Dual Immunohistochemical Staining Research Articles

Exploring p53 protein expression and its link to TP53 mutation in myelodysplasia-related malignancies-Interpretive challenges and potential field of applications.

TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.

Abstract B016: Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining

Abstract Using gene expression profiling data, we previously identified three immune subtypes (low-TIL, high-TIL, high-ISG) of breast cancer (BC) among Chinese BC patients in Hong Kong (HK) (Zhu et al., Breast Cancer Res, 2017). To investigate if standard in situ immunohistochemical (IHC) staining could capture the BC immune landscape, we analyzed archival tissue sections from 148 HKBC patients who were previously characterized based on gene expression. Dual IHC staining for 8 immune markers (CD3, CD8, CD20, FOXP3, CD68, CD163, PDL1, and IDO1) and PanCK was performed. Digitized whole slide images for each marker were analyzed using high-accuracy machine learning algorithms trained to quantify the percentage of positively stained regions. Strong correlations were found between IHC immune markers and gene expression-based immune and intrinsic subtypes. Compared to patients with lower level of tumor-infiltrating lymphocytes (low-TIL) classified based on gene expression data, those in the high-TIL subtype (characterized by high T cell and immune checkpoint gene expression) showed higher proportions of CD3, CD8, CD20, FOXP3, CD68, and PDL1 IHC positively stained cells. Similarly, the high-ISG subtype (with increased interferon-stimulated gene expression and TP53 somatic mutations) had higher proportions of CD3, CD8, CD20, FOXP3, and CD68 stained cells than the low-TIL subtype and in particular exhibited high CD163 staining, indicative of a proinflammatory tumor microenvironment. CD8, CD20, CD163, PDL1, and IDO1 expression varied significantly by PAM50 subtypes, with luminal (A and B) subtypes showing lower staining than non-luminal (HER2 and Basal) subtypes. A distinctive feature, however, of luminal B tumors was the higher infiltration by CD163-expressing cells when compared with the luminal A subtype. Except for FOXP3, all examined IHC markers showed significantly higher expression in patients with somatic TP53 mutations (CD3: P= 0.003, CD8: P= 0.005, CD20: P= 0.003, CD68: P= 0.002, CD163: P= 0.002, PDL1: P= 0.003, and IDO1: P= 0.013). On the other hand, expression of these markers did not significantly vary by PIK3CA mutation or germline APOBEC3A/B deletion polymorphism, which is more prevalent among East Asians than other populations and was previously linked to BC risk and immune environment. Of the BC risk factors assessed, only body mass index (BMI) demonstrated associations with immune cell densities, with higher BMI associated with lower expression of CD3, CD8, and CD20. This study sheds further light on the in situ immune landscape of gene expression-based BC subtypes, suggesting that standard IHC assessment of immune markers could refine BC subtype classification for clinical and epidemiological use. Citation Format: Mustapha Abubakar, Hela Koka, Priscilla Lee, Scott Lawrence, Karun Mutreja, Difei Wang, Bin Zhu, Shelly Tse, Xiaohong R Yang. Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B016.

Increased CD16a (FcγRIIIA) Expression in The Tumor Microenvironment of Atypical Neurofibromatous Neoplasms of Uncertain Biologic Potential May Be Associated with Progression from Neurofibromas to Atypical Neurofibromas.

Neurofibroma (NF) is a benign tumor in the peripheral nervous system, but it can infiltrate around structures and cause functional impairment and disfigurement. We incidentally found that the expression of CD16a (Fc gamma receptor IIIA) was increased in NFs compared to in non-neoplastic nerves and hypothesized that CD16 could be relevant to NF progression. We evaluated the expressions of CD16a, CD16b, CD68, TREM2, Galectin-3, S-100, and SOX10 in 38 cases of neurogenic tumors (NF, n = 18; atypical neurofibromatous neoplasm of uncertain biologic potential (ANNUBP), n = 14; and malignant peripheral nerve sheath tumor (MPNST), n = 6) by immunohistochemical staining. In the tumor microenvironment (TME) of the ANNUBPs, CD16a and CD16b expression levels had increased more than in the NFs or MPNSTs. CD68 and Galectin-3 expression levels in the ANNUBPs were higher than in the MPNSTs. Dual immunohistochemical staining showed an overlapping pattern for CD16a and CD68 in TME immune cells. Increased CD16a expression was detected in the ANNUBPs compared to the NFs but decreased with malignant progression. The CD16a overexpression with CD68 positivity in the ANNUBPs potentially reflects that the TME immune modulation could be associated with NF progression to an ANNUBP. Further studies should explore the role of CD16a in immunomodulation for accelerating NF growth.

