. ConclusionCombined evaluation of WEE1 and phosphorylated CDK1 LI in specimens of OSCC may predict the OSCC recurrence and progression.

Identification of potential biomarkers and novel therapeutic targets through genomic analysis of small cell bladder carcinoma and associated clinical outcomes.

Small cell bladder carcinoma (SCBC) represents a rare histologic variant with a poor prognosis and for which no routine biomarkers exist. Limited reports of genomic sequencing in SCBC have demonstrated a high prevalence of TP53 and RB1 gene mutations, though the prognostic value of these and other gene variants in SCBC remains undefined. In this study, we performed targeted genomic sequencing on a cohort of SCBC patients and correlated genomic findings with clinical outcomes to identify potential novel biomarkers. Thirty-one patients with SCBC and available treatment-naïve tumor specimens were identified from an institutional database (23 limited stage [LS], 8 extensive stage [ES]). Small cell carcinoma specimens were microdissected and subjected to tumor next-generation whole-exon sequencing with a 592 gene panel. Kaplan-Meier techniques and Cox proportional hazards models were used to evaluate genomic aberration association with relapse-free survival (RFS) and overall survival (OS) in the limited stage cohort. The most common pathogenic gene variants included ARID1A (48%), TP53 (48%) and RB1 (48%). Mutations in genes with potential therapeutic targets not routinely evaluated in SCBC included BRCA1/2 (16%), POLE (13%), JAK2 (13%), PDGFB (13%) and FGFR3 (3%). Multiple novel biomarker candidates showed trends for improvements in OS in the LS subset including ERCC2 (HR 0.322, P = 0.122) and RB1 (HR 0.481, P = 0.182), while LS patients with TP53 mutations (HR 2.730, P = 0.056), and MCL1 gene amplification (HR 4.183, P = 0.018) suggested inferior OS. Additionally, gene or copy number variants with potential prognostic benefit included UBR5 and DAXX (P = 0.02, [hazard ratios nonestimable due to zero events in biomarker positive groups]). These results support the role for tumor genomic profiling in SCBC and identify multiple potential novel biomarkers and therapeutic targets in this rare disease. Efforts to validate these findings should lead to improved decision-making and treatment outcomes in SCBC.