Phenotypic immune characterization of gastric and esophageal adenocarcinomas reveals profound immune suppression in esophageal tumor locations.

Background: Tumors in the distal esophagus (EAC), gastro-esophageal junction including cardia (GEJAC), and stomach (GAC) develop in close proximity and show strong similarities on a molecular and cellular level. However, recent clinical data showed that the effectiveness of chemo-immunotherapy is limited to a subset of GEAC patients and that EACs and GEJACs generally benefit less from checkpoint inhibition compared to GACs. As the composition of the tumor immune microenvironment drives response to (immuno)therapy we here performed a detailed immune analysis of a large series of GEACs to facilitate the development of a more individualized immunomodulatory strategy. Methods: Extensive immunophenotyping was performed by 14-color flow cytometry in a prospective study to detail the immune composition of untreated gastro-esophageal cancers (n=104) using fresh tumor biopsies of 35 EACs, 38 GEJACs and 31 GACs. The immune cell composition of GEACs was characterized and correlated with clinicopathologic features such as tumor location, MSI and HER2 status. The spatial immune architecture of a subset of tumors (n=30) was evaluated using multiplex immunohistochemistry (mIHC) which allowed us to determine the tumor infiltration status of CD3+, CD8+, FoxP3+, CD163+ and Ki67+ cells. Results: Immunophenotyping revealed that the tumor immune microenvironment of GEACs is heterogeneous and that immune suppressive cell populations such as monocytic myeloid-derived suppressor cells (mMDSC) are more abundant in EACs compared to GACs (p<0.001). In contrast, GACs indicated a proinflammatory microenvironment with elevated frequencies of proliferating (Ki67+) CD4 Th cells (p<0.001), Ki67+ CD8 T cells (p=0.002), and CD8 effector memory-T cells (p=0.024). Differences between EACs and GACs were confirmed by mIHC analyses showing lower densities of tumor- and stroma-infiltrating Ki67+ CD8 T cells in EAC compared to GAC (both p=0.021). Discussions: This comprehensive immune phenotype study of a large series of untreated GEACs, identified that tumors with an esophageal tumor location have more immune suppressive features compared to tumors in the gastro-esophageal junction or stomach which might explain the location-specific responses to checkpoint inhibitors in this disease. These findings provide an important rationale for stratification according to tumor location in clinical studies and the development of location-dependent immunomodulatory treatment approaches.

Prediction of the Ki-67 expression level in head and neck squamous cell carcinoma with machine learning-based multiparametric MRI radiomics: a multicenter study.

BACKGROUND: This study aimed to develop and validate a machine learning (ML)-based fusion model to preoperatively predict Ki-67 expression levels in patients with head and neck squamous cell carcinoma (HNSCC) using multiparametric magnetic resonance imaging (MRI). METHODS: A total of 351 patients with pathologically proven HNSCC from two medical centers were retrospectively enrolled in the study and divided into training (n = 196), internal validation (n = 84), and external validation (n = 71) cohorts. Radiomics features were extracted from T2-weighted images and contrast-enhanced T1-weighted images and screened. Seven ML classifiers, including k-nearest neighbors (KNN), support vector machine (SVM), logistic regression (LR), random forest (RF), linear discriminant analysis (LDA), naive Bayes (NB), and eXtreme Gradient Boosting (XGBoost) were trained. The best classifier was used to calculate radiomics (Rad)-scores and combine clinical factors to construct a fusion model. Performance was evaluated based on calibration, discrimination, reclassification, and clinical utility. RESULTS: Thirteen features combining multiparametric MRI were finally selected. The SVM classifier showed the best performance, with the highest average area under the curve (AUC) of 0.851 in the validation cohorts. The fusion model incorporating SVM-based Rad-scores with clinical T stage and MR-reported lymph node status achieved encouraging predictive performance in the training (AUC = 0.916), internal validation (AUC = 0.903), and external validation (AUC = 0.885) cohorts. Furthermore, the fusion model showed better clinical benefit and higher classification accuracy than the clinical model. CONCLUSIONS: The ML-based fusion model based on multiparametric MRI exhibited promise for predicting Ki-67 expression levels in HNSCC patients, which might be helpful for prognosis evaluation and clinical decision-making.