FLI-1/Melan-A dual stain is an alternative to PRAME in differentiating metastatic melanoma from nodal nevus: A monocentric retrospective study.

Melanocytic nevi existing in lymph nodes create a diagnostic challenge by mimicking metastases. PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemical (IHC) stain can differentiate one from another. FLI-1 IHC expression has been shown in malignant melanoma with variable sensitivity while melanocytic nevi were reported to be negative. We hypothesized that FLI-1/Melan-A dual IHC staining may be used in the distinction of metastatic melanoma from nodal nevi and can be an alternative and/or complementary to PRAME. In this study, we examined 13 lymph nodes with metastatic melanoma and 13 lymph nodes with benign deposits. We stained all of the lymph nodes with FLI-1, FLI-1/Melan-A dual, and PRAME IHC stains. In addition, we stained paired skin samples of the metastatic lymph nodes with FLI-1 and PRAME. In primary cutaneous melanomas, 11 of 13 were positive for FLI-1 and PRAME expression (85%). Malignant cells in 12 and 13 lymph nodes showed positive expression of PRAME and FLI-1, respectively. Only one case with a nevic cell deposit was weakly positive for FLI-1 and the remaining benign cases were negative for both FLI-1 and PRAME. Our results show that FLI-1/Melan-A dual stain is as sensitive and specific as PRAME in distinguishing lymph nodes with metastatic melanoma from nodal nevi. Further studies with larger case numbers are needed to support our significant results.

The Prognostic Reliability of Lymphovascular Invasion for Patients with T3N0 Colorectal Cancer in Adjuvant Chemotherapy Decision Making

Simple SummaryThis retrospective analysis evaluated the prognostic implications of lymphovascular invasion (LVI) associated with adjuvant chemotherapy in 1634 patients with pT3N0 colorectal cancer. Extensive pathologic review and dual immunohistochemical (IHC) staining with CD31 and D2-40 were undertaken in a subset of 242 patients to determine the reliability of LVI as a prognostic factor. The diagnosis of LVI and PNI changed in 82 (33.9%) and 61 (25.2%) patients, respectively, after central pathologic review (mean follow up duration, 50 (1–114) months). Five-year recurrence-free survival (RFS) and overall survival (OS) rates were 92% and 94.8%, respectively. Before and after pathologic review, LVI was not associated with OS but was associated with RFS after reviewing patients with pT3N0 colorectal cancer. In this patient cohort, the prognostic implications of LVI may have been underrecognized when using hematoxylin and eosin staining slides only for pathologic diagnoses, possibly leading to low recurrence prediction rates.Lymphovascular invasion (LVI) is a high-risk feature guiding decision making for adjuvant chemotherapy. We evaluated the prognostic importance and reliability of LVI as an adjuvant chemotherapy indicator in 1634 patients with pT3N0 colorectal cancer treated with curative radical resection between 2012 and 2016. LVI and perineural invasion (PNI) were identified in 382 (23.5%) and 269 (16.5%) patients, respectively. In total, 772 patients received adjuvant chemotherapy. The five-year recurrence-free survival (RFS) and OS rates were 92% and 94.8%, respectively. Preoperative obstruction, PNI, and positive margins were significantly associated with RFS and OS; however, adjuvant chemotherapy and LVI were not. Pathologic slide central reviews of 242 patients using dual D2-40 and CD31 immunohistochemical staining was performed. In the review cohort, the diagnosis of LVI and PNI was changed in 82 (33.9%) and 61 (25.2%) patients, respectively. Reviewed LVI, encompassing small vessel invasion, lymphatic invasion, and large vessel invasion, was not an independent risk factor associated with OS but was related to RFS. The prognostic importance of LVI and adjuvant chemotherapy was not defined because LVI may be underrecognized in pathologic diagnoses using hematoxylin and eosin staining slides only, leading to low recurrence rate predictions. Using LVI as a guiding factor for adjuvant chemotherapy requires further consideration.

HPV Status as Prognostic Biomarker in Head and Neck Cancer-Which Method Fits the Best for Outcome Prediction?

