True Tumor Cells Research Articles

The utilization of artificial intelligence to produce clinically relevant scores on PD-L1 immunostained non-small cell lung cancer biopsies.

e14576 Background: Immunotherapies targeting complex programmed death-ligand 1 (PD-L1) interactions have been groundbreaking in the treatment of non-small cell lung cancer (NSCLC), offering new strategies for patients who are likely to respond. PD-L1 binds to the checkpoint PD-1 to regulate T cells, which has an important role in boosting the immune response. Identifying patients who may benefit from PD-L1 focused care is dependent on the interpretation of the drug’s associated companion diagnostic (CDx) immunohistochemistry (IHC) assay. PD-L1 IHC assays stratify NSCLC patients using a Tumor Proportion Score (TPS), a manual scoring paradigm which must be applied by a clinical pathologist. Application of the score is complex as it requires the reader to visually exclude stromal and tumor-infiltrating immune cells across a full sample, retaining PD-L1 expression information for true tumor cells only. Methods: In this study, we utilized the PD-L1 [28-8] immunohistochemistry (IHC) assay to stain 10 NCSLC specimens. Stained slides were then imaged using the AT2 scanner (Leica Biosystems, Buffalo Grove, IL) scanner and analyzed using the Visiopharm Image Analysis platform. Results: The intricacy of TPS application is time consuming for the pathologist and introduces opportunity for inter- and intra-reader variability. Our novel image analysis approach utilizes artificial intelligence (AI) to automatically denote tumor nests and exclude tumor-infiltrating immune cells. The remaining true tumor cells may then be quantified for PD-L1 expression across the full biopsy, producing a TPS for each sample. Conclusions: This method allows for great accuracy and time-efficiency in providing a TPS than traditional methods, which may result in improved patient care.

Differences in PD-L1-Expressing Macrophages and Immune Microenvironment in Testicular Germ Cell Tumors.

To characterize the tumor microenvironment of testicular germ cell tumors (GCTs) using immunohistochemical markers. Seventy-seven orchiectomies, including 36 nonmetastatic (NM) seminomas, 15 metastatic (M) seminomas, 13 nonmetastatic nonseminomatous germ cell tumors (NSGCTs), and 13 metastatic NSGCTs, were studied with PD-1, PD-L1, FOXP3, CD68, CD163, and mismatch repair (MMR) immunohistochemistry. FOXP3+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) expressing CD68 and CD163 were enumerated. PDL-1 expression was evaluated on tumor cells and macrophages. GCTs primarily express PD-L1 on TAMs, except choriocarcinoma, where true tumor cell positivity was noted. Seminomas reveal increased intratumoral PD-L1+ TAMs compared with NSGCTs (P < .05). Activated TILs are increased in NM-seminomas compared with M-seminomas (P < .05). All GCTs retained MMR expression. Robust PD-L1+ TAMs are significantly expanded in seminomas compared with NSGCTs. Among all GCTs, only choriocarcinoma cells reveal true positivity for PD-L1. These findings expand the realm of potentially targeted treatments for GCTs.

Cellular localization of PD-L1 expression in mismatch-repair-deficient and proficient colorectal carcinomas

Blockade of the interaction between PD-1 and its ligands PD-L1 has shown clinical efficacy across several tumor types, especially in mismatch-repair-deficient colorectal carcinoma. The aim of this study was to examine the pattern and cellular localization of PD-L1 expression in the different molecular subtypes of mismatch-repair-deficient colorectal cancers vs. their mismatch-repair-proficient counterparts. PD-L1/SATB2 double-antibody-immunohistochemistry was utilized to distinguish tumor cell from immune cell staining. We observed in our series of 129 colorectal adenocarcinomas that PD-L1 expression occurred primarily in tumor-associated-immune cells and most prominently at the tumor-stroma-interface of the invasive front. The level of invasive front immune cell staining was significantly higher in mismatch-repair-deficient tumors compared to mismatch-repair-proficient tumors (p < 0.001), but no difference was observed among the different subtypes of mismatch-repair-deficient tumors: Lynch syndrome-associated vs. MLH1-methylated vs. unexplained. While selected mismatch-repair-proficient tumors exhibited unusually high tumor-infiltrating-lymphocytes and had high level immune cell PD-L1 expression, a positive correlation between PD-L1 expression and high lymphocyte count was detected only in mismatch-repair-deficient tumors (r = 0.39, p < 0.001) and not in mismatch-repair-proficient tumors. Notably, true tumor cell PD-L1 expression in colorectal carcinoma was rare, present in only 3 of 129 tumors (2.3%): 2 MLH1-methylated and 1 mismatch-repair-proficient with high tumor-infiltrating-lymphocytes; and the staining in the tumor cells in all 3 was diffuse (>=50% of the tumor). These findings may serve to inform further efforts aiming to evaluate PD-L1 immunohistochemistry vis-à-vis molecular sub-classification as predictive biomarkers in the treatment of colorectal carcinoma.

