Additional Immunohistochemistry Research Articles

Sindrom iznenadne smrti uzrokovan tumorom srca

Cardiac tumors are not a common pathology. However, due to their occurrence in the vital organ, they bare an extremely important clinical significance. According to biological behavior, they are divided into 2 groups, primary (benign and malignant), which are less frequent and secondary (metastatic), which are more common. Among all primary benign cardiac tumors, myxoma is the one that occurs most often. A great number of studies indicate that it originates from multipotent mesenchymal cells of the septum of the left atrium. It is due to its localization that some authors consider it potentially malignant. This paper presents a case of cardiac myxoma which is echocardiographically diagnosed in a 34-year-old woman who died suddenly, several days before the scheduled surgery. The diagnosis of the disease was confirmed after the autopsy, by a routine haematoxylin-eosin (H&E) staining method and an additional immunohistochemistry method (IHC). By presenting a rare clinical case, we emphasize the fact that every diagnosed cardiac tumor requires immediate cardiac surgery without delay.

How Valuable Is the RT-qPCR of Pituitary-Specific Transcription Factors for Identifying Pituitary Neuroendocrine Tumor Subtypes According to the New WHO 2017 Criteria?

The classification of pituitary neuroendocrine tumors (PitNETs) subtypes continues generating interest. In 2017, the World Health Organization (WHO) proposed considering the immunohistochemical (IHC) analysis of pituitary-specific transcription factors (TF) for their typification. The present study targeted the quantification of pituitary-specific TF (TPIT, PIT-1, SF-1, GATA2, ESR1) gene expression by RT-qPCR to overcome the shortcomings of IHC and to complement it. We analyzed 251 tumors from our collection of PitNETs and performed additional IHC studies in a subset of 56 samples to analyze the concordance between gene and protein expression of the TF. The molecular and IHC studies allowed us to significantly reduce the percentage of null cell tumors in our series, most of which were reclassified as gonadotroph tumors. The concordance between the molecular and the immunohistochemical studies was good for tumors coming from the corticotroph and Pit-1 lineages but worsened for the rest of the tumors. Indeed, the RT-qPCR helped to improve the typification of plurihormonal Pit-1 and unusual tumors. Overall, our results suggest that the RT-qPCR of pituitary-specific TF and hormone genes could help pathologists, endocrinologists, and neurosurgeons to improve the management of patients with pituitary tumors.

Comparative pathologic analysis of mediastinal B-cell lymphomas: selective expression of p63 but no GATA3 optimally differentiates primary mediastinal large B-cell lymphoma from classic Hodgkin lymphoma

BackgroundInterpretation of mediastinal biopsy is often challenging even for experienced pathologists especially when a hematolymphoid neoplasm is suspected. Primary mediastinal large B-cell lymphoma (PMLBCL) and classic Hodgkin lymphoma (CHL) represent two major types of mature B-cell lymphomas of the mediastinum. Although PMLBCL and mediastinal CHL share many clinicopathologic characteristics, their treatment strategies and responses are remarkably different. We therefore aimed to find distinctive histologic or protein markers to better differentiate these two lesions.MethodsSearch for primary mediastinal B-cell lymphomas found 52 consecutive cases from 3 university hospitals of Korea during 2005 to 2012. Among them, 32 cases that were available for additional immunohistochemistry (IHC) assessment were enrolled in this study. These cases consisted of the following: CHL (N = 13), PMLBCL (N = 16), and B-cell lymphoma unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL (gray zone lymphoma, N = 3). Along with the clinicopathologic findings, the expression of p63, GATA3 and cyclin E was investigated by IHC in the three categorized lesions mentioned above.ResultsMost clinical features overlapped between PMLBCL and CHL except for the increased disease progression and mortality found in PMLBCL. In the pathologic review, the presence of epithelioid granuloma favored a diagnosis of CHL, whereas reticulated or alveolar patterns of fibrosis were characteristic of PMLBCL. For protein markers, p63 was predominantly positive in PMLBCL (15/16) compared with CHL (2/13), which indicates that p63 is a marker of the highest diagnostic accuracy when calculated by the area under the ROC curve. GATA3 was expressed in the majority of CHL cases (10/13) compared with PMLBCL (0/16), while the expression of cyclin E was only rarely present in a minor population of PMLBCL.ConclusionsP63 expression in tumor cells, even focal expression, and no GATA3 is the most helpful feature in distinguishing PMLBCL from mediastinal CHL.