Simple SummaryHead and neck cancer belongs to the six most common cancers worldwide, with the majority of cases being associated with chronic alcohol and/or tobacco consumption. Over the past years, a rising proportion of those cancer cases are rather caused by a mucosal infection with high-risk human papillomavirus (HPV). In general, those patients are younger and respond better to different treatment options, which results in an overall better prognosis. Despite this high importance of HPV status in head and neck cancer, there is no established standard biomarker for the detection of HPV-related tumor biology. In our study, we tested six different detection methods that are currently used and compared their diagnostic and prognostic validity in a collective of 153 head and neck cancer patients. Thereby, immunohistochemical staining of p16—a protein biomarker that is overexpressed in HPV-related cancers—showed the best performance as prognostic biomarker and should be combined with a direct detection of HPV-DNA in case of therapeutic consequences. The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

Modified arteriosclerosis score predicts the outcomes of diabetic kidney disease

BackgroundThe significance of renal arteriosclerosis in the prediction of the renal outcomes of diabetic kidney disease (DKD) remains undetermined.MethodsWe enrolled 174 patients with DKD from three centres from January 2010 to July 2017. The severity and extent of arteriosclerosis were analysed on sections based on dual immunohistochemical staining of CD31 and α-smooth muscle actin. An X-tile plot was used to determine the optimal cut-off value. The primary endpoint was renal survival (RS), defined as the duration from renal biopsy to end-stage renal disease or death.ResultsThe baseline estimated glomerular filtration rate (eGFR) of 135 qualified patients was 45 (29 ~ 70) ml/min per 1.73 m2, and the average 24-h urine protein was 4.52 (2.45 ~ 7.66) g/24 h. The number of glomeruli in the biopsy specimens was 21.07 ± 9.7. The proportion of severe arteriosclerosis in the kidney positively correlated with the Renal Pathology Society glomerular classification (r = 0.28, P < 0.012), interstitial fibrosis and tubular atrophy (IFTA) (r = 0.39, P < 0.001), urine protein (r = 0.213, P = 0.013), systolic BP (r = 0.305, P = 0.000), and age (r = 0.220, P = 0.010) and significantly negatively correlated with baseline eGFR (r = − 0.285, P = 0.001). In the multivariable model, the primary outcomes were significantly correlated with glomerular class (HR: 1.72, CI: 1.15 ~ 2.57), IFTA (HR: 1.96, CI: 1.26 ~ 3.06) and the modified arteriosclerosis score (HR: 2.21, CI: 1.18 ~ 4.13). After risk adjustment, RS was independently associated with the baseline eGFR (HR: 0.97, CI: 0.96 ~ 0.98), urine proteinuria (HR: 1.10, CI: 1.04 ~ 1.17) and the modified arteriosclerosis score (HR: 2.01, CI: 1.10 ~ 3.67), and the nomogram exhibited good calibration and acceptable discrimination (C-index = 0.82, CI: 0.75 ~ 0.87).ConclusionsThe severity and proportion of arteriosclerosis may be helpful prognostic indicators for DKD.

Optimizing Detection of Lymphatic Invasion in Primary Cutaneous Melanoma With the Use of D2-40 and a Paired Melanocytic Marker.

Dual immunohistochemical (IHC) staining with D2-40 and S100 improves detection of lymphatic invasion (LI) in primary cutaneous melanoma. However, limited data exist evaluating this technique using other melanocytic markers, and thus, the optimal marker for detection of LI is unestablished. To address this knowledge gap, a case-control study was performed comparing melanoma specimens from 22 patients with known lymphatic spread (LS) with a control group of 11 patients without LS. Specimens underwent dual IHC staining with D2-40 and MART-1, SOX-10, and S100 to evaluate for LI. Receiver operating characteristic analysis was used to estimate each stain's accuracy for detection of LI. The LS group was more likely to be ≥65 years (P = 0.04), have a tumor thickness of ≥1 mm (P < 0.01), and have ulcerated tumors (P = 0.02). Detection of LI with D2-40/MART-1 significantly correlated with LS (P = 0.03), and the D2-40/MART-1 stain was most accurate for LI based on receiver operating characteristic curve analysis (area under the curve [AUC] 0.705) in comparison with D2-40/SOX-10 (AUC 0.575) and D2-40/S100 (AUC 0.633). These findings suggest that MART-1 may be the optimal melanocytic marker to combine with D2-40 for detection of LI in melanoma. Further studies are needed to determine the utility of routinely performing these stains for histopathologic analysis of melanoma.