P2.01-001 Enrichment-Free, Rapid Metabolic Assay for Detection of Tumor Cells in Pleural Effusion and Pheripheral Blood

Current methods for circulating tumor cell (CTC) detection are mostly include an enrichment step and the subsequent immunostaining-based identification of CTCs by epithelial and leukocytes markers. These methods are limited by loss and damage of CTCs during the enrichment and fail to determine the malignancy and drug targets of putative CTCs. We describe an enrichment-free, metabolic-based assay for rapid detection of tumor cells in the pleural effusion and peripheral blood samples. All nucleated cells are plated on microwell chips that contain 200,000 addressable microwells. These cells are labeled with a fluorescent anti-CD45 antibody (leukocyte marker), a fluorescent glucose analog (2-NBDG) and a dead cell marker (EthD-1). The microwell chips are imaged by a computerized high-speed fluorescent microscope in three colors and the bright filed. A computation algorithm analyzes the images and identify candidate tumor cells that are viable, CD45 negative, and exhibit high glucose uptake (EthD-1-/CD45-/2-NBDGhigh). A micromanipultor is then utilized to retrieve single tumor cells based on recorded addresses for single-cell sequencing. EthD-1-/CD45-/2-NBDG>100 cells are identified as candidate tumor cells. Single-cell sequencing based on a small panel of driver oncogenes (EGFR, KRAS, PIK3CA) shows that >60% of candidate tumor cells are true tumor cells harboring mutations in the panel. Single-cell whole exome sequencing results show all candidate tumor cells have high mutation frequency in dirver oncogenece and tumor suppressors from Qiagen's Human Lung Cancer Panel. Meanwhile, CD45-/EthD-1-/2-NBDG>100 tumor cells show heterogenieity in cytokeratin (CK) expression, and only ∼40% of these tumor cells are found CK positive. We have developed a simple and functional-based method to rapidly identify tumor cells with high glucose uptake in the clinical liquid samples without enrichment. These tumor cells are addressable, enabling single-cell manipulation and sequencing. Clinical feasibility of this assay has been established by testing samples from a cohort of patients.

PCA3 and PSA gene activity correlates with the true tumor cell burden in prostate cancer lymph node metastases.

Extent of pelvic lymph node (LN) dissemination is a critical prognostic feature for patients with prostate cancer (PCa) maintaining extended pelvic lymphadenectomy (LAD) as the gold standard for LN-staging. Unfortunately, conventional histopathological assessment may miss micrometastasis and recently presented immunocytochemical approach of the single cell analysis is still intricate. To comparatively assess the potential of Prostate cancer gene 3 (PCA3) and prostate specific antigene (PSA) to perform as markers for tumor cell load. Patients with high risk PCa for LN metastasis undergoing either a sentinel LN-guided staging LAD or retropubic radical prostatectomy with sentinel-guided pelvic LN dissection were included. LNs were investigated by routine histopathology. Tumor cell load was quantified by %immunocytochemistry. immunocytochemical single cell analysis. Gene activity was determined by qRT-PCR. Twenty four out of 226 LNs were positive in routine histopathology and 51 in single cell analysis. PSA mRNA level correlated with tumor cell density in patients with a positive immunocytochemistry. Gene activity of PCA3 was upregulated in metastatic LNs and correlated with tumor cell density in patients with tumor-invaded LNs as detected by immunocytochemistry. PCA3 gene expression discriminates LN metastasis and might outperform PSA gene activity in reflecting tumor cell burden in pelvic LNs of PCa patients.

