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The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.
In gastric cancer (GC), there are four molecular subclasses that indicate whether patients respond to chemotherapy or immunotherapy, according to the TCGA. In clinical practice, however, not every patient undergoes molecular testing. Many laboratories have used well-implemented in situ techniques (IHC and EBER-ISH) to determine the subclasses in their cohorts. Although multiple stains are used, we show that a staining approach is unable to correctly discriminate all subclasses. As an alternative, we trained an ensemble convolutional neuronal network using bagging that can predict the molecular subclass directly from hematoxylin–eosin histology. We also identified patients with predicted intra-tumoral heterogeneity or with features from multiple subclasses, which challenges the postulated TCGA-based decision tree for GC subtyping. In the future, deep learning may enable targeted testing for molecular subtypes and targeted therapy for a broader group of GC patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Background: Routine human papillomavirus (HPV) testing is performed in cervival cancer and is required for classification of some head and neck cancers. In penile cancer a statement on HPV association of the carcinoma is required. In most cases p16 immunohistochemistry as a surrogate marker is applied in this setting. Since differing clinical outcomes for HPV positive and HPV negative tumors are described we await HPV testing to be requested more frequently by clinicians, also in the context of HPV vaccination, where other HPV subtypes are expected to emerge.
Method: Therefore, a cohort of archived, formalin-fixed paraffin embedded (FFPE) penile neoplasias was stained for p16 and thereafter tested for HPV infection status via PCR based methods. Additionally to Sanger sequencing, we chose LCD-Array technique (HPV 3.5 LCD-Array Kit, Chipron; LCD-Array) for the detection of HPV in our probes expecting a less time consuming and sensitive HPV test for our probes.
Results: We found that LCD-Array is a sensitive and feasible method for HPV testing in routine diagnostics applicable to FFPE material in our cohort. Our cohort of penile carcinomas and carcinomas in situ was associated with HPV infection in 61% of cases. We detected no significant association between HPV infection status and histomorphological tumor characteristics as well as overall survival.
Conclusions: We showed usability of molecular HPV testing on a cohort of archived penile carcinomas. To the best of our knowledge, this is the first study investigating LCD-Array technique on a cohort of penile neoplasias.
Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell–derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1− B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.
Penile squamous cell carcinomas are rare tumor entities throughout Europe. Early lymphonodal spread urges for aggressive therapeutic approaches in advanced tumor stages. Therefore, understanding tumor biology and its microenvironment and correlation with known survival data is of substantial interest in order to establish treatment strategies adapted to the individual patient. Fifty-five therapy naïve squamous cell carcinomas, age range between 41 and 85 years with known clinicopathological data, were investigated with the use of tissue microarrays (TMA) regarding the tumor-associated immune cell infiltrate density (ICID). Slides were stained with antibodies against CD3, CD8 and CD20. An image analysis software was applied for evaluation. Data were correlated with clinicopathological characteristics and overall survival. There was a significant increase of ICID in squamous cell carcinomas of the penis in relation to tumor adjacent physiological tissue. Higher CD3-positive ICID was significantly associated with lower tumor stage in our cohort. The ICID was not associated with overall survival. Our data sharpens the view on tumor-associated immune cell infiltrate in penile squamous cell carcinomas with an unbiased digital and automated cell count. Further investigations on the immune cell infiltrate and its prognostic and possible therapeutic impact are needed.
Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. The nodular sclerosing subtype of cHL (NS cHL) is characterised by a proliferation of fibroblasts in the tumour microenvironment, leading to fibrotic bands surrounding the lymphoma infiltrate. Several studies have described a crosstalk between the tumour cells of cHL, the Hodgkin- and Reed-Sternberg (HRS) cells, and cancerassociated fibroblasts (CAF). However, to date a deep molecular understanding of these fibroblasts is lacking. Aim of the present study therefore was a comprehensive
characterisation of these fibroblasts. Moreover, only a few studies describe the interplay of HRS cells and CAF. The paracrine communication and direct interaction of these two
cellular fractions have been investigated within this study. Finally, the influence of a few HRS cells within a lymph node orchestrate the mere alteration of its architecture and
morphology. Gene expression and methylation profiles of fibroblasts isolated from primary lymph node suspensions revealed persistent differences between fibroblasts obtained from NS cHL and lymphadenitis. NS cHL derived fibroblasts exhibit a myofibroblastic - inflammatory phenotype characterised by MYOCD, CNN1 and IL-6 expression. TIMP3, an inhibitor of matrix metalloproteinases, was strongly upregulated in NS cHL fibroblasts, likely contributing to the accumulation of collagen in sclerotic bands of NS cHL. Treatment by luteolin could reverse this fibroblast phenotype and decrease TIMP3 secretion. NS cHL fibroblasts showed enhanced proliferation when they were exposed to soluble factors released from HRS cells. For HRS cells, soluble
factors from fibroblasts were not sufficient to protect them from Brentuximab-Vedotin(BV) induced cell death. However, HRS cells adherent to fibroblasts were protected from BV-induced injury. The cHL specific interaction of both cell fractions reveals an initiation of inflammatory key regulators such as IL13 and IL4. Among important adhesion molecules known from literature the blocking of integrin beta 1 solely interrupted the adhesion of HRS cells to CAF. In summary, this study proves the stable reprograming of CAF phenotype and expression derived from NS cHL. It presents a suitable in vitro model for studying the interaction of HRS cells and CAF by paracrine factors and adherence. Most importantly the observations confirm the importance of fibroblasts for HRS cells´ inflammatory niche and cell survival associated with TIMP3 which probably acts as a major factor to the typical accumulation of fibrosis observed in NS cHL.
Background: To study neoadjuvant chemoradiotherapy (nCRT) and potential predictive factors for response in locally advanced oral cavity cancer (LA-OCC).
Methods: The INVERT trial is an ongoing single-center, prospective phase 2, proof-of-principle trial. Operable patients with stage III-IVA squamous cell carcinomas of the oral cavity were eligible and received nCRT consisting of 60 Gy with concomitant cisplatin and 5-fluorouracil. Surgery was scheduled 6-8 weeks after completion of nCRT. Explorative, multiplex immunohistochemistry (IHC) was performed on pretreatment tumor specimen, and diffusion-weighted magnetic resonance imaging (DW-MRI) was conducted prior to, during nCRT (day 15), and before surgery to identify potential predictive biomarkers and imaging features. Primary endpoint was the pathological complete response (pCR) rate.
Results: Seventeen patients with stage IVA OCC were included in this interim analysis. All patients completed nCRT. One patient died from pneumonia 10 weeks after nCRT before surgery. Complete tumor resection (R0) was achieved in 16/17 patients, of whom 7 (41%, 95% CI: 18-67%) showed pCR. According to the Clavien-Dindo classification, grade 3a and 3b complications were found in 4 (25%) and 5 (31%) patients, respectively; grade 4-5 complications did not occur. Increased changes in the apparent diffusion coefficient signal intensities between MRI at day 15 of nCRT and before surgery were associated with better response (p=0.022). Higher abundances of programmed cell death protein 1 (PD1) positive cytotoxic T-cells (p=0.012), PD1+ macrophages (p=0.046), and cancer-associated fibroblasts (CAFs, p=0.036) were associated with incomplete response to nCRT.
Conclusion: nCRT for LA-OCC followed by radical surgery is feasible and shows high response rates. Larger patient cohorts from randomized trials are needed to further investigate nCRT and predictive biomarkers such as changes in DW-MRI signal intensities, tumor infiltrating immune cells, and CAFs.
Introduction and Objective: Identifying patients that benefit from cisplatin-based adjuvant chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC). The purpose of this study is to correlate “luminal” and “basal” type protein expression with histological subtypes, to investigate the prognostic impact on survival after adjuvant chemotherapy and to define molecular consensus subtypes of “double negative” patients (i.e., without expression of CK5/6 or GATA3).
Materials and Methods: We performed immunohistochemical (IHC) analysis of CK5/6 and GATA3 for surrogate molecular subtyping in 181 MIBC samples. The mRNA expression profiles for molecular consensus classification were determined in CK5/6 and GATA3 (double) negative cases using a transcriptome panel with 19.398 mRNA targets (HTG Molecular Diagnostics). Data of 110 patients undergoing radical cystectomy were available for survival analysis.
Results: The expression of CK5/6 correlated with squamous histological subtype (96%) and expression of GATA3 was associated with micropapillary histology (100%). In the multivariate Cox-regression model, patients receiving adjuvant chemotherapy had a significant survival benefit (hazard ratio [HR]: 0.19 95% confidence interval [CI]: 0.1–0.4, p < 0.001) and double-negative cases had decreased OS (HR: 4.07; 95% CI: 1.5–10.9, p = 0.005). Double negative cases were classified as NE-like (30%), stroma-rich (30%), and Ba/Sq (40%) consensus molecular subtypes and displaying different histological subtypes.