[Comparative immunohistochemistry study of different antibodies clones for detection of breast cancer markers (estrogen receptor, progesterone receptor, HER2/neu, Ki-67)]. / Sravnitel'noe issledovanie antitel razlichnykh klonov dlya vyyavleniya markerov raka molochnoi zhelezy (retseptorov estrogenov, progesterona, HER2/neu, Ki-67) immunogistokhimicheskim metodom.

OBJECTIVE: A comparative study of detection of breast cancer markers (estrogen receptors, progesterone receptors, HER2/neu, Ki-67) by immunohistochemical method with antibodies produced by PrimeBioMed (Russia) and antibodies produced by Roche Ventana (USA). MATERIAL AND METHODS: Surgical specimens and biopsies from 37 patients with invasive breast cancer were used. Sections were stained with antibodies of clones ER SP1 and GM030, PR 1E2 and PBM-5B8, HER2/neu 4B5 and PBM-46A6, Ki-67 30-9 and GM010. RESULTS: There was a high positive and significant correlation between the immunohistochemistry results and antibodies of the clones ER-SP1 and GM030, PR1E2 and PBM-5B8, HER2/neu4B5 and PBM-46A6, Ki-67 30-9 and GM010. CONCLUSION: The study showed the possibility of using antibodies of clones GM030, HER2/neu 4B5, PBM-46A6, GM010 (PrimeBioMed) on the Ventana Bench Marck Ultra automatic immunostainer using the detection system UltraView Universal DAB Detection Kit.

The Predictive Role of Serum Lipid Levels, p53 and ki-67, According to Molecular Subtypes in Breast Cancer: A Randomized Clinical Study.

Dyslipidemia is a component of metabolic syndrome, having an important role in the carcinogenesis of different tumor types, such as prostate, ovarian, or renal cancer. The number of studies on the predictive potential of the different components of the lipid profile with a predictive potential in breast cancer is quite low. The evaluation of the lipid profile was carried out for the 142 patients who benefited from neoadjuvant therapy (NAC) in order to identify a potential predictive biomarker. The serological sample collection was performed sequentially according to a standardized protocol, pre-NAC, post-NAC and 6 months post-NAC after a 6-h pre-collection fast. We also investigated in the general group the presence or absence of the p53 mutation (TP53) and of the mitotic index ki-67, respectively, in relation to the molecular subtypes. The menopausal status, tumor size, family history, grading, Ki-67, p53 and LN metastases have a predictive nature regarding overall survival (OS) (p < 0.05), while for disease free survival (DFS), only tumor size, tumor grading, Ki-67 > 14, and p53+ are of predictive nature. The genetic and molecular analysis carried out in our group indicates that 71.67% have a Ki-67 score higher than 14%, and 39% of the patients have the positive P53 mutation. The multivariate analysis in the case of patients included in the TNBC subtype showed that the increased tumor volume (p = 0.002) and increased level of HDL (p = 0.004) represent predictive factors for the tumor response rate to NAC. High HDL-C levels before NAC and increased LDL-C levels after NAC were associated with the better treatment response in ER-positive and HER2+ breast cancer patients. Increased HDL-C values and tumor volume represent predictive factors as to the response rate to NAC in the case of patients included in the TNBC subtype. Regarding the ER+ and HER2+ subtypes, increased levels of HDL-C pre-NAC and increased levels of LDL-C post-NAC were associated with a better therapeutic response rate. Tumor grading, Ki-67, p53, and LN metastases have a predictive nature for OS, while tumor size, tumor grading, and Ki-67 > 14, and p53+ are predictive for DFS.