Unexpected PAX8 Immunoreactivity in Metastatic High-grade Breast Cancer.

Immunohistochemistry (IHC) is often critical for distinction between metastatic carcinomas of Mullerian organ and breast origin. Paired box family protein 8 (PAX8) has been described as a transcription factor highly specific to neoplasms derived from Mullerian organs, thyroid, and kidney. PAX8 IHC with polyclonal and monoclonal antibody reagents was performed on 27 primary and 22 metastatic breast carcinomas. Eight of 27 primary breast carcinomas (30%) were positive for PAX8 with the monoclonal antibody reagent only; 0 of 22 were polyclonal anti-PAX8 immunoreactive. Substantial numbers of metastases had positive immunoreactivity for polyclonal anti-PAX8 (23%). Each of these metastases and additional cases (45% total) also had positive immunoreactivity for monoclonal anti-PAX8, including 5 of 7 brain metastases. IHC with monoclonal anti-PAX8 was positive on 6 of 7 primary breast carcinomas corresponding to PAX8-positive metastases. Together, these results indicate a significant fraction of breast carcinoma metastases and corresponding primary neoplasms have immunoreactivity for PAX8, and positivity rates depend on the antibody used. Diagnoses of metastatic breast carcinoma were achieved with the aid of clinical history and additional IHC in cases of PAX8 immunoreactivity. Contextual interpretation is imperative for PAX8 IHC, particularly when the differential diagnosis includes metastatic breast carcinoma with limited diagnostic material available.

Malignant Perivascular Epithelioid Cell Tumor Mimicking Renal Cell Carcinoma: A Diagnostic Pitfall

Abstract Introduction Perivascular epithelioid cell tumors (PEComas) are defined by the World Health Organization (WHO) as mesenchymal tumors arising from perivascular epithelioid cells with myomelanocytic immunophenotype. In the kidney, they are often referred to as epithelioid angimyolipoma (AML). Most are benign but some can be malignant. Their rarity and wide differential render diagnosis challenging. Case Presentation A 56-year-old male presented with a 3-month history of flank pain. Computed tomography (CT) identified bilateral necrotic heterogeneous enhancing kidney masses, with right significantly larger than left. Initially, the patient underwent a right radical nephrectomy. On gross examination, no tumor infiltration was observed. Histology demonstrated sheets of highly atypical cells with clear cytoplasm and easily identifiable nucleoli. Areas of spindle and rhabdoid differentiation were present. The initial diagnosis was renal clear cell (RCC) carcinoma, Fuhrman grade 4, with extensive rhabdoid and focal sarcomatoid features. A follow-up CT demonstrated a hypodense area within the right hepatic lobe. Status post liver resection and histomorphological examination, the lesion was identical to the renal mass except for focal areas of prominent epithelioid cells with eosinophilic cytoplasm. Immunohistochemical (IHC) stains of both masses were positive for Melan-A, HMB-45, and EMA but negative for PAX8, SOX10, S100, SMA, desmin, and cytokeratin. The initial diagnosis was amended to malignant AML with hepatic metastasis. Discussion and Conclusion Renal PEComa (epithelioid AML) is a rare tumor with a prevalence of 0.44%. While most are benign, some are malignant with metastatic potential. The differential diagnosis for PEComa is broad and includes more common tumors like RCC, melanoma, clear cell sarcoma, and liposarcoma. The presence of spindle and epithelioid histology with eosinophilic cytoplasm in RCC should prompt additional IHC workup (HMB45, Melan-A, SMA, and EMA) in order to exclude PEComa/AML.

P04.14 Loss of oligodendroglial features at recurrence in five diffuse low-grade glioma patients treated with repeated surgery

