•Immune checkpoint inhibitors are the most promising treatment for advanced NSCLC.•Primary and acquired resistance to immune checkpoint inhibitors is common.•We examined biopsies taken from sites of ...acquired resistance or mixed responses.•Transformation from NSCLC to SCLC was identified as a mechanism of resistance.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death world-wide. Immune checkpoint inhibitors (ICI) have become the most promising type of treatment in oncology in general, and significantly so in NSCLC. Limited data is available about mechanisms of primary resistance. Data is lacking about mechanisms involved in acquired resistance or mixed responses in NSCLC. We aimed to identify mechanisms of resistance by studying biopsies taken from sites of secondary progression.
We identified all cases of NSCLC that have received ICI for advanced disease in our institute. Of these cases, those that have demonstrated acquired resistance or mixed responses, and have underwent a biopsy from a progressive lesion were analyzed. Selected specimens were subjected to next-generation sequencing (NGS; Oncomine™ Solid Tumour Fusion Transcript Kit).
Out of 664 lung cancer cases, 249 were NSCLC that have received ICI. Of these, eight cases matched our search criteria. Two of them demonstrated transformation to small cell lung cancer (SCLC; 2/8, 25%). NGS verified a common origin to a matched pre-treatment NSCLC specimen and an on-treatment progressive SCLC specimen. In two cases no tumor cells were found and in the remaining four the pathology was similar to the initial biopsy. In one of the cases of SCLC transformation platinum-etoposide chemotherapy was administered, with short-term benefit only and further disease progression.
Mechanisms of acquired resistance to ICI include SCLC transformation. Repeat biopsies of progressing lesions after initial response or in cases of mixed response can shed light on mechanisms of resistance.
One of the major hurdles for the advancement of cancer immunotherapy is lack of robust, accessible experimental models. We aimed to produce an ex-vivo organ culture (EVOC) model of immunotherapy for ...non-small cell lung cancer (NSCLC). Freshly resected early stage tumors were collected from the operating room, fragmented to clusters < 450 µm and cultured with fetal calf serum and human autologous serum. The resulting EVOC includes cancer epithelial cells within tumor tissue clusters and immune cells. Original tissue features are reflected in the EVOCs. The response to immune checkpoint inhibitors (ICI) was assessed by IFNγ gene induction. Interestingly, IFNγ EVOC induction was numerically higher when anti-CTLA4 was added to anti-PD-L1 treatment, supporting the notion that anti-CTLA4 impacts cancer partly through tumor-resident immune cells. In parallel, immunohistochemistry (IHC) for key immune-related proteins was performed on the formalin-fixed paraffin embedded (FFPE) corresponding tumors. EVOC IFNγ induction by ICI correlated with basal non-induced IFNγ, CD8, CD4 and FOXP3 mRNA levels within EVOCs and with tumor-FFPE-IHC for CD8 and granzyme B. A weaker correlation was seen with tumor-FFPE-IHC for CD3, CD4, CD68, FOXP3 and tumor-PD-L1. Tertiary lymphoid structure density was also correlated with the ICI response. Our study provides novel data about biomarkers that correlate with ICI-induced response of early stage NSCLC. Retention of the microenvironment and minimal addition of exogenous factors suggest this model to reliably represent the original tumor. The cluster-based EVOC model we describe can provide a valuable, yet simple and widely applicable tool for the study of immunotherapy in NSCLC.
Abstract In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic ...pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection.
BackgroundSoluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of ...their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases.MethodsCleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients’ effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay.ResultsMass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients’ HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8+ T cells.ConclusionsPleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.
Insulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine ...approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells.
IPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the "building blocks" of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture.
Co-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINβα, which play a key role in pancreatic differentiation.
Vasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.
High-grade ovarian cancer accounts for higher mortality rates because of ineffective biomarkers for early diagnosis. Deep proteome profiling of the microvesicles from a total of 187 liquid biopsies ...of Utero-tubal Lavage, combined with support vector machine algorithms, extracted a 9-protein classifier with high accuracy. The signature predicted all the early stage lesions, and outperformed the known markers CA125 and HE4 with 70‥ sensitivity and 76.2‥ specificity. Our study reveals UtL-microvesicle proteomics as the potential biomarker source for early diagnosis of HGOC.
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Highlights
•Microvesicle proteomics of 187 utero-tubal lavage samples for early diagnosis of HGOC.•Machine learning-based classification of a 9-protein signature with high predictive power.•Signature has 70‥ sensitivity and 76.2‥ specificity, predicting stage I lesions.