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

Evaluation of histological and ultrastructural changes provoked by prenatal tramadol on postnatal cortical cerebellar neuronal development in rats: possible implication of Ki67, GFAP and MicroRNA-7/P53 signalling trajectories.

Tramadol is a novel centrally acting analgesic. Despite, its implementation during pregnancy may impair neuronal survival and synaptic development in neonatal cerebella. The current investigation assessed the histological and ultrastructural alterations in postnatal cortical cerebellar neuronal development induced by prenatal tramadol. 30 offsprings were divided to control group I: fifteen pups born to mothers given saline from D10 till D21 of gestation. Tramadol-treated group II: fifteen pups born to mothers received tramadol HCL (50 mg/kg/day) from D10 till D21 of gestation. Pups were categorized into three subgroups (a, b, and c) and offered for sacrifice on the seventh, fourteenth and twenty-first post-natal days. Light microscopic examination revealed the overcrowding and signs of red degeneration affecting purkinje cell layer. Neurodegenerative signs of both purkinje and granule cell neurons were also confirmed by TEM in form of chromatin condensation, dilated Golgi channels, disrupted endoplasmic reticulum, marked infolding of the nuclear envelope and decrease in granule cell precursors. In addition, the astrocytic processes and terminal nerve axons appeared with different degrees of demyelination and decreased number of oligodendrocytes and degenerated mitochondria. Furthermore, group II exhibited an increase in P53 immune expression. The area percentage of apoptotic cells detected by TUNEL assay was significantly increased. Besides to the significant decrease of Ki67 immunoreactivity in the stem neuronal cell progenitors. Quantitative PCR results showed a significant decline in micro RNA7 gene expression in tramadol treated groups resulting in affection of multiple target genes in P53 signaling pathways, improper cortical size and defect in neuronal development.

The association between calreticulin and glucagon-like peptide-1 expressions with prognostic factors in high-grade gliomas.

OBJECTIVE: The aim of this study is to present the expressions of Calreticulin (CALR) and Glucagon-like peptide-1 (GLP-1) in high-grade gliomas and to further show the relation between the levels of these molecules and Ki-67 index, presence of Isocitrate dehydrogenase (IDH)-1 mutation, and tumor grade. PATIENTS AND METHODS: A total of 43 patients who underwent surgical resection due to high-grade gliomas (HGG) (grades III and IV) were included. The control group comprised 27 people who showed no gross pathology in the brain during the autopsy procedures. Adequately sized tumor samples were removed from each patient during surgery, and cerebral tissues were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. RESULTS: Patients with high-grade gliomas showed significantly higher levels of CALR and significantly lower levels of GLP-1 when compared to control subjects (P = 0.001). CALR levels were significantly higher, GLP-1 levels were significantly lower in grade IV gliomas than those in grade III gliomas (P = 0.001). Gliomas with negative IDH-1 mutations had significantly higher CALR expressions and gliomas with positive IDH-1 mutations showed significantly higher GLP-1 expressions (P = 0.01). A positive correlation between Ki-67 and CALR and a negative correlation between Ki-67 and GLP-1 expressions were observed in grade IV gliomas (P = 0.001). CONCLUSIONS: Our results showed that higher CALR and lower GLP-1 expressions are found in HGGs compared to normal cerebral tissues.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Immunohistochemical and molecular evaluation of TUSC2 expression in breast cancer.