Abstract BACKGROUND Diffuse low-grade gliomas (DLGG) are characterized by a continuous growth and an unavoidable anaplastic transformation. IDH mutation and 1p19q codeletion have been integrated to the 2016 WHO classification to define the oligodendroglioma entity. Whenever feasible, neurosurgical resection is the first treatment option. At recurrence, a second surgical resection is proposed in selected cases. The consistency of molecular patterns (IDH mutation, 1p19q codeletion) at recurrence has been poorly studied in DLGG. MATERIAL AND METHODS We conducted a retrospective study on consecutive DLGG patients treated at our institution with repeated surgery (2006–2017). Clinical and biological data were collected for both the initial and subsequent surgery. Additional immunohistochemistry (including tumor morphology, ATRX and p53) and genetic analyses (TERT promoter mutation, CIC mutation, CGHa) were also performed on tumors with joint loss of IDH mutation and of 1p19q codeletion at recurrence. RESULTS A total of 71 patients were identified. Analyses were carried out on 56 patients (molecular data missing: n=15). Nine patients (16.1%) presented with a loss of their IDH mutation at second surgery. Five of them (8.9%) had an additional loss of their 1p19q codeletion. These five cases (3 men, median age 36.6 years) were all treated with surgery as the first oncological treatment. The first surgery revealed in all cases tumors with morphological oligodendroglial features, IDH mutation and 1p19q codeletion. Further molecular analysis strengthened the diagnosis of oligodendroglioma (TERT promoter and CIC mutations, no ATRX loss, no expression of p53). Four patients were followed-up after the first surgery; one patient received Temozolomide 14 months later due to FLAIR tumor volume growth. Because of the regrowth of the residual FLAIR tumor volume, a second surgery was performed in all patients, after a median time of 38.9 months. The morphological oligodendroglial features were lost, and the genetic analyses revealed in all cases no IDH mutation, no 1p19q codeletion, no ATRX loss and no expression of p53. No evidence of anaplasia was found histologically or by CGHa analysis in these recurrent tumors. CONCLUSION We describe five DLGG patients with a shared histo-molecular evolution characterized by the loss of the initial IDH mutation and of oligodendroglial features at second surgery. While rare, this possible evolution must be acknowledged as it can impact the subsequent therapeutic strategy. This observation is the first of a loss of founder alterations in DLGG genesis (i.e. IDH mutation and 1p19q codeletion); the involved mechanism likely differs from the previously described oligoclonal selection caused by spontaneous tumor genetic drift and/or pressure of chemotherapy, and could be linked with the Darwinian selection of a subpopulation of tumor cells after the first surgery.

Abstract CT050: Expansion cohort of Phase I study of oral sapacitabine and oral seliciclib in patients with metastatic breast cancer and BRCA1/2 mutations

Abstract Introduction: Sapacitabine, a nucleoside analog, and seliciclib, a cyclin-dependent kinase 2/9 inhibitor, constitute a novel oral regimen aimed at augmenting DNA damage and impairing cell cycle checkpoints. The initial phase I cohort investigating this combination demonstrated a 25% response rate in BRCA carriers. Hence, we developed an expansion cohort to assess the safety and efficacy of this regimen in patients with metastatic breast cancer and BRCA1/2 mutations. Methods: We enrolled 20 patients with HER2-negative metastatic breast cancer and germline or somatic BRCA1/2 mutations, who were treated with sapacitabine 50 mg twice daily for days 1-7 followed by seliciclib 800 mg twice daily for days 8-10 of a 21-day cycle. Baseline or archival biopsies underwent RAD51 immunohistochemistry to assess for functional homologous recombination proficiency. Available tissue was sent for whole exome and transcriptome sequencing, and pre- and post-treatment blood was submitted for cell-free DNA sequencing to assess for genomic correlates of response. Results: Participants received a median of 2 prior lines of chemotherapy for metastatic disease. Of the 9 patients who received a prior platinum agent, 6 progressed on this therapy. In addition to chemotherapy, 7 patients received and progressed on a prior PARP inhibitor. The overall response rate for sapacitabine and seliciclib in this cohort was 10%, consisting of 2 patients with partial responses lasting 4.7 and 9.0 months, respectively. The clinical benefit rate (CR + PR + SD ≥ 6 months) was 30%, and durations of stable disease ≥ 6 months ranged from 7.4 to 11.7 months. For all patients, median PFS was 3.7 months. The most frequent grade 3/4 adverse events were neutropenia (25% of patients), transaminitis (20%), and rash (10%). No patients who progressed on prior PARP inhibitor therapy and 6 of 13 patients (46%) with no history of PARP inhibitor resistance experienced clinical benefit (p = 0.052 by Fisher’s exact test). In contrast, 1 of 6 patients (17%) who progressed on prior platinum chemotherapy and 5 of 14 patients (36%) with no history of platinum resistance experienced clinical benefit (p = 0.61 by Fisher’s exact test). Notably, the tumors of some resistant patients harbored BRCA reversion mutations. Additional genomic analyses and RAD51 immunohistochemistry will be presented. Conclusions: The combination of sapacitabine and seliciclib was safe and led to durable clinical benefit in some patients with metastatic breast cancer and BRCA1/2 mutations. Prior progression on PARP inhibitors predicted resistance to this combination, associated in some cases with BRCA reversion mutations. Based on these results, the combination of sapacitabine and the PARP inhibitor olaparib is now being investigated in patients with PARP-naïve metastatic HER2-negative breast cancer and germline BRCA1/2 mutations. Citation Format: Tanya Keenan, David Liu, Haitham Elmarakeby, Daniel Stover, Bose Kochupurakkal, Adam Tracy, Elaine Danielczyk, Leilani Anderson, Chelsea Andrews, Brendan Reardon, Beth Overmoyer, Eric Winer, Daniella Zheleva, Judy Chiao, David Blake, Eliezer Van Allen, Geoffrey I. Shapiro, Sara Tolaney. Expansion cohort of Phase I study of oral sapacitabine and oral seliciclib in patients with metastatic breast cancer and BRCA1/2 mutations [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 CT050.