Von Hippel-Lindau Disease: A Genetic and Clinical Review

Background: Von Hippel–Lindau Disease (VHL) is an autosomal dominant inherited systemic cancer syndrome that gives rise to cystic and highly vascularized tumors in many organs, including the eye. Recent studies have contributed to the understanding of VHL pathophysiology, genetics, and the role of the VHL protein. This article reviews recent studies on VHL clinical findings, genetics and tumorigenesis. Methods: Literature review of articles on VHL genetics with correlation to clinical findings. Results: Genotype-phenotype correlation studies show that patients with a complete deletion mutation of the VHL gene, relative to participants with a missense or protein-truncating mutation, had better visual acuity and decreased tumorigenesis incidence of retinal hemangioblastomas. It has also been documented that higher levels of vascular endothelial growth factor (VEGF), hypoxia induced factor (HIF), and ubiquitin are found in ocular hemangioblastomas. The stromal foamy vacuolated cells seem to be the true tumor cells of the disease acting on the surrounding endothelial cells in ocular hemangioblastomas. Tumor cells and ocular lesions have shown increased levels of Erythropoietin (Epo), Epo receptor (EpoR), and CD133. Also, CXCR4, a CXC chemokine receptor, is expressed in retinal VHL hemangioblastomas. Recent studies suggest that the VHL mutation alone may not be sufficient to develop VHL-associated neoplasms. Studies suggest that targeting various proteins along with anti-angiogenesis molecules may be a better therapeutic approach than targeting VEGF alone. Conclusion: Understanding of the mechanisms and genetics underlying VHL and its associated retinal hemangioblastomas has increased substantially in recent years. This knowledge suggests that future advances may include better identification of individuals at higher risk of vision loss and the development of novel individualized therapies.

50 Years Ago in CORR®: Synovial Sarcoma Kirk J. Anderson, MD and Orliss Wildermuth, MD CORR 1961;29:55-70

50 Years Ago in CORR®: Synovial Sarcoma Kirk J. Anderson, MD and Orliss Wildermuth, MD CORR 1961;29:55-70

Value of clusterin expression in pathologic diagnosis and histogenesis of giant cell tumor of tendon sheath

Analyze the immunophenotype of the different cells in the various subtypes of giant cell tumor of tendon sheath (GCTS) and investigate the value of clusterin in pathological diagnosis and histogenesis of giant cell tumor of tendon sheath. A total of 104 cases of GCTS from the surgical pathology files of Shanghai Jiaotong university affiliated the sixth people's hospital were identified. Immunohistochemistry (IHC) for clusterin, desmin, CD163, CD68, p63, p53, Ki-67 and CD35 was performed on all cases, using EnVision technique. All cases of GCTS were researched, including 44 cases of localized type (L-GCTS), 32 cases of diffused type (D-GCTS), 26 cases of pigmented villonodular synovitis (PVNS) and 2 cases of malignant type. There was a slight female predominance in all these subtypes, and the male to female ratio was about 38:66. L-GCTS usually occured within the small joints (90.9%, 40/44), while D-GCTS, PVNS and M-GCTS commonly occured within the large weight-bearing joints [68.8% (22/32), 100% (26/26) and 2/2 respectively]. Of 74 cases with follow-up, the recurrence rates of L-GCTS, D-GCTS, PVNS and M-GCTS respectively were 30.3% (10/33), 30.4% (7/23), 18.8% (3/16) and 2/2. The different subtypes of GCTS had the same cell components, including the large synovial-like mononuclear cells, the small histiocytoid cells, foamy histiocytes cells, inflammatory cells, fibroblasts and the osteoclast-like multinucleated giant cells. There were obvious differences among immunophenotype of the various cell components in GCTS: the large synovial-like mononuclear cells were strong positive for clusterin, partly positive for desmin and Ki-67, and negative for CD163. The small histiocytoid cells were strong positive for CD163 but negative for clusterin and desmin. The osteoclast-like multinucleated giant cells were strong positive for CD68 but negative for clusterin, CD163 and desmin. Normal synoviocytes were strong positive for clusterin, partly positive for desmin. The number of the large synovial-like mononuclear cells that were positive for clusterin in D-GCTS were more than that in L-GCTS (P < 0.01) and PVNS (P < 0.05). GCTS was synovial tumors, not belonged to the category of fibrohistiocytic lesions. The true tumor cells may be the large synovial-like mononuclear cells, and the number of the cells in the D-GCTS was obviously more than that in L-GCTS and PVNS. This may be the reason that the biological behavior of D-GCTS was more aggressive, destructive and recurrent. Clusterin was an useful marker in pathological differential diagnosis of GCTS.