Epithelial-mesenchymal plasticity and lymph node metastases in invasive breast carcinoma of no special type

Background: Breast cancer is the commonest cancer among women with distant metastases being the leading cause of mortality. Epithelial-mesenchymal plasticity (EMP) is a proposed mechanism involved in establishment of metastasis. E-cadherin and vimentin can be used as epithelial and mesenchymal markers, respectively, to identify EMP. Aims: To compare E-cadherin and vimentin expressions between node-negative and node-positive invasive breast carcinoma–no special type (IBC-NST) and their lymph node metastases. Methods: Forty node negative tumours and 44 axillary node positive tumours of IBC-NST with their metastatic lymph nodes were retrieved from patients who had undergone mastectomy at the Teaching Hospital, Peradeniya. Samples were subjected to dual immunohistochemical staining with anti-E-cadherin and anti-vimentin primary antibodies. Any cytoplasmic staining for vimentin and 70% or more of membranous staining for E-cadherin were taken as positive and cells counted in 10 high power fields. Intensity of staining was graded as high=3, moderate=2, low=1 and absent=0 by comparing with internal controls. Percentage of positive cells was multiplied by the intensity to obtain the immuno-score (IS) for both protein expressions. IS was compared using independent and paired t-test. Results and conclusions: A lower E-cadherin (p=0.001) and higher vimentin (p=0.008) expression was seen in node positive group compared to node negative group. A higher E-cadherin (p<0.001) and lower vimentin (p<0.001) expression was seen in lymph nodes compared to the corresponding primary tumours. A significant loss of epithelial properties and acquisition of mesenchymal properties in the node positive group indicates epithelial to mesenchymal transition of tumour cells, contributing to metastasis. In the lymph nodes, higher E-cadherin and lower vimentin expression suggests a reverse mechanism, i.e., mesenchymal to epithelial transition. These Results are suggestive of epithelial-mesenchymal plasticity of tumour cells in establishment of metastasis in IBC-NST.

Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development.

SLFN11 has recently been reported to execute cancer cells harboring replicative stress induced by DNA damaging agents. However, the roles of SLFN11 under physiological conditions remain poorly understood. Germinal center B-cells (GCBs) undergo somatic hypermutations and class-switch recombination, which can cause physiological genotoxic stress. Hence, we tested whether SLFN11 expression needs to be suppressed in GCBs during B-cell development. To clarify the expression profile of SLFN11 in different developmental stages of B-cells and B-cell-derived cancers. We analyzed the expression of SLFN11 by mining cell line databases for different stages of normal B-cells and various types of B-cell-derived cancer cell lines. We performed dual immunohistochemical staining for SLFN11 and B-cell specific markers in normal human lymphatic tissues. We tested the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in GCB-derived lymphoma cell lines. We also examined the therapeutic efficacy of these drugs in combination with cytosine arabinoside and the effects of SLFN11 on the efficacy of cytosine arabinoside in SLFN11-overexpressing cells. SLFN11 mRNA level was found low in both normal GCBs and GCB-DLBCL (GCB like-diffuse large B-cell lymphoma). Immunohistochemical staining showed low SLFN11 expression in GCBs and high SLFN11 expression in plasmablasts and plasmacytes. The EZH2 and HDAC epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphoma cells and made them more susceptible to cytosine arabinoside. SLFN11 overexpression further sensitized GCB-derived lymphoma cells to cytosine arabinoside. The expression of SLFN11 is epigenetically suppressed in normal GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.

