Expression In Malignant Lesions Research Articles

Heterogeneity of HLA-G Expression in Cancers: Facing the Challenges.

Phenotypic heterogeneity has been observed in most malignancies, which represents a considerable challenge for tumor therapy. In recent decades, the biological function and clinical significance of the human leukocyte antigen (HLA)-G have been intensively explored. It is now widely accepted that HLA-G is a critical marker of immunotolerance in cancer cell immune evasion and is strongly associated with disease progress and prognosis for cancer patients. Moreover, it has recently been emphasized that the signaling pathway linking HLA-G and immunoglobulin-like transcripts (ILTs) is considered an immune checkpoint. In addition, HLA-G itself can generate at least seven distinct isoforms, and intertumor and intratumor heterogeneity of HLA-G expression is common across different tumor types. Furthermore, HLA-G heterogeneity in cancers has been related to disease stage and outcomes, metastatic status and response to different therapies. This review focuses on the heterogeneity of HLA-G expression in malignant lesions, and clinical implications of this heterogeneity that might be relevant to personalized treatments are also discussed.

A comparative study of p53 expression in premalignant and malignant cervical lesions at a tertiary care institute of Rohilkhand region, India

Background: Cervical cancer ranks as the 2nd most common cancer among Indian women between 15 and 44 years of age. The secondary biomarker p53 has been studied at various centers in the world and has been found to be deregulated in cervical cancers. Objective: This study was undertaken to observe the expression and association of p53 and grades of p53 in premalignant and malignant cervical lesions and also with their histopathological subtypes. Materials and Methods: The present prospective study was conducted, from January 2016 to December 2016. Patients with premalignant and malignant cervical lesions of epithelial origin were included, while those with other pathological entities were excluded. A total of 53 patients were finally enroled for this study. Cases with p53 expression in more than 5% cells were considered positive for p53 overexpression. Results: The mean of p53 expression was higher in malignant lesions (13.29 ± 16.00) when compared to premalignant lesions (1.12 ± 2.23) and the result was statistically significant (P = 0.019). No statistically significant association (P = 0.875) was found between types of premalignant lesions and p53 overexpression as well as types of malignant lesions and p53 expression. Conclusion: In the present study, much higher percentage of p53 overexpression is observed in malignant than premalignant lesions, the difference being statistically significant (P = 0.009). Mean p53 expression in the malignant lesions is also higher than premalignant lesions. p53 expression is therefore a suggested marker for differentiating malignant from premalignant lesions.

Abstract 5656: Quantitative measurement of PDL1 expression across tumor types using laser capture microdissection and reverse phase protein microarray

Abstract Background: The efficacy of immunotherapy, including therapeutic strategies capable of modulating innate/adaptive immune resistance, varies greatly across tumor types. As the number of available immunotherapies accelerates, the study of predictive markers by IHC (e.g. PDL1 expression) is under intense investigation. However, staining protocol inconsistency, variation across scoring systems, and subjective interpretation of the immunostaining have produced conflicting results thus far. This work explored the role of Laser Capture Microdissection (LCM) coupled with Reverse Phase Protein Microarray (RPPA) as an alternative high throughput, quantitative, operator independent platform for measuring PDL1 expression across tumor types. Material and Methods: Pure epithelial cells were isolated via LCM from 178 samples including: 72 ovarian cancers (OC), 57 lung adenocarcinomas (LC), 30 metastatic breast cancers (MBC), and 19 pancreatic cancers (PC). PDL1 expression was measured on a continuous scale using quantitative RPPA based analysis. Each tumor type was processed and arrayed independently. Experimental samples and reference standards used for inter-assay normalization were printed in triplicates. Results: PDL1 expression varied greatly across tumor types. LC were characterized by the greatest intra-tumor fold dynamic range (> 35-fold), followed by OC (< 13-fold), MBC (< 4-fold), and PC (< 2-fold). PDL1 expression of 46/57 (80.7%) LC, 17/30 (56.7%) MBC, 6/19 (31.5%) PC, and 20/72 (27.8%) OC was greater than the population median of all tumors combined. Within the LC samples with PDL1 expression equal to the top quartile of the population, 10 (71.4%) were KRAS mutant lesions and 4 (28.6%) were WT tumors. Finally, amongst LC and PC harboring a KRAS mutation, PC showed an overall lower expression of PDL1 with only 2/19 (10.5%) cases been above the population median and none within the top quartile of the population. Conclusions: The LCM-RPPA workflow has the unique ability to capture immune checkpoint expression on a continuous quantitative scale as well as capture its broad dynamic range. Because RPPA is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate more accurate cut-point of therapeutic response prediction. Overall the dynamic range of PDL1 was broader in LC compared to other solid tumors, and LC had a much higher proportion of patients with tumors expressing high levels of PDL1. These quantitative differences may explain therapeutic efficacy of PDL1 inhibition across tumor types. Such speculative hypothesis should be further validated in prospective clinical trials. Finally, these preliminary data suggest that organ specific microenvironments more than specific driving mutations (e.g. KRAS) may strongly influence PDL1 expression in malignant lesions. Citation Format: Elisa Baldelli, Valerie Calvert, K. Alex Hodge, Maria Isabella Sereni, Guido Gambara, Eric B. Haura, Lucio Crino', Bryant Dunetz, Sergio Pecorelli, David J. Perry, Stephen P. Anthony, Nicholas Robert, Donald W. Northfelt, Mohammad Jahanzeb, Emanuel F. Petricoin, Mariaelena Pierobon. Quantitative measurement of PDL1 expression across tumor types using laser capture microdissection and reverse phase protein microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5656. doi:10.1158/1538-7445.AM2017-5656