Deep learning-based predictive biomarker for immune checkpoint inhibitor response in metastatic non-small cell lung cancer.

9094 Background: In the era of immunotherapy, immune checkpoint inhibitor (ICI) has changed the treatment paradigm in metastatic non-small cell lung cancer (NSCLC). Along with clinical trials, there is an ongoing investigation to discover the predictive biomarker of ICI which so far has unsatisfactory reliability. As an effort to enhance the predictive value of ICI treatment, we applied deep learning and developed artificial intelligent (AI) score (range from 0 to 1) to analyze the specific context of immune-tumor microenvironment (TME) extracted by scanned images from H&E slides. Methods: As a ground work, deep learning-based H&E image analyzer, Lunit SCOPE, has been trained with H&E images (n = 1824) from ICI naive NSCLC samples. For the calculation of AI score, training was conducted using responder/non-responder labeled ICI treated samples from the exploratory cohort. The ICI responder was defined as the patient with a best overall response of partial or complete response and stable disease for more than 6 months. The positivity of PD-L1 immunohistochemistry (IHC) was assessed manually by pathologists. Results: The exploratory cohort is composed of NSCLC patients treated with ICI (n = 189) in Samsung Medical Center, and response to ICI was observed in 72 (38.1%) patients. Median follow-up duration was 6.8 months (6.6~8.2). Samples with PD-L1 IHC positive, defined by ≥ 1%, was observed in 138 (73.0%) patients. AI score was significant higher in the responder group (median: 0.391 vs 0.205, P = 6.14e-5), and the patients with AI score above the cut-off (0.337) showed a better response to ICI (odds ratio [OR] 3.47 P = 7.34e-5) which is higher than patients with PD-L1 ≥ 1% (OR 1.92, P = 0.069). High AI score group (n = 83) showed significantly favorable PFS compared to low AI score group (n = 106, median PFS: 5.1m vs 1.9m, hazard ratio [HR] 0.51, P = 9.6e-5) and this outcome was independent with PD-L1 status (P = 6.0e-5). In subgroup analysis, PFS of PD-L1 high / AI score high group (n = 63) had longer median PFS (6.7m) compared to both PD-L1 high / AI score low group (n = 70, 4.0m, P = 0.001) and PD-L1 low/AI score low group (n = 35, 1.9m, P = 4.0e-6). Tumor infiltrating lymphocyte (TIL) density of cancer epithelium was significantly correlated with AI score (Pearson’s r = 0.310, P = 1.43e-5), which suggests that AI score may partly reflect TME represented by TIL. Conclusions: The AI score by machine-learned information, extracted from H&E images without additional IHC stain, could predict responsiveness and PFS of ICI treatment independent of PD-L1 IHC positivity.

Improvement in neurogenesis and memory function by administration of Passiflora incarnata L. extract applied to sleep disorder in rodent models