Endothelial to Mesenchymal Transition and the Reverse Contributes to Heart Failure and Recovery Thereafter

Introduction Previous studies have demonstrated Endothelial to Mesenchymal Transition (EndMT) in heart failure (HF) and it likely contributes to cardiac fibrosis. The reverse phenomenon of Mesenchymal to Endothelial Transition (MET) has not been well described in the setting of reversal of fibrosis. We aim to evaluate the occurrence of such cell transitions in mouse model of non-ischemic HF and recovery from HF. Hypothesis Endothelial to Mesenhcymal transitions contribute to heart failure while the reverse contributes to the recovery from HF. Methods HF is induced in C57BL/6 mice by exposure to water containing L-NAME and NaCL and implantation of an osmotic pump delivering Angiotensin II, which induces HF in 5 weeks. At 5 weeks the pump runs out of Angiotensin II and the water is changed back to normal water. Previous observations have shown a reduction of heart failure markers over an additional 14 weeks (19 week total). In the current experiment we decided to focus during the maximum reversal of fibrosis based on previous data and hence stopped the recovery phase at Week 9. Dual immunohistochemical staining was applied using antibodies to α-SMA (fibroblast cell marker) and CD31 (endothelial cell marker) to identify cells under transition. Results In line with previous observations the HF phenotype peaks at week 5 and reverses through week 9. (Figure 1B). Dual staining cells have a double peak during Week 3 and Week7 suggesting cell transitions to contribute to both heart failure and recovery. Conclusions While endothelial to mesenchymal transition contributes to the fibrotic state of heart failure, the reverse transitions could be playing a role in the remodeling of the heart in recovery.

Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer.

The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.

Utility of pVHL, maspin, IMP3, S100P and Ki67 in the distinction of autoimmune pancreatitis from pancreatic ductal adenocarcinoma

Morphology plays an important role in the distinction of autoimmune pancreatitis (AIP) from pancreatic ductal adenocarcinoma (PDAC). However, we aimed to determine the utility of immunohistochemical tumor markers to contribute in the distinction of these entities. In surgical specimens with AIP (n = 20), PDAC (n = 20) and normal pancreas (n = 20), the expression of pVHL, maspin, IMP3, S100P and Ki67 was examined. We evaluated intralobular reactive ducts / acinoductal metaplasia (ILDs) and extralobular ducts (ELDs) in AIP, neoplastic glands in PDAC, and ductal epithelium in the normal pancreas, using a five-tiered scoring system. The Ki67 hot spot index (Ki67-HSPI) was determined manually and using automated digital imaging analysis of virtual double stains of Ki67 and CK8. Besides, sequential dual-immunohistochemical staining of maspin/pVHL, maspin/IMP3 and Ki67/maspin was performed in a subset of the specimens. Strong overexpression of IMP3, maspin, S100P and Ki67 and loss of pVHL was observed in PDAC compared to AIP and normal pancreas. In AIP however, focal and weak aberrant expression was observed with the following proportions in ILDs/ELDs: pVHL in 45 %/85 %, maspin in 30 %/70 %, IMP3 in 55 %/5%, S100P in 10 %/35 % and Ki67-HSPI >20 % in 15 %/70 %. At least two markers were aberrantly expressed in ILDs/ELDs in 45 %/60 %. The aberrant expression was more pronounced in type 2 AIP compared to type 1. In conclusion, our data indicate that pVHL, maspin, IMP3, S100P and Ki67 can be focal and weak aberrantly expressed in AIP. However, when used as a panel, these markers seem to be useful for the differentiation of AIP from PC.

Age-Related Changes in Immune Cells of the Human Cochlea.

Age-related hearing loss is a chronic degenerative disorder affecting one in two individuals above the age of 75. Current population projections predict a steady climb in the number of older individuals making the search for interventions to prevent or reverse this disorder even more critical. There is growing acceptance that aberrant activity of resident or infiltrating immune cells, such as macrophages, is a major factor contributing to the onset and progression of age-related degenerative diseases. However, how macrophage populations and their functionally-driven morphological characteristics change with age in the human cochlea remains largely unknown. In this study, we employed immunohistochemical approaches along with confocal and super-resolution imaging, three-dimensional reconstructions, and quantitative analysis to determine age-related changes in macrophage numbers and morphology as well as interactions with other cell-types and structures of the auditory nerve and lateral wall in the human cochlea. In the cochlea of human ears from young and middle aged adults those macrophages in the auditory nerve assumed a worm-like structure in contrast to those in the spiral ligament or associated with the dense microvascular network in the stria vascularis which exhibited a highly ramified morphology. Macrophages in both the auditory nerve and cochlear lateral wall showed morphological alterations with age. The population of activated macrophages in the auditory nerve increased in cochleas obtained from older donors. Dual-immunohistochemical staining with macrophage, myelin, and neuronal markers revealed increased interactions of macrophages with the glial and neuronal components of the aged auditory nerve. These findings implicate the involvement of abnormal macrophage-glia interactions in age-related physiological and pathological alterations in the human cochlea. There is clearly a need to further investigate the contribution of macrophage-associated inflammatory dysregulation in human presbyacusis.