OBJECTIVE: Tumor suppressor candidate 2 has shown to be deleted in lung, colon, and bladder cancer types. In the present study, we aimed to investigate the expression of TUSC2 in breast cancer. MATERIALS AND METHODS: A total of thirty patients with breast cancer were included in the study. Normal and tumor tissue samples from fresh mastectomy materials were stored at -80 C until the number of cases was completed for gene expression analysis. Histopathological examination was carried out with routine hematoxylin & eosin method. TUSC2 staining was performed for immunohistochemical analysis. RESULTS: The tumors of thirteen patients were Luminal A, fourteen patients were Luminal B, one patient was cerbB2(+), and tumors of two patients were triple-negative. Ki67 proliferation index was less than 14% in fifteen cases and tumor size was less than 2 cm in seven cases. Lymphovascular invasion and lymph node metastasis were present in thirteen cases. Statistically, TUSC2 expression significantly decreased or was lost in breast tumor tissues compared to normal tissues (p < 0.0001). TUSC2 expression decreased as the Ki67 proliferation index increased (p = 0.0003), and TUSC2 expression decreased as tumor size increased (p = 0.0483). The loss or decrease in the TUSC2 expression was significant as the tumor grade increased (p = 0.3740). Gene expression analysis correlated with immunohistochemistry results. CONCLUSION: The results of the present study demonstrated a decrease or loss of TUSC2 expression in breast cancer tissue compared to normal tissue. A correlation was found between TUSC2 expression and Ki67 proliferation index and tumor size.

Study of morphology with assessment of expression of proliferation marker Ki67 antigen and P53 protein in lesions of gall bladder.

OBJECTIVE: To study the spectrum and distribution of histopathological changes and evaluate immunohistochemistry markers p53 protein and Ki67 antigen in various lesions of gall bladder. MATERIALS AND METHODS: A total of 804 consecutive gall bladder specimens were evaluated. Forty cases were selected for immunohistochemical analysis to evaluate expression of p53 and ki67 proliferation index, including 20 carcinoma gall bladder cases and 20 cases of inflammatory pathology associated with metaplasia, atypia, hyperplasia, dysplasia, and adenoma. p53 immunostaining was categorized as wild type and mutant type. ki67 of >20% was considered high expression. RESULTS: The majority of the gall bladder lesions were inflammatory in origin, most common being chronic cholecystitis. In the group of 20 gall bladder carcinoma cases, 65% were p53 mutant and the remaining 35% cases had a p53 wild-type immunophenotype. 55% cases showed high expression for ki67 labeling. However, significant correlation ( P < 0.05) was seen with lympho-vascular invasion. Among non-malignant lesions, normal/wild-type p53 expression was seen with increasing intensity and positivity in lesions with atypia and intra-epithelial neoplasms. Ki67 index also showed the same trend in all cases. CONCLUSIONS: p53 and ki-67 expression increases in inflammation, and further increment occurs in premalignant and malignant lesions of the gall bladder epithelium and can be used as a marker of aggression of histopathological lesions. The results emphasize the potential of Ki-67 and p53 as biomarkers of carcinogenesis in gall bladder carcinoma.

Assessment of Ki-67 expression in cases of prostatic carcinoma and its correlation with clinical outcomes.

BACKGROUND: Treatment decisions after diagnosis of clinically localized prostate cancer are difficult due to variability in tumor behavior. As there is a high prevalence of low-grade prostate cancer with an indolent course, we need improved markers of prostate cancer lethality in order to reduce the overtreatment. In the current study, we assessed Ki-67 expression in cases of prostate carcinoma and correlated its expression with clinical outcomes. MATERIALS AND METHODS: It was a single-center retrospective descriptive type of study. A total of 50 cases were included. Diagnosed cases of adenocarcinoma on Transurethral Resection of the Prostate (TURP) chips and Trucut prostatic biopsies (Archival biopsy specimens) for whom five years follow-up was available from record files and/or telephonic interviews were included. The clinical outcomes (rate of distant metastases, disease specific survival, and overall survival) over a period of five years were recorded. RESULTS: In the current study, 78% of the cases of carcinoma prostate were positive for Ki-67 expression. The mean Ki-67 staining index was 15.22% among the cases. The cases with High Ki-67 Staining index had a significantly higher rate of distant metastasis, poor disease-specific survival, and overall survival compared to cases with low Ki-67 staining index. CONCLUSION: Ki-67 can be used along with the other established prognostic parameters to assess the lethality of prostate cancer.