Recently, there have been reports that chronic insomnia acts as an insult in the brain, causing memory loss through the production of ROS, inflammation, and, Alzheimer’s disease if persistent. Insomnia remains the leading cause of sleep disturbance and as such has serious implications for public health. Patients with Alzheimer's disease are also known to suffer from severe sleep disturbance. Meanwhile, vitexin is a key ingredient in Passiflora incarnata L (passion flower, PF) extract, which is known to help with sleep. This medicinal plant has been used as a folk remedy for sedation, anxiety and sleep since centuries ago, but the standardization work has not been done and the extent of the effect has not been clearly demonstrated.For this reason, we tried to test the possibility that repeated administration of PF could improve the memory by promoting hippocampal neurogenesis at the DBA/2 mice known have inherited sleep disorders, as well as preventive effects of Alzheimer's disease.Here, we found that vitexin, which is the main bioactive component of ethanol extracts from leaves and fruits (ratio; 8:2) of PF, confirmed the improvement of neurogenesis (DCX) of DBA/2 mice repeated PF oral administration by immunohistochemistry (IHC) and western blot analysis. PF-treated group showed increased the neurotrophic factor (BDNF) in the hippocampus compared with that of vehicle-treated group, but the inflammation markers Iba-1 (microglial marker) and COX-2 were inconsistent between the groups. However, we found COX-2 signal is essential for hippocampal neurogenesis according to the additional IHC experiments using COX-2 inhibitor and pIkappaB have shown. In addition, although prescription sleeping pills have been reported to show significant changes in appetite and metabolic rate from time to time, no changes in the feeding behavior, body weight, metabolic rate and body composition of the animals were observed by administration of PF. Interestingly, we found that short-term oral administration of PF displayed improved memory according to the water maze test. Quantitative analysis of Tau protein, which is a marker of Alzheimer's disease, was performed in the SD rats and DBA/2 mice by repeated PF oral administration and pTau/Tau values were significantly decreased in PF-treated group than vehicle-treated group.In conclusion, our results suggest that PF lead high hippocampal neurogenesis in the animals even in inherited sleep-disturbed animals. The increased hippocampal neurogenesis functionally enhanced memory and learning functions by repeated PF oral administration. These results identify PF as a potential therapy for enhancing memory functions and prevention of Alzheimer's disease through actions on the hippocampus.

Study the effect of indomethacin administration on breast tumor growth and iNOS gene expression in tumor-bearing mice

Breast cancer is a public health concern among the women. iNOS is stated by the effect of various inflammatory factors and is thus called inducible NOS. Investigating iNOS expression is a potent tool for understanding effective molecular parameters in tissue and cellular responses to external factors. In this research study, iNOS expression in mice with breast cancer was investigated and the effects of various doses of indomethacin on the iNOS gene expression in breast tumors was evaluated.4T1 cells were grown in RPMI 1640 Medium. 200 µl of cell suspension (107 cells)were injected into the mice right flank, subcutaneously. 35 Balb/C albino mice divided into five groups each group consist of 7 mice. First group was healthy control group, second group was tumor control, and the 3rd, 4th and 5th groups were treated by 25μg, 50μg and 100μg of indomethacin, respectively. The rate of breast tumor growth was measured in treated groups for two months. The levels of iNOS gene expression was determined using real time RT-PCR technique.Our results demonstrated, iNOS expression in tumor tissue increased with the growth of breast cancer cells. Furthermore, Administration of 25μg and 100μg indomethacin neither had significant effect on breast tumor growth nor effect on iNOS gene expression. While 50μg indomethacin decreased the tumor growth (P>0.05) and decreased iNOS gene expression. Further studies such as evaluating the effects of indomethacin on the other anti-tumor immunosuppressing factor and additional tests western blot, flow cytometry and immunohistochemistry recommended in order to obtain more practical findings.

Anatomical Pathology Laboratory Activity in Meknes, Morocco

Cancer continues to be a major health problem in the world. During 2018, there were 9.6 million deaths and 18.1 million new cases registered across the globe. It is estimated that one in 5 men and one in 6 women will develop cancer during their lifetime. This disease kills one in 8 men and one in 11 women. Developing countries have seen an increase in cancer occurrence and change in type of cancers due to change in social and economic conditions. The objective of this research was to further evaluate 1659 specimens for anatomical pathology testing. The data was collected from the records of the pathological anatomy laboratory at the Mohamed V Hospital in Meknes, Morocco. It is one-year study period (January-December 2013). Results of the anatomy pathology exam showed cancer positive for 9.6% of specimens; inconclusive for 1%; and negative for 66.2%. For all specimens, the most analyzed organs are breast with 29.7%; skin with 21.3%; cervical with 2.6%. For cancer patients, the most affected organs are skin (21.3%) and breast (29.7%). For cancer patients, Neoplasm is present in 9.3% of patients. Patients with metastatic cancer represent 0.3%. Patients with non-neoplastic lesions represent 66.2%. 1% of patients represent uncertain results. Additional immunohistochemistry research is needed before conclusive recommendations are made.