Abstract LB-224: Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer

Abstract PURPOSE: Cdk4 inhibitors (CDK4i), such as palbociclib, are approved in combination with hormonal therapy as a front line treatment for metastatic HR+, Her2- breast cancer. However, despite these advances, many patients demonstrate either de novo or acquired resistance. This highlights the need to identify biomarkers to pinpoint patients who would respond to this therapy. In Rb+ tumors, which comprise the majority of HR+ breast cancers, Ki67, Cyclin D, cdk4, and p16 levels do not identify responsive subgroups. Cyclin D or cdk4 levels themselves are not reliable because tyrosine phosphorylated p27Kip1 (pY88) is required for activation of the cyclin D-cdk4 (DK4) complex. We have previously shown that the level of pY88 correlates with cdk4 activity: the more pY88 detected, the greater the specific activity of cdk4 (Patel, et. al. MCR, 2018). We developed dual immunohistochemical staining for p27 and pY88 and demonstrated that pY88 status, which is negative in benign breast epithelium, stratified tumors across all hormone receptor groups (Gottesman, et.al, MCR, 2018). Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the active cdk4 target. We have grown tumor resection material in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were non-responsive, while explants from the low or high pY88 group responded to drug. Thus, we hypothesized the pY88-p27 status may serve as a biomarker for patients that can respond to cdk4i therapy. RESULTS: We analyzed paraffin-embedded archival breast cancer biopsies from a 13 patient cohort of HR+, Her2- patients who had received Palbociclib/Letrozole in the front line metastatic setting. 62% of these patients progressed within 8 months of treatment, while 38% were on drug for 12-38 months. Using our staining method, we stratified patients into three groups based on pY88 status: 23% had no staining (Group 0), 38% had a low percentage of pY88+ cells (Group 1), and 38% had a high percentage of pY88+ cells (Group 2, &amp;gt;30% of cells pY88+). We then compared the time until disease progression, when they were removed from drug, for each group. Patients with a pY88 score of 0 had disease progression within 6-8 months, suggesting that they exhibited primary resistance. Patients with a pY88 score of 2 also showed disease progression within 6 months. This suggested that the Y88 high group may have too much active cdk4 target to respond to pharmacological drug dose, a hypothesis supported by our breast cancer cell line data. However, patients with a low pY88 score were all still on drug after 12+ months with either reduced tumor or stable disease. Additional patients continue to be recruited. CONCLUSION: Our data suggest that pY88-p27 status, as a surrogate marker for cdk4 activity, associates with responsiveness to CDK4i treatment. Clinical use of the pY88 biomarker may identify patients responsive or resistant to Cdk4 targeting drugs. Citation Format: Stacy W. Blain, Susan R.S. Gottesman, Jonathan Somma, Vladislav Tsiperson, Julia McGuinness, Matthew Ingham, Kevin Kalinsky, Gary Schwartz, Elina Tress, Hanina Hibshoosh, Mohamed Mh Kahila, Bachar Samra Samra, Evelyn Taiwo, Steve Xie. Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-224.

Abstract PD5-09: Immune parameters associated with survival in triple negative and HER2-positive breast cancer patients with 10 years of follow-up