Effects of different KRAS mutants and Ki67 expression on diagnosis and prognosis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a prevalent form of non-small cell lung cancer with a rising incidence in recent years. Understanding the mutation characteristics of LUAD is crucial for effective treatment and prediction of this disease. Among the various mutations observed in LUAD, KRAS mutations are particularly common. Different subtypes of KRAS mutations can activate the Ras signaling pathway to varying degrees, potentially influencing the pathogenesis and prognosis of LUAD. This study aims to investigate the relationship between different KRAS mutation subtypes and the pathogenesis and prognosis of LUAD. A total of 63 clinical samples of LUAD were collected for this study. The samples were analyzed using targeted gene sequencing panels to obtain sequencing data. To complement the dataset, additional clinical and sequencing data were obtained from TCGA and MSK. The analysis revealed significantly higher Ki67 immunohistochemical scores in patients with missense mutations compared to controls. Moreover, the expression level of KRAS was found to be significantly correlated with Ki67 expression. Enrichment analysis indicated that KRAS missense mutations activated the SWEET_LUNG_CANCER_KRAS_DN and CREIGHTON_ENDOCRINE_THERAPY_RESISTANCE_2 pathways. Additionally, patients with KRAS missense mutations and high Ki67 IHC scores exhibited significantly higher tumor mutational burden levels compared to other groups, which suggests they are more likely to be responsive to ICIs. Based on the data from MSK and TCGA, it was observed that patients with KRAS missense mutations had shorter survival compared to controls, and Ki67 expression level could more accurately predict patient prognosis. In conclusion, when utilizing KRAS mutations as biomarkers for the treatment and prediction of LUAD, it is important to consider the specific KRAS mutant subtypes and Ki67 expression levels. These findings contribute to a better understanding of LUAD and have implications for personalized therapeutic approaches in the management of this disease.

Ki-67 Change in Anthracyline-containing Neoadjuvant Chemotherapy Response in Breast Cancer.

OBJECTIVE: Anthracycline-containing regimens are irreplaceable in neoadjuvant chemotherapy (NAC) for breast cancer (BC) at present. However, 30% of early breast cancer (EBC) patients are resistant to anthracycline-containing chemotherapy, leading to poor prognosis and higher mortality. Ki-67 is associated with the prognosis and response to therapy, and it changes after NAC. METHODS: A total of 105 BC patients who received anthracycline-containing NAC were enrolled. Then, the optimal model of Ki-67 was selected, and its predictive efficacy was analyzed. Immunohistochemistry (IHC) was used to determine the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) status and Ki-67 level. Fluorescent in situ hybridization (FISH) was used to verify the HER-2 when the IHC score was 2+. RESULTS: The post-NAC Ki67 level after treatment with anthracycline drugs was lower than pre-NAC Ki-67 (19.6%±23.3% vs. 45.6%±23.1%, P<0.001). Furthermore, patients with the Ki-67 decrease had a border line higher pathological complete response (pCR) rate (17.2% vs. 0.0%, P=0.068), and a higher overall response rate (ORR) (73.6% vs. 27.8%, P<0.001), when compared to patients without the Ki-67 decrease. The ΔKi-67 and ΔKi-67% were valuable markers for the prediction of both the pCR rate and ORR. The area under the curve (AUC) for ΔKi-67 on pCR and ORR was 0.809 (0.698-0.921) and 0.755 (0.655-0.855), respectively, while the AUC for ΔKi-67% on pCR and ORR was 0.857 (0.742-0.972) and 0.720 (0.618-0.822), respectively. Multivariate logistic regression model 1 revealed that ΔKi-67 was an independent predictor for both pCR [odds ratio (OR)=61.030, 95% confidence interval (CI)=4.709-790.965; P=0.002] and ORR (OR=10.001, 95% CI: 3.044-32.858; P<0.001). Multivariate logistic regression model 2 revealed that ΔKi-67% was also an independent predictor for both pCR (OR=408.922, 95% CI=8.908-18771.224; P=0.002) and ORR (OR=5.419, 95% CI=1.842-15.943; P=0.002). CONCLUSIONS: The present study results suggest that ΔKi67 and ΔKi67% are candidate predictors for anthracycline-containing NAC response, and that they may provide various information for further systematic therapy after surgery in clinical practice.