Centromere protein F (CENPF), a microtubule binding protein, modulates cancer metabolism by regulating pyruvate kinase M2 phosphorylation signaling

ABSTRACT Prostate cancer (PC) is the most commonly diagnosed cancer in men and is the second leading cause of male cancer-related death in North America. Metabolic adaptations in malignant PC cells play a key role in fueling the growth and progression of the disease. Unfortunately, little is known regarding these changes in cellular metabolism. Here, we demonstrate that centromere protein F (CENPF), a protein associated with the centromere-kinetochore complex and chromosomal segregation during mitosis, is mechanically linked to altered metabolism and progression in PC. Using the CRISPR-Cas9 system, we silenced the gene for CENPF in human PC3 cells. These cells were found to have reduced levels of epithelial-mesenchymal transition markers and inhibited cell proliferation, migration, and invasion. Silencing of CENPF also simultaneously improved sensitivity to anoikis-induced apoptosis. Mass spectrometry analysis of tyrosine phosphorylated proteins from CENPF knockout (CENPFKO) and control cells revealed that CENPF silencing increased inactive forms of pyruvate kinase M2, a rate limiting enzyme needed for an irreversible reaction in glycolysis. Furthermore, CENPFKO cells had reduced global bio-energetic capacity, acetyl-CoA production, histone acetylation, and lipid metabolism, suggesting that CENPF is a critical regulator of cancer metabolism, potentially through its effects on mitochondrial functioning. Additional quantitative immunohistochemistry and imaging analyzes on a series of PC tumor microarrays demonstrated that CENPF expression is significantly increased in higher-risk PC patients. Based on these findings, we suggest the CENPF may be an important regulator of PC metabolism through its role in the mitochondria.

Anti-ARHGAP26 Autoantibodies Are Associated With Isolated Cognitive Impairment.

Autoantibodies against the RhoGTPase-activating protein 26 (ARHGAP26) were originally identified in the context of subacute autoimmune cerebellar ataxia. Further studies identified a wider clinical spectrum including psychotic, affective, and cognitive symptoms. Only a few patients reported so far had evidence of a tumor association. A prospective analysis between January 2015 and December 2017 at the Dept. of Neurology at Charité—Universitätsmedizin Berlin identified 14 patients with ARHGAP26 autoantibodies on a cell-based assay, of which three patients had additional brain immunohistochemistry staining of cerebellar molecular layer and Purkinje cells, who were therefore considered antibody-positive. In all three patients, ARHGAP26 autoantibodies were associated with tumors. In two patients, an isolated cognitive impairment without additional neurological deficits was observed. These cases thus further extend the clinical spectrum associated with ARHGAP26 autoantibodies and strengthen a potential paraneoplastic context.

Abrupt Dyskeratotic and Squamoid Cells in Poorly Differentiated Carcinoma: Case Study of Two Thoracic NUT Midline Carcinomas with Cytohistologic Correlation.

Cytologic diagnosis of nuclear protein in testis (NUT) midline carcinoma (NMC) is important due to its aggressive behavior and miserable prognosis. Early diagnosis of NMC can facilitate proper management, and here we report two rare cases of thoracic NMC with cytohistologic correlation. In aspiration cytology, the tumor presented with mixed cohesive clusters and dispersed single cells, diffuse background necrosis and many neutrophils. Most of the tumor cells had scanty cytoplasm and medium-sized irregular nuclei, which had fine to granular nuclear chromatin. Interestingly, a few dyskeratotic cells or squamoid cell clusters were present in each case. Biopsy specimen histology revealed more frequent squamous differentiation, and additional immunohistochemistry tests showed nuclear expression of NUT. Because this tumor has a notorious progression and has been previously underestimated in terms of its prevalence, awareness of characteristic findings and proper ancillary tests should be considered in all suspicious cases.

Interobserver variation in CD30 immunohistochemistry interpretation; consequences for patient selection for targeted treatment.

CD30 immunohistochemistry (IHC) in malignant lymphoma is used for selection of patients in clinical trials using brentuximab vedotin, an antibody drug-conjugate targeting the CD30 molecule. For reliable implementation in daily practice and meaningful selection of patients for clinical trials, information on technical variation and interobserver reproducibility of CD30 immunohistochemistry (IHC) staining is required. We conducted a three-round reproducibility assessment of CD30 scoring for categorised frequency and intensity, including a technical validation, a 'live polling' pre- and post-instruction scoring round and a web-based round including individual scoring with additional IHC information to mimic daily diagnostic practice. Agreement in all three scoring rounds was poor to fair (κ = 0.12-0.35 for CD30-positive tumour cell percentage and κ = 0.16-0.41 for staining intensity), even when allowing for one category of freedom in percentage of tumour cell positivity (κ = 0.30-0.61). The first round with CD30 staining performed in five independent laboratories showed objective differences in staining intensity. In the second round, approximately half the pathologists changed their opinion on CD30 frequency after a discussion on potential pitfalls, highlighting hesitancy in decision-making. Using fictional cut-off points for percentage of tumour cell positivity, agreement was still suboptimal (κ = 0.35-0.60). Lack of agreement in cases with heterogeneous expression is shown to influence patient eligibility for treatment with brentuximab vedotin, both in clinical practice and within the context of clinical trials, and limits the potential predictive value of the relative frequency of CD30-positive neoplastic cells for clinical response.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye

BackgroundAbsence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. MethodsWestern blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-β1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. ResultsIHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-β1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. ConclusionsThe collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-β1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

Increased Expression of Programmed Death-Ligand 1 in Infiltrating Immune Cells in Hepatocellular Carcinoma Tissues after Sorafenib Treatment

Objective: Programmed death-ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has been reported to be related to prognosis in patients with hepatocellular carcinoma (HCC) after hepatectomy. The impact of sorafenib on PD-L1 expression in the TME of advanced HCC is unclear. Patients and Methods: Patients with HCC who received sorafenib for advanced disease at National Taiwan University Hospital, Taipei, Taiwan, and who had paired HCC tissues obtained before and after sorafenib treatment were included in the study group. HCC patients not treated with sorafenib who had paired primary and recurrent or metastatic tissues were identified as the reference group. The membrane PD-L1 staining, detected by immunohistochemistry (IHC) using SP142 antibody, was semiquantitatively scored in tumor cells (TCs) or tumor-infiltrating immune cells (ICs). Additional IHC assays were employed to characterize the PD-L1-expressing ICs. Results: Twenty-three advanced HCC patients with pre- and post-sorafenib paired HCC tissues were included in the study group. The median duration of sorafenib treatment was 4.3 months (range: 1.3–18.7). PD-L1 expression in ICs was significantly higher in post-sorafenib HCC tissues than in pre-sorafenib HCC tissues (pre-sorafenib vs. post-sorafenib IHC 0/1/2/3: 11/5/5/2 vs. 5/5/2/11, p = 0.016). However, PD-L1 expression in TCs was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs. 14/5/0/4, p = 0.094). In the reference group of 44 patients not treated with sorafenib, PD-L1 expression in ICs and TCs was not significantly different between the paired primary and metastatic HCC tissues. By performing IHC double staining with PD-L1 and CD68, we found the PD-L1-expressing ICs were mainly CD68-positive macrophages. PD-L1 expression levels of pre- and post-sorafenib tissues were not associated with patients’ overall survival or duration of sorafenib treatment. Conclusions: PD-L1 expression in ICs was significantly increased in post-sorafenib HCC tissues. The mechanisms and clinical significance of this observation warrants further investigation.

The histomorphological spectrum of restrictive chronic lung allograft dysfunction and implications for prognosis

AbstractChronic lung allograft dysfunction continues to be the main contributor to poor long-term allograft survival after lung transplantation. The restrictive phenotype of chronic lung allograft dysfunction carries a particularly poor prognosis. Little is known about the pathogenetic mechanisms involved in restrictive chronic lung allograft dysfunction. In this study, we performed histomorphological and immunohistochemical analysis of restrictive chronic lung allograft dysfunction lungs. Explant lung tissue from 21 restrictive chronic lung allograft dysfunction patients was collected and histopathologic patterns of rejection, fibrosis and vascular changes were scored after routine histochemical stains and additional immunohistochemistry for endothelial markers and C4d. In all, 75% of cases showed evidence of acute cellular rejection; lymphocytic bronchiolitis was absent in most lungs, whereas in 55% there was obliterative bronchiolitis. Almost half of the cases showed a pattern consistent with pleuroparenchymal fibro-elastosis (n=10), and a subset showed nonspecific interstitial pneumonia (n=5) or irregular emphysema (n=5). Fibrinous alveolar exudates were frequently seen in association with fibrosis (n=6), but no diffuse alveolar damage was found. Evidence of microvascular damage was present in most cases. An emphysematous pattern of fibrosis was associated with a better survival (P=0.0030), whereas fibrinous exudates were associated with a worse survival (P=0.0007). In addition to the previously described nonspecific interstitial pneumonia and pleuroparenchymal fibro-elastosis patterns in restrictive chronic lung allograft dysfunction, we are the first to describe a pattern of fibrosis-induced subpleural/paraseptal emphysema. This pattern confers a better survival, whereas fibrinous exudates are associated with a worse survival. We believe that our findings offer a pathogenetic theory for pleuroparenchymal fibro-elastosis in restrictive chronic lung allograft dysfunction, and show that restrictive chronic lung allograft dysfunction is an increasingly heterogeneous disease with presumably different mechanisms of subpattern formation.