Immunohistochemical analysis of tumor-associated stroma in oral squamous cell carcinoma with and without preexisting oral submucous fibrosis

Context: It has been proposed that oral squamous cell carcinoma (OSCC) arising in preexisting oral submucous fibrosis (OSMF) constitutes a clinicopathologically distinct disease than the OSCC arising without OSMF. Expression of myofibroblasts is an early event in tumorogenesis, and its expression in malignant lesions has been suggested to represent an important participant in invasion. Aim: To evaluate the expression of myofibroblasts in OSCC arising in OSMF and OSCC without OSMF. Settings and Design: Thirty each histopathologically proven cases of OSMF, OSCC with OSMF, and OSCC without OSMF were included in the study. The tissue sections were studied for histopathological evaluation and alpha-smooth muscle actin (α-SMA) expression. Statistical Analysis Used: Descriptive and inferential statistics using Kruskal–Wallis test, Mann–Whitney test, and Chi-square test. Results: The expression of α-SMA was found to be high in OSCC with OSMF and OSCC without OSMF group as compared to OSMF group. However, the difference between α-SMA expression in OSCC with OSMF and OSCC without OSMF group was found statistically nonsignificant. Comparison of α-SMA score with histopathological grading and tumor, node, metastasis staging in OSCC with OSMF and OSCC without OSMF groups showed statistically nonsignificant difference, whereas comparison of α-SMA score with lymph node metastasis was statistically significant. Conclusion: The altered extracellular matrix in OSCC with OSMF could be responsible for modified epithelial–mesenchymal transition, which is further proven by reduced intensity and distinct pattern of α-SMA staining in OSCC with OSMF.

Expression of FoxP3 Protein Plays a Key Role in Thyroid Tumors in Children

The expression of FoxP3 in tumor cells might play an important role in cancer progression. We evaluated the immunoexpression of FoxP3 in thyroid tumors in children. Studies revealed high nuclear FoxP3 expression in follicular adenoma, papillary carcinoma, follicular carcinoma and low in goiter. Malignant tumors and adenomas, revealed a statistically significant higher expression of FoxP3 compared with the thyroid goiter. High FoxP3 expression in malignant lesions compared with low expression in goiter, may be indirect evidence of its role in carcinogenesis. Revealed high expression of FoxP3 in benign tumor, may suggest a strong activation of oncogenic processes in this lesion.

HLA antigen and NK cell activating ligand expression in malignant cells: a story of loss or acquisition

Malignant transformation of cells is often associated with changes in classical and non-classical HLA class I antigen, HLA class II antigen as well as NK cell activating ligand (NKCAL) expression. These changes are believed to play a role in the clinical course of the disease since these molecules are critical to the interactions between tumor cells and components of both innate and adaptive immune system. For some time, it has been assumed that alterations in the expression profile of HLA antigens and NKCAL on malignant cells represented loss of classical HLA class I antigen and induction of HLA class II antigen, non-classical HLA class I antigen and/or NKCAL expression. In contrast to these assumptions, experimental evidence suggests that in some cases dysplastic and malignant cells can acquire classical HLA class I antigen expression and/or lose the ability to express HLA class II antigens. In light of the latter findings as well as of the revival of the cancer immune surveillance theory, a reevaluation of the interpretation of changes in HLA antigen and NKCAL expression in malignant lesions is warranted. In this article, we first briefly describe the conventional types of changes in HLA antigen and NKCAL expression that have been identified in malignant cells to date. Second, we discuss the evidence indicating that, in at least some cell types, classical HLA class I antigen expression can be acquired and/or the ability to express HLA class II antigens is lost. Third, we review the available evidence for the role of immune selective pressure in the generation of malignant lesions with changes in HLA antigen expression. This information contributes to our understanding of the role of the immune system in the control of tumor development and to the optimization of the design of immunotherapeutic strategies for the treatment of cancer.