High-grade ovarian cancer (HGOC) is the leading cause of mortality from gynecological malignancies, because of diagnosis at a metastatic stage. Current screening options fail to improve mortality because of the absence of early-stage-specific biomarkers. We postulated that a liquid biopsy, such as utero-tubal lavage (UtL), may identify localized lesions better than systemic approaches of serum/plasma analysis. Further, while mutation-based assays are challenged by the rarity of tumor DNA within nonmutated DNA, analyzing the proteomic profile, is expected to enable earlier detection, as it reveals perturbations in both the tumor as well as in its microenvironment. To attain deep proteomic coverage and overcome the high dynamic range of this body fluid, we applied our method for microvesicle proteomics to the UtL samples. Liquid biopsies from HGOC patients (n = 49) and controls (n = 127) were divided into a discovery and validation sets. Data-dependent analysis of the samples on the Q-Exactive mass spectrometer provided depth of 8578 UtL proteins in total, and on average ∼3000 proteins per sample. We used support vector machine algorithms for sample classification, and crossed three feature-selection algorithms, to construct and validate a 9-protein classifier with 70% sensitivity and 76.2% specificity. The signature correctly identified all Stage I lesions. These results demonstrate the potential power of microvesicle-based proteomic biomarkers for early cancer diagnosis.
Tumor-host interactions play a major role in malignancies' initiation and progression. We have reported in the past that tumor cells attenuate genotoxic stress-induced p53 activation in neighboring ...stromal cells. Herein, we aim to further elucidate cancer cells' impact on signaling within lung cancer stroma. Primary cancer-associated fibroblasts were grown from resected human lung tumors. Lung cancer lines as well as fresh cultures of resected human lung cancers were used to produce conditioned medium (CM) or cocultured with stromal cells. Invasiveness of cancer cells was evaluated by transwell assays, and
tumor growth was tested in Athymic nude mice. We found CM of a large variety of cancer cell lines as well as
-cultured lung cancers to rapidly induce protein levels of stromal-MDM2. CM of nontransformed cells had no such effect. Mdm2 induction occurred through enhanced translation, was mTORC1-dependent, and correlated with activation of AKT and p70 S6 Kinase.
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knockdown in fibroblasts reduced the invasion of neighboring cancer cells, independently of stromal-p53.
overexpression in fibroblasts enhanced cancer cells' invasion and growth of inoculated tumors in mice. Our results indicate that stromal-MDM2 participates in a p53-independent cancer-host feedback mechanism. Soluble cancer-originated signals induce enhanced translation of stromal-MDM2 through AKT/mTORC1 signaling, which in turn enhances the neighboring cancer cells' invasion ability. The role of these tumor-host interactions needs to be further explored. IMPLICATIONS: We uncovered a novel tumor-stroma signaling loop, which is a potentially new therapeutic target in lung cancer and possibly in additional types of cancer.
AimsKlotho is a trans-membrane protein that serves as a tumour suppressor in a wide array of malignancies. Recent data suggest it as an epigenetically silenced tumour suppressor in cervical cancer. ...Yet, the expression pattern of klotho in cervical cancer has not been determined. We aimed to study the expression of klotho in squamous cell carcinomas (SQCC) and adenocarcinoma (ADC) of the cervix.MethodsKlotho expression was analysed by immunohistochemistry in 44 SQCC samples, 38 ADC samples and the adjacent normal tissue. For each sample, percentage of positive stained cells, staining intensity and a combined staining score were recorded. Staining was validated by measuring klotho mRNA levels, using quantitative RT-PCR, in 18 of the samples.ResultsKlotho expression was high in all endocervical and exocervical normal tissues adjacent to tumour. No expression of klotho was noted in 7 out of 38 (18.4%) ADC samples and in 2 out of 44 (4.5%) SQCC samples. Staining intensity, number of positively stained cells and combined intensity score were all lower in tumours compared with normal adjacent tissues in ADC and SQCC. Klotho mRNA levels highly correlated with immunohistochemical (IHC) staining (p=0.008).ConclusionsWe found reduced klotho expression in cervical carcinoma, especially in ADC, compared with normal adjacent tissue. Our results support the role of klotho as a potential tumour suppressor in cervical cancer. Further studies are required in order to establish the therapeutic role of klotho in cervical carcinoma and identify patients who may benefit from it.
Delayed diagnosis of ovarian cancer, as well as high recurrence rates and lack of personalized therapy options, are among the causes for poor survival figures. Much effort is made towards developing ...new therapeutic possibilities, however predictive biomarkers are still unavailable. CCNE1 amplification, occurring in ∼20% of the high grade serous ovarian tumors, was previously proposed as a marker for platinum resistance and poor prognosis as well as for CDK2 inhibition. The current study aimed to examine the role of CCNE1 positive-immunostain as a predictor of first-line taxane-platinum chemoresistance. We evaluated matched pre- vs. post-neoadjuvant chemotherapy tumor samples and correlated the degree of pathological response to treatment with CCNE1 expression levels. Our results indicate that CCNE1 immunohistochemistry does not predict taxane-platinum chemoresistance in ovarian cancer patients. Further research is required in order to enable personalized adjuvant treatment, in cases where poor pathological response is achieved after the neoadjuvant phase.
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