Abstract The clinical utility of tumor-infiltrating lymphocytes (TIL) is actively being investigated in breast cancer (BC). It is unclear whether TIL spatial location and organization in tertiary lymphoid structures (TLS) have an impact on prognosis. Additionally, the significance of PD-1 and PD-L1 expression is being debated due to conflicting data from several studies. We hypothesize that the presence, extent and spatial location of multiple immune biomarkers, reflecting ongoing immune responses, will be consistently associated with a good prognosis in highly infiltrated BC [triple-negative (TNBC) and HER2+]. The relationship between these immune biomarkers and clinical outcome was examined in the TNBC and HER2+ cohorts of node-positive BC patients enrolled in the BIG 02-98 adjuvant phase III trial with available material for immunohistochemical (IHC) labeling (N=113 and N=136, respectively). HER2+ patients did not receive trastuzumab. Dual IHC staining was performed on full-face consecutive tissue sections. Scoring was independently performed by two pathologists, blinded to the clinical data, and included: global, intratumoral and stromal TIL and TLS, assessed on CD3/CD20 slides; the percentage and location of PD-1 and PD-L1 expression, assessed on PD-1/PD-L1 slides. TIL were considered as a categorical variable with different cut-offs used for each parameter and for each cohort (TNBC and HER2+). Invasive disease-free survival (I-DFS) and overall survival (OS) were analyzed (median follow-up: 10 years). Cox proportional hazard models were used for survival analyses. The TNBC cohort revealed an association between global TIL and outcome [adjusted hazard ratio (HR) for I-DFS: 0.27 (0.15-0.51); OS: 0.26 (0.13-0.53)]. Similar results were observed for stromal and intratumoral TIL. PD-L1 expression within TLS was an independent predictor of OS, after adjustment for tumor size and age [HR: 0.30 (0.09-0.99)]. Multivariate analysis reveals this effect was principally driven by high stromal TIL (&amp;gt;17.5% based on CD3/CD20 assessment) (χ2 OS: p=0.009). In contrast, no significant prognostic associations were found in the overall HER2+ cohort. However high T cell TIL were associated with improved I-DFS and OS in the ER-/HER2+ group [I-DFS: 0.34 (0.14-0.80); OS: 0.32 (0.12-0.86)] and stromal TIL were associated with improved I-DFS in the ER+/HER2+ group [HR: 0.29 (0.09-0.94)] (univariate analyses). No significant associations between the number of TLS nor the expression of PD-1 with outcomes were observed in either cohorts. The presence of PD-L1+ TLS, driven by high baseline TIL, was associated with an excellent prognosis in node-positive TNBC. This observation might reflect specific immune activities taking place in these mini lymph node-like structures adjacent to the tumor bed where specific antitumor memory immune responses could be generated. No different prognostic impact was observed when analyzing TIL spatial location. Although the statistical power of the study might be limited, in line with previous findings our data reveal that, among the immune parameters evaluated, TIL are the strongest predictor of outcome in TNBC, while PD-L1+ TLS could be a new and important parameter that requires further investigation. Citation Format: Solinas C, de Wind A, Van den Eynden G, Ameye L, Garaud S, De Silva P, Boisson A, Noel G, Langouo Fontsa M, Buisseret L, de Azambuja E, Francis PA, Di Leo A, Crown JP, Sotiriou C, Larsimont D, Paesmans M, Piccart-Gebhart M, Willard-Gallo K. Immune parameters associated with survival in triple negative and HER2-positive breast cancer patients with 10 years of follow-up [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-09.

A Quantitative Study of Empty Baskets in Essential Tremor and Other Motor Neurodegenerative Diseases.

The underlying biology of essential tremor (ET) is poorly understood. Purkinje cell (PC) loss has been observed in some studies, although this finding remains somewhat controversial. Basket cells are interneurons whose axonal collaterals form a plexus around PC soma. When there is PC loss, this basket plexus appears empty. We used dual immunohistochemical staining for calbindin D28k and glutamic acid decarboxylase to quantify "empty baskets" as an indirect and alternative method of detecting PC loss. Microscopic analyses on 127 brains included ET and a spectrum of motor neurodegenerative diseases (50 ET, 27 spinocerebellar ataxias [SCAs], 25 Parkinson disease, 25 controls). The median percentage of empty baskets in ET patients was 1.5 times higher than controls (48.8% vs 33.5%, p < 0.001) but lower in ET than in SCA1 (59.7%, p = 0.011), SCA2 (77.5%, p = 0.003), and SCA6 (87.0%, p < 0.001). PC loss is not a feature of SCA3, and the median percentage of empty baskets (30.1%) was similar to controls (p = 0.303). These data provide support for PC loss in ET and are consistent with the notion that ET could represent a mild form of cerebellar degeneration with an intermediate degree of PC loss.