Real-world use of multigene signatures in early breast cancer: differences to clinical trials.

PURPOSE: In Italy, Lombardy was the first region to reimburse multigene assays (MGAs) for patients otherwise candidates for chemotherapy. This is a real-world experience of MGAs usage in six referral cancer centers in Lombardy. METHODS: Among MGAs, Oncotype DX (RS) was used in 97% of cases. Consecutive patients tested with Oncotype DX from July 2020 to July 2022 were selected. The distribution of clinicopathologic features by RS groups (low RS: 0-25, high RS: 26-100) was assessed using chi-square and compared with those of the TAILORx and RxPONDER trials. RESULTS: Out of 1,098 patients identified, 73% had low RS. Grade and Ki67 were associated with RS (p < 0.001). In patients with both G3 and Ki67 > 30%, 39% had low RS, while in patients with both G1 and Ki67 < 20%, 7% had high RS. The proportion of low RS in node-positive patients was similar to that in RxPONDER (82% vs 83%), while node-negative patients with low RS were significantly less than in TAILORx (66% vs 86%, p < 0.001). The distribution of Grade was different from registration trials, with more G3 and fewer G1 (38% and 3%) than in TAILORx (18% and 27%) and RxPONDER (10% and 24%) (p < 0.001). Patients ≤ 50 years were overrepresented in this series (41%) than in TAILORx and RxPONDER (31% and 24%, respectively) (p < 0.001) and, among them, 42% were node positive. CONCLUSIONS: In this real-world series, Oncotype DX was the test almost exclusively used. Despite reimbursement being linked to pre-test chemotherapy recommendation, almost 3/4 patients resulted in the low-RS group. The significant proportion of node-positive patients ≤ 50 years tested indicates that oncologists considered Oncotype DX informative also in this population.

Molecular features of luminal breast cancer defined through spatial and single-cell transcriptomics.

BACKGROUND: Intratumour heterogeneity is a hallmark of most solid tumours, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing on patient-derived xenografts (PDXs) to profile spatially resolved cell populations within oestrogen receptor-positive (ER+ ) breast cancer and to elucidate their importance in oestrogen-dependent tumour growth. METHODS: Two PDXs of 'ER-high' breast cancers with opposite oestrogen-mediated growth responses were investigated: oestrogen-suppressed GS3 (80-100% ER) and oestrogen-dependent SC31 (40-90% ER) models. The observation was validated via single-cell analyses on an 'ER-low' PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by a public ER+ breast cancer single-cell dataset and protein-based dual immunohistochemistry (IHC) of SC31 examining important luminal cancer markers (i.e., ER, progesterone receptor and Ki67). The translational implication of our findings was assessed by clinical outcome analyses on publicly available cohorts. RESULTS: Our space-gene-function study revealed four spatially distinct compartments within ER+ breast cancers. These compartments showed functional diversity (oestrogen-responsive, proliferative, hypoxia-induced and inflammation-related). The 'proliferative' population, rather than the 'oestrogen-responsive' compartment, was crucial for oestrogen-dependent tumour growth, leading to the acquisition of luminal B-like features. The cells expressing typical oestrogen-responsive genes like PGR were not directly linked to oestrogen-dependent proliferation. Dual IHC analyses demonstrated the distinct contribution of the Ki67+ proliferative cells toward oestrogen-mediated growth and their response to a CDK4/6 inhibitor. The gene signatures derived from the proliferative, hypoxia-induced and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the oestrogen-responsive signature showed better prognoses, suggesting that this compartment would not be directly associated with oestrogen-dependent tumour progression. CONCLUSIONS: Our study identified the gene signature in our 'proliferative' compartment as an important determinant of luminal cancer subtypes. This 'proliferative' cell population is a causative feature of luminal B breast cancer, contributing toward its aggressive behaviours.