Reporting the Capture Efficiency of a Filter-Based Microdevice: A CTC Is Not a CTC Unless It Is CD45 Negative—Response

The basis for identification of disseminated tumor cells (DTC) and circulating tumor cells (CTC) is evaluation of antigenic differences [for epithelial tumors, generally antigens which identify their epithelial origin, such as cytokeratins (CK)] coupled with the morphology of the CTC (reviewed in ref. 1). In fact, morphologic examination is fundamental in distinguishing “background” staining from the true tumor cells. We agree with the basic premise of de Bono and colleagues that there is value to identify absence of CD45 staining in CK-positive epithelial cells as qualitative differentiator for DTCs or CTCs. But this is particularly useful in the cases in which cell morphology is not optimal (as is the case with CellSearch assessment), which was not the problem we faced with our microfilter platform. We believe that CD45 staining is not necessary when the morphology can be adequately assessed by cytology or historically used in defining a tumor cell in previous studies that have identified DTCs and showed their clinical relevance. As identifying features in our studies in the present study, the positive staining for CK cells was supplemented by morphologic features, such as nucleocytoplasmic ratios and cell size, which we believe are adequate for distinguishing CTCs from the background hematopoietic cells. Besides, given that the microfilter platform is better at enriching the CTCs on the hematopoietic background, the distinction of CTCs from the lymphocytes is far less problematic. It is also noteworthy here that many nonlymphocytic hematopoietic cells are CD45 negative, clearly showing that dependence on CD45 staining as a method for defining CTCs can be suboptimal. Nevertheless, our microfilter can be employed as a platform for staining for both the CK and the CD45; indeed, we have incorporated such dual staining in our subsequent studies. We agree that incorporation of more selective biomarkers going forward will help in better characterization of CTCs.See the original Letter to the Editor, p. 3048R.J. Cote has patents on described technology. He is the consultant and founder of the Filtini, Inc., which has licensed the patents. In addition, H. Lin and R. Datar are named inventors on the patents. A. Williams disclosed no potential conflicts of interest.

Morphological and immunohistochemical characterization of isolated tumor cells by p53 status in gastrointestinal tumors

Isolated tumor cells (ITCs) in cancer patients are retrieved mostly using immunohistochemistry with antibodies directed against antiepithelial antigens (for example Ber-EP4), which are supposed not to be present in metastatic-free tissue. To date, there has been ongoing controversy whether those cells have biologic significance and are linked with tumor progression and impaired patient's prognosis. Therefore, the aim of this study was to further characterize Ber-EP4-positive cells in various tissues, with special emphasis on their tumorigenic origin. The frequency and prognostic impact of ITCs in lymph nodes displayed by means of monoclonal antibody Ber-EP4 were evaluated in retrospective (n = 292) and prospective (n = 100) collectives of various gastrointestinal carcinomas free of metastatic disease in conventional histopathology (pN0). Furthermore, the frequency of ITCs in the peritoneal cavity and bone marrow was analyzed in case of absence of overt distant metastasis (pM0) in the prospective collective. Ber-EP4-immunoreactive cells were further characterized for tumorigenic origin using morphological criteria and immunohistochemical double staining for Ber-EP4 and p53. Ber-EP4-positive cells could be revealed in lymph nodes in 44.3% of pN0-gastrointestinal carcinomas, in the peritoneal cavity in 19%, and in the bone marrow in 10%. In lymph nodes, BerEP4-immunoreactive cells exhibited a metastatic-atypical morphology in 59%; however, it was always typical for true tumor cells in the peritoneal cavity or bone marrow. The cumulative 5-year survival rate was adversely affected by Ber-EP4-immunoreactive cells in uni- and multivariate analysis, irrespective of the underlying cell morphology (68% for Ber-EP4 negative, 41% for Ber-EP4 positive with atypical and typical morphology each). In the case of a p53-positive primary tumor, 70% of the corresponding ITCs also overexpressed p53, while the remainder was deemed p53 negative (p = 0.002). ITCs detected by the antiepithelial antibody Ber-EP4 are present in a substantial proportion of apparently tumor-free lymph nodes. These cells impair patients' prognoses, irrespective of the underlying cell morphology. As approximately one third of Ber-EP4-positive cells in p53-positive primary tumors do not overexpress p53; their true tumorigenic origin needs to be further investigated.