Proto‐oncogene, Pim‐3 with serine/threonine kinase activity, is aberrantly expressed in human colon cancer cells and can prevent Bad‐mediated apoptosis

We previously observed that Pim-3 with serine/threonine kinase activity, was aberrantly expressed in malignant lesions of endoderm-derived organs, liver and pancreas. Because Pim-3 protein was not detected in normal colon mucosal tissues, we evaluated Pim-3 expression in malignant lesions of human colon, another endoderm-derived organ. Pim-3 was detected immunohistochemically in well-differentiated (43/68 cases) and moderately differentiated (23/41 cases) but not poorly differentiated colon adenocarcinomas (0/5 cases). Moreover, Pim-3 proteins were detected in adenoma (35/40 cases) and normal mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim-3 was constitutively expressed in SW480 cells and the transfection with Pim-3 short hairpin RNA promoted apoptosis. In the same cell line, a pro-apoptotic molecule, Bad, was phosphorylated at Ser(112) and Ser(136) sites of phosphorylation that are representative of its inactive form. Ser(112) but not Ser(136) phosphorylation in this cell line was abrogated by Pim-3 knockdown. Furthermore, in human colon cancer tissues, Pim-3 co-localized with Bad in all cases (9/9) and with phospho-Ser(112)Bad in most cases (6/9). These observations suggest that Pim-3 can inactivate Bad by phosphorylating its Ser(112) in human colon cancer cells and thus may prevent apoptosis and promote progression of human colon cancer.

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.

A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections

Abnormalities in HLA class I antigen expression are frequently found in malignant tumors. Their potential role in the clinical course of the disease and in the outcome of T cell-based immunotherapy has stimulated interest in the characterization of the molecular mechanisms underlying HLA class I antigen abnormalities in malignant cells. Multiple mechanisms have been identified. Among them are abnormalities in antigen processing machinery (APM) component expression. In spite of this information, APM component expression in malignant lesions has been investigated only to a limited extent because of the lack of availability, for most APM components, of monoclonal antibodies (mAb) which stain formalin-fixed, paraffin-embedded tissues. The latter are the substrate of choice in immunohistochemical (IHC) reactions. To overcome this limitation, we have developed a simple and reproducible method to generate APM component-specific mAb which stain formalin-fixed, paraffin-embedded tissue sections. This method involves five steps: (i) immunogenic amino acid sequences, which display low homology with their mouse counterparts when possible, are identified in APM components and utilized to synthesize peptides; (ii) BALB/c mice are immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides and with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant APM component proteins; (iii) immunized mice, which develop high titer APM component-specific antibodies, are utilized to generate hybridomas which are screened for APM component-specific antibody production by Western blotting assays, with lymphoid cell lysates; (iv) identified APM component-specific mAb are characterized in their specificity and in their reactivity with permeabilized cells in ELISA and/or flow cytometry; and (v) mAb, with the appropriate reactivity pattern, are tested in IHC reactions with formalin-fixed, paraffin-embedded tissue sections. The use of the methodology we have developed resulted in the generation of a panel of APM component-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections in IHC reactions. These reagents will facilitate the analysis of APM component expression in tissues under physiological and pathological conditions. In addition, the methodology we have described is likely to be applicable to other antigenic systems to develop mAb capable of detecting protein components of interest in formalin-fixed, paraffin-embedded tissue sections.

HLA class I defects in malignant lesions: what have we learned?

Depending on the tumor types, HLA class I antigen downregulation or loss has been found in 16% to 50% of malignant lesions in many malignancies with a clinical association with histopathological markers of poor prognosis of the disease and with reduced free interval and survival. These findings may reflect the escape of tumor cells with HLA class I abnormalities from recognition and destruction by HLA class I-restricted, tumor-associated antigen-specific cytotoxic T lymphocytes. This possibility has stimulated investigations on the mechanisms underlying HLA class I antigen abnormalities in malignant cells. Distinct molecular defects underlying an abnormal HLA class I phenotype have been identified and characterized. These defects range from structural alterations of the genes which encode HLA class I antigen subunits to deregulation of antigen processing machinery components responsible for a functional HLA class I antigen expression. These findings, in conjunction with those of clinical recurrence of lesions with HLA class I antigen loss following T cell-based immunotherapy in patients, suggests that immunoselection may play a role in the generation of malignant lesions with HLA class I antigen abnormalities. This possibility has stressed the need to effectively monitor functional HLA class I antigen expression in malignant lesions in the application of T cell-based immunotherapy as well as to develop strategies to circumvent the negative impact of immunoselection.

Identification and location of human papillomavirus type 16 antisense early promoter and characterisation of antisense RNA.

Antisense RNA sequences of various regions of human papillomavirus type 16 (HPV 16) were previously found in a number of cervical lesions, but the viral or cellular promoter has not been identified. HPV 16 E7 oncogene antisense transcripts expressed from an antisense promoter in viral DNA were found in the present study by RNase protection assays for total and cytoplasmic RNA. The antisense promoter for these transcripts was located within HPV 16 nt 4030-4230 by deletion analyses. The results also suggested that most of the antisense RNA was relatively short. The antisense promoter of HPV 16 was functional for expression of antisense RNA of a heterologous gene. Antisense-sense double-stranded E7 RNA was detected, and the sense RNA of this duplex was apparently inefficient for splicing or cleavage/poly(A) addition. These results show that HPV 16 can produce early region antisense RNA, which is from a promoter within a defined region of the viral genome. The possible importance of these transcripts for the regulation of episomal HPV 16 gene expression in infected and premalignant lesions and the possible importance of their deregulation for expression in malignant lesions are discussed.