BRCA1 expression, proliferative and apoptotic activities in ovarian epithelial inclusions

BackgroundThe purpose of this study was to examine proliferative and apoptotic activity in relation with BRCA1 expression in ovarian epithelial inclusions (OEIs), the putative precursor lesions of ovarian epithelial cancer (OEC).MethodsBenign ovaries from 95 patients were examined. Dual immunohistochemical staining for both BRCA1 and MIB-1 were performed to examine the relationship between BRCA1 and MIB-1 in OEI cells. Apoptotic activity was assessed on the parallel tissue sections by using TUNEL assay. Patients’ age, menstrual phase and menopausal status were analyzed.ResultsOEIs were present in the ovaries of 53% of the patients. OEIs were less frequently found in premenopausal (45%) than postmenopausal women (58%). BRCA1 and MIB-1 were found in 27 and 47% of the OEI-containing ovaries, respectively. All BRCA1 positive OEI cells are MIB-1 positive with dual staining method, although overall the percentage of positive cells was small. No significant difference was found for BRCA1 and MIB-1 expression in OEIs between menopausal status and menstrual phases. Apoptosis containing OEIs were seen in 70% of the ovaries. Compared to OEIs in proliferative menstrual phase and premenopausal status, significantly more apoptosis was found in OEIs from secretory phase and postmenopausal women. A small fraction of the epithelial cells within OEIs are proliferating or dying.ConclusionsLow estrogen and/or high progesterone levels may promote OEI cell turnover via induction of apoptosis. Imbalance between cell proliferation and death within OEIs under influence of hormones may play a role in the ovarian epithelial tumorigenesis.

Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage

The claudin family comprises four-pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin-25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult. For immunostaining, an anti-CLDND1 antibody that recognizes the specific epitope in the extracellular first loop was prepared. Dual immunohistochemical staining with CD31 using coronal cryosections of intact murine cerebellum tissue revealed that CLDND1 is expressed on endothelial cells. We therefore performed an in vitro permeability test using a human brain endothelial cell (HBEC) line to reveal whether CLDND1 contributes to cell adhesion like other claudins. CLDND1 was expressed on HBECs as well as in murine cerebellum tissue, and a strong signal was observed at TJs. RNA interference against CLDND1 decreased both the mRNA and protein levels without cytotoxicity. The permeability to small molecules, but not to large ones, across confluent HBECs increased on CLDND1 knockdown compared with mock-treated cells. These results suggest that the transient decrease of CLDND1 after cerebellar hemorrhage is responsible for low-molecular-weight selective vascular hyperpermeability. © 2017 Wiley Periodicals, Inc.

Microtubule-associated protein 1B: Novel paraneoplastic biomarker.

To report the identification of microtubule-associated protein (MAP) 1B as the antigen of the previously described Purkinje cell cytoplasmic antibody type 2 (PCA-2) antibody, its frequency, and clinical, oncological, and serological associations. Archival serum or cerebrospinal fluid (CSF) specimens were available from 96 of 118 consecutive PCA-2-IgG-seropositive patients identified during 1993-2016. The autoantigen, defined in mouse brain lysate by Western blot and mass spectrometry, was confirmed by dual immunohistochemical staining using commercial antibodies. The major antigenic region was defined by Western blot using recombinant protein fragments. IgG in 95 of 96 patients' serum or CSF (but in none of 98 healthy or disease control subjects' serum specimens) bound to recombinant MAP1B. A minority (17.5%) of patients' IgG also bound to MAP1A. PCA-2 was often accompanied by additional neural autoantibody markers of small-cell carcinoma, including collapsin response-mediated protein 5 (CRMP5) IgG (26%) or antineuronal nuclear antibody type 1 (ANNA-1) IgG (also known as anti-Hu; 13%). Neurological manifestations in 95 patients were (in decreasing frequency): peripheral neuropathy, 53%; cerebellar ataxia, dysmetria, or dysarthria, 38%; and encephalopathy, 27%. Cancer (majority small-cell lung carcinoma [SCLC]) was detected in 66 of 84 evaluated patients (79%). The MAP1B (PCA-2) autoantibody detection rate, among approximately 70,000 patients undergoing service neural autoantibody evaluation in 2015, was 0.024%, equaling amphiphysin IgG (0.026%) and more common than ANNA-2 (also known as anti-Ri; 0.016%) and PCA-Tr (also known as delta/notch-like epidermal growth factor-related receptor [DNER]; 0.006%). MAP1B, the PCA-2 autoantigen, represents a novel target in paraneoplastic neurological disorders and has high predictive value for SCLC. Its relatively high prevalence, compared with other recognized paraneoplastic neural autoantibodies, justifies its testing in comprehensive paraneoplastic neural autoantibody evaluation. Ann Neurol 2017;81:266-277.