Expression of E-cadherin, β-catenin and p120ctn in so-called pulmonary sclerosing hemangioma

So-called pulmonary sclerosing hemangioma (PSH) is a rare kind of pulmonary tumor. Its histological origin and nature, which have become research hot spots for many years, are still uncertain. The aim of this study is to investigate the immunophenotype of cuboidal cells and polygonal cells through observing the expression of E-cadherin, β-catenin and p120ctn in cuboidal cells and polygonal cells of PSH. Expression of E-cadherin, β-catenin and p120 ctn was detected in 25 cases of PSH samples and 8 cases of pulmonary inflammatory pseudotumor samples by immunohistochemistry. Immunohistochemistry results showed that the surface cuboidal cells of PSH were strongly positive on membrane for E-cadherin, β-catenin and p120 ctn , with cytoplasmic positive expression of β-catenin. However,the polygonal cells were negative for E-cadherin, cytoplasmic positive for β-catenin and predominantly cytoplasmic positive and weakly membranous positive for p120ctn . In polygonal cells, all the three adhesion molecules showed heterogenicity staining. The cytoplasm and membrane of inner covering cells in the hemorrhagic pattern were positively stained for E-cadherin, β-catenin and p120ctn . The expression of the three adhesion molecules in hyperplastic type II alveolar cells of pulmonary inflammatory pseudotumor was similar to cuboidal cells of PSH. It is concluded that cuboidal cells of PSH may be the hyperplastic type II alveolar cells, whereas polygonal cells are true tumor cells lacking the E-cadherin/catenin complex which is expressed in well differentiated epithelial cells. The inner covering cells in the hemorrhagic pattern of PSH are perhaps epithelial cells as cuboidal cells rather than vascular endothelial cells.

Tumor cell formation by normal somatic cells fusing and cancer prevention prospects

Malignant transformation might be ascribed to one of the rather widespread processes taking place in the organism daily and being genetically inherited in a normal somatic cell. In order that normal cells are transformed into malignant ones, they should undergo the following two stages: (1) initiation - the precancerous cell (first-stage synkarion) formation, and (2) promotion - the true tumor cell (second-stage synkaryon) formation. For promoting the tumor process, i.e. transformation into a true tumor synkaryon, first - stage synkaryon (precancerous cell) should go through the next stage/stages of development at the molecular and subcellular level. In a tumor synkaryon, in particular in its plasma membrane, most probably the antigenic mosaics of normal precursor cells is inherited and, therefore, such cells "escape" the effect of the immune forces of the body. In other words, antigenic determinants existing on the surface of normal initial (parent) precursor cells condition inaction of the so-called immune surveillance. The cancer prevention concept implies synchronous conversion of a tumor cell into the stage of nonviable polykaryocytes. In addition, for preventing this fatal disease the inhibition of the process of approaching and further adhesion of normal somatic cells would suffice. The expected outcome would be the creation in the body of the conditions when somatic cells are incapable of close contacts with each other (not injurious for organism). This will completely exclude the fusion of cells and then nuclei and reduce thus to a minimum the possibility of cancer cell development.

Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma

We report the morphological characteristics of 30 cases of sclerosing hemangioma (SH) of the lung and explore the histological origin of the major cells in these tumors. In addition to routine light and electron microscopy, immunohistochemistry was performed by using 12 monoclonal primary and 5 polyclonal primary antibodies. These included surfactant protein B (SP-B), thyroid transcription factor-1 (TTF-1), mast cell trypsin, CD68, epithelial antigen markers (high molecular weight cytokeratin, low molecular weight cytokeratin [CK-L], epithelial membrane antigen [EMA], cancer embryonic antigen), mesothelial antigen, neuroendocrine markers (neuron-specific enolase [NSE], chromogranin A, synaptophysin, calcitonin, adrenocorticotropic hormone, human growth hormone [hHG]), vimentin, and CD34. Surface cuboidal cells have short microvilli and have lamellar bodies in their cytoplasm. They can sometimes merge into multinuclear giant cells. Immunohistochemical results showed that these cells are strongly positive for SP-B, TTF-1, CK-L, EMA, and cancer embryonic antigen, whereas polygonal cells, previously also described as round or pale cells, were strongly positive for vimentin and TTF-1, and positive or weakly positive for 2 to 3 kinds of neuroendocrine markers. Sparse neuroendocrine granules and abundant microfilaments were observed in their cytoplasm. Some cell clusters in the solid regions were positive for SP-B and EMA. Mast cells existed sparsely in almost every field. Both cuboidal and polygonal cells were negative to CD34 and mesothelial antigen staining. We conclude that cuboidal cells of SH originate from reactive proliferating type II pneumocytes, which can fuse into multinuclear giant cells. Polygonal cells, as true tumor cells, likely originate from multipotential primitive respiratory epithelium and possess the capability for multipotential differentiation. The antibodies of SP-B, TTF-1, vimentin, and CK-L are very helpful to diagnosis and differential diagnosis of SH.

Osteoclast-like Giant Cell Tumor of the Pancreas with Metastases to Gallbladder and Lymph Nodes. A Case Report

Osteoclast-like giant cell tumor of the pancreas (OGTP) is a rare neoplasm, of which the histogenesis is still controversial. Here we report a case of OGTP involving the head of the pancreas in a 71-year-old woman with metastases to the gallbladder and lymph nodes. The primary and metastatic tumors had identical histopathological, immunohistochemical, ultrastructural and molecular biological features. Microscopically, the tumors were characterized by atypical, often pleomorphic mononuclear cells associated with the proliferation of benign-appearing osteoclast-like giant cells (OGCs). Electron microscopic observation provided ultrastructural evidence of epithelial differentiation of the mononuclear cells, including microvilli and desmosomes, which was not obtained for OGCs. On immunohistochemical study, OGCs stained for CD68 (KP-1), LCA and HAM56, whereas mononuclear cells only reacted with PCNA. These findings clearly suggest that mononuclear cells are capable of differentiation and proliferation and may have been the only true tumor cells in this neoplasm, and that OGCs may have been a paraneoplastic product of this rare tumor. On examination of DNA from dewaxed sections of the tumor, we found no p53 mutation in the tumor tissue, but found two K-ras mutations in codon 12; this pattern of mutation commonly occurs in pancreatic carcinoma, indicating a somewhat genetic relationship of OGTP to pancreatic carcinoma. Although OGTP often has a favorable prognosis, the outcome in the present case was poor due to early tumor spread, with less than two years postoperative survival.

Medulloblastoma

Twenty medulloblastomas were studied comparatively through clinical and light and electron microscopic observations. Electron microscopy (EM) seemed important and, at times, essential in the structural characterization of these tumors. By light microscopic criteria alone, there were 12 “classical” undifferentiated cases, 7 cases showing astrocytic differentiation, and 1 “differentiated” but unclassifiable case. After reclassification by EM criteria, there were 7 undifferentiated cases, 10 cases with astrocytic differentiation, and 2 cases with neuronal differentiation; 1 case remained unclassified. The astrocytic tumor cells, showing formation of bundles of 8- to 10-nm glial fibrils, were considered true tumor cells and not “reactive” cells, whereas the neuronal tumors contained cells showing axonal processes, dense core granules, and synaptic-like structures. The authors conclude that medulloblastomas are neoplasms of a neuroepithelial embryonic origin and as such they may remain undifferentiated or may differentiate in either an astrocytic or a neuronal direction. The glial route is common, whereas the neuronal route is rare. Medulloblastomas should be subclassified by EM criteria into three groups: I, undifferentiated; II, astrocytic; and III, neuronal. This subclassification will be of value in predicting the potential for short vs. long term survival of the patient. Patients with astrocytic tumors (Subgroup II) had a longer survival than those with undifferentiated tumors (Subgroup I): 4.6 vs. 3.5 years. The undifferentiated tumors were located predominantly in the midline, whereas tumors showing astrocytic differentiation were located equally in the midline or in the cerebellar hemisphere. (Neurosurgery, 6: 398-411, 1980)