BRCA1 and BRCA2 germline mutations in Chinese Hakka breast cancer patients.

OBJECTIVE: To investigate the prevalence of BRCA1/2 gene variants and evaluate the clinical and pathological characteristics associated with these variants in Chinese Hakka breast cancer patients. METHODS: A total of 409 breast cancer patients were analyzed based on next-generation sequencing results, with 337 categorized as non-carriers and 72 as carriers of BRCA1/2 variants. Data on the patients' BRCA1/2 gene mutation status, clinical and pathological characteristics, as well as menstrual and reproductive information, were collected, analyzed, compared, and tabulated. Logistic regression analysis was performed to explore the relationship between clinical characteristics and pathogenic variants. RESULTS: Among the patients, 72 were identified as carriers of pathogenic or likely pathogenic variants in BRCA1/2, while 337 had likely benign or benign mutations. The BRCA1 c.2635G > T (p. Glu879*) variant was detected at a high frequency, accounting for 12.5% (4/32) of the BRCA1 mutations, while the c.5164_5165del (p.Ser1722Tyrfs*4) variant was common among the BRCA2 mutations, accounting for 17.5% (7/40). It was observed that a higher proportion of BRCA1 carriers had the triple-negative breast cancer subtype, whereas more BRCA2 carriers exhibited estrogen receptor (ER) + and progesterone receptor (PR) + subtypes. Multivariate logistic regression analysis revealed that a family history of cancer (OR = 2.36, 95% CI = 1.00-5.54), bilateral cancer (OR = 4.78, 95% CI 1.61-14.20), human epidermal growth factor receptor 2 (HER2)- (OR = 8.23, 95% CI 3.25-20.84), and Ki67 ≥ 15% (OR = 3.88, 95% CI 1.41-10.65) were associated with BRCA1/2 mutations, with the age at diagnosis, age at menarche, and premenopausal status serving as covariates. CONCLUSIONS: The most common pathogenic variant of the BRCA1 and BRCA2 in breast cancer patients was c.2635G > T and c.5164_5165del, respectively. Additionally, a family history of cancer, bilateral cancer, HER2-, and Ki67 ≥ 15% were identified as independent predictors of BRCA1/2 pathogenic variants.

DNA content and clinicopathological features aid in distinguishing ameloblastic carcinoma from ameloblastoma.

BACKGROUND: Ameloblastoma and ameloblastic carcinoma are epithelial odontogenic tumors that can be morphologically similar. In the present study, we evaluated the DNA content and Ki-67 index in the two tumors. METHODS: The paraffin blocks of the tumors were selected to obtain sections for the immunohistochemical reactions and preparation of the cell suspension for acquisition in a flow cytometer. The Random Forest package of the R software was used to verify the contribution of each variable to classify lesions into ameloblastoma or ameloblastic carcinoma. RESULTS: Thirty-two ameloblastoma and five ameloblastic carcinoma were included in the study. In our sample, we did not find statistically significant differences in Ki-67 labeling rates. A higher fraction of cells in 2c (G1) was correlated with the diagnosis of ameloblastoma, whereas higher rates of 5c-exceeding rate (5cER) were correlated with ameloblastic carcinoma. The Random Forest model highlighted histopathological findings and parameters of DNA ploidy study as important features for distinguishing ameloblastoma from ameloblastic carcinoma. CONCLUSION: Our findings suggest that the parameters of the DNA ploidy study can be ancillary tools in the classification of ameloblastoma and ameloblastic carcinoma.

Discordance of Oncotype DX scores in synchronous bilateral and unilateral multifocal breast cancers.