Implementation of Anatomic Pathology Services in Mirebalais, Haiti

Abstract 97 Purpose Despite great barriers, implementing pathology services in resource-limited rural settings is achievable. Challenges include the lack of infrastructure, trained personnel, supply chain, and decentralized services, and have hindered the appropriate provision of cancer care to those who live in resource-limited settings. In Haiti, we began treating cancer at University Hospital Mirebalais (HUM) before pathology services existed, sending samples to the United States for diagnoses. This model allowed patients to receive treatment, but turnaround time was often delayed. Our goal is to implement pathology services at HUM that can provide high-quality diagnostics to patients who are enrolled in the cancer program. We demonstrate that, by rolling out a phased implementation of local pathology services with appropriate commitment from local and international partners, accurate pathologic diagnoses can exist in low- and middle-income countries. Methods Phase I—infrastructure and personnel—occurred fall 2016 with the construction of the laboratory and the hiring of a pathologist and laboratory technicians, which allowed samples to be grossed into paraffin blocks before being sent to Boston. Phase II—technology transfer and training—occurred fall 2017 with the procurement of additional equipment and hands-on histology training, which allowed samples to be cut, stained, and diagnosed on-site. In this phase, only complex samples that needed additional immunohistochemistry or a second opinion were sent to Boston. Phase III—testing capacity expansion—will occur summer 2018 and involves procuring immunohistochemistry and training, which will further limit the need to send cases to the United States. Phase IV—routine testing and quality assurance—will occur in tandem with phase III and involves implementing a telepathology system on-site in the HUM laboratory. Results Outcomes of this phased implementation will result in decentralized pathology services in the public sector and ultimately decreased turnaround times, producing better clinical outcomes. Our model demonstrates that it is possible to build local pathology capacity in rural areas. Since the opening of our laboratory, more than 1,000 cases have been processed into paraffin blocks before being sent to Boston. In September 2017, the first cases were diagnosed in country, and more than 100 cases have been processed and reported in Haiti. Conclusion Building on the foundational phases, the next phases of implementation involve long-term diagnostic support that will correlate directly with a substantial decrease in turnaround time. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST No COIs from the authors.

Assessment of Ki67 in relation to radiotherapy (RT) fractionation and prognosis in the CHHiP (CRUK/06/016) trial.

98 Background: A uniform fractionation schedule is used to deliver external beam RT for localised prostate tumours Individualising fractionation according to tumour biology may improve outcomes. In addition recurrence rates after RT vary considerably and better prognostic markers are needed to guide treatment choices. This study aims to identify if the cell proliferation marker Ki67 predicts response to RT fractionation in CHHiP, a randomised trial of 3 RT fractionation schedules. It also aims to identify if Ki67 predicts prognosis. Methods: A matched case:control study design was used, patients with biochemical or clinical failure > 2 years after RT (BCR) were matched to patients without recurrence according to established prognostic factors (Gleason score, PSA, Tumour-stage) and fractionation schedule. Immunohistochemical (IHC) staining of diagnostic biopsy sections was carried out using the MIB1 Ki67 antibody. Two independent investigators scored Ki67 using the unweighted global method (1) to derive a mean and maximum percentage of cells staining positive (mean Ki67 and maximum Ki67 respectively). Conditional logistic regression models were fitted with interaction terms between the biomarker and the fractionation schedules to determine whether Ki67 predicted BCR according to fractionation. Secondly models were fitted using the entire case:control study sample to estimate the prognostic value of Ki67 on risk of BCR. Results: Ki67 results were available for 173 matched pairs. The interaction terms between Ki67 and the fractionation schedules were not significant. However mean and maximum Ki67 were significant prognostic markers for BCR in a model adjusted for established prognostic factors. Conditioning on matching variables and age, the odds of BCR is estimated to increase by 9% per 1 point increase in mean Ki67 (OR = 1.09, 95%CI:1.04–1.15, p = 0.001). Conclusions: Ki67 did not predict BCR according to fractionation schedule in CHHiP, however it did predict prognosis independently of established prognostic factors. Additional IHC biomarkers are under evaluation.