PURPOSE: Oncotype DX, a 21-gene expression profiling test, has become standard of care in the management of estrogen receptor (ER)-positive breast cancer. In multifocal tumors, it is unclear whether testing of the different foci is necessary. We evaluated the concordance of Oncotype DX recurrence scores (RS) between 2 tumor foci in synchronous bilateral or unilateral multifocal tumors and characterized pathological predictors of discordance. METHODS: We reviewed 713 ER+, HER2- primary invasive breast cancer patients with Oncotype RS and identified 17 bilateral synchronous patients (34 tumors) and 13 unilateral multifocal patients (26 tumors) with available Oncotype RS on all foci. Discordance in Oncotype RS between synchronous tumors was recorded and associations with clinicopathologic features including tumor size, histology, Nottingham histologic grade, progesterone receptor staining, and Ki67 index were analyzed. RESULTS: Bilateral synchronous tumors were present in older patients (median age 59 years) and had larger tumor (median size 17 mm) and more discordant histology (10/17, 59%) as compared to unilateral multifocal tumors (median age 49 years, p < 0.01; median tumor size 12 mm, p = 0.01; discordant histology 2/13, 15%, p = 0.03). Oncotype RS were discordant in 47% (8/17) of bilateral and 54% (7/13) of unilateral multifocal tumors. Concordant Oncotype RS was associated with similar histologic grade and Ki67 index in 78% (7/9) of bilateral and 100% (6/6) of multifocal tumors. In contrast, only 25% (2/8) of bilateral (p = 0.06) and 14% (1/7) of unilateral multifocal (p < 0.01) cases with discordant Oncotype RS had concordant histology grades and Ki67 levels. In synchronous tumors with discordant Oncotype RS and Ki67 index, all (4/4) foci with higher RS had higher Ki67 index. CONCLUSION: Discordance of Oncotype RS is common in both bilateral and unilateral multifocal breast cancer and is likely associated with discordant histologic grade or Ki67.

Nodal pCR and overall survival following neoadjuvant chemotherapy for node positive ER+/Her2- breast cancer.

PURPOSE: The role of neoadjuvant chemotherapy (NAC) in node-positive (N+) ER+/HER2- breast cancer (BC) is debated, given low total pathologic complete response (pCR) rates. However, the rate and impact of nodal pCR is unknown. We sought to evaluate nodal pCR rates and the impact on overall survival (OS). Further, we sought to validate the association between nodal pCR with age and Ki67. METHODS: We queried the National Cancer Database for cN + ER+/HER2- BC patients treated with NAC and surgery. Data from 2010 to 2018 were used to evaluate nodal pCR and OS, with multivariable Cox proportional hazards modeling for OS, as well as Ki67 for the years 2018-2019. RESULTS: From 2010 to 2018, we identified 19,611 cN + ER+/HER2- BC patients treated with NAC. While total pCR occurred in only 7.4%, nodal pCR rates were nearly double (14.3%). Nodal pCR (+/- breast pCR) was seen in 21.7% and associated with 5-year OS rate of 86.1% (95% CI: 84.9-87.4%) versus 77.1% (95% CI: 76.3-77.9%) in patients without nodal pCR (p < 0.001). On multivariable analysis, nodal pCR had better OS (adjusted HR 0.57, 95% CI 0.52-0.63, p < 0.001) across all age groups. Of 2,444 patients with available Ki67, those with age < 50 and Ki67 ≥ 20% had the highest nodal pCR at 31.6%. CONCLUSION: In cN + ER+/HER2- BC treated with NAC, nodal pCR is common, associated with age and Ki67, and prognostic for OS. These data strongly suggest that for cN + patients, eradication of nodal disease is critical for OS, and total pCR may not be the optimal measure of NAC benefit.

RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/ß-catenin signaling in melanoma cells and tumor growth in vivo.

PURPOSE: The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/ß-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. METHODS: We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active ß-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. RESULTS: Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active ß-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and ß-catenin, as manifested by a decrease in the transcriptional activity of ß-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on ß-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. CONCLUSION: RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.