To assess the predictive value of multiparametric MRI for treatment response evaluation of induction chemo-immunotherapy in locally advanced head and neck squamous cell carcinoma.
Twenty-two patients ...with locally advanced, histologically confirmed head and neck squamous cell carcinoma who were enrolled in the prospective multicenter phase II CheckRad-CD8 trial were included in the current analysis. In this unplanned secondary single-center analysis, all patients who received contrast-enhanced MRI at baseline and in week 4 after single-cycle induction therapy with cisplatin/docetaxel combined with the immune checkpoint inhibitors tremelimumab and durvalumab were included. In week 4, endoscopy with representative re-biopsy was performed to assess tumor response. All lesions were segmented in the baseline and restaging multiparametric MRI, including the primary tumor and lymph node metastases. The volume of interest of the respective lesions was volumetrically measured, and time-resolved mean intensities of the golden-angle radial sparse parallel-volume-interpolated gradient-echo perfusion (GRASP-VIBE) sequence were extracted. Additional quantitative parameters including the T1 ratio, short-TI inversion recovery ratio, apparent diffusion coefficient, and dynamic contrast-enhanced (DCE) values were measured. A model based on parallel random forests incorporating the MRI parameters from the baseline MRI was used to predict tumor response to therapy. Receiver operating characteristic (ROC) curves were used to evaluate the prognostic performance.
Fifteen patients (68.2%) showed pathologic complete response in the re-biopsy, while seven patients had a residual tumor (31.8%). In all patients, the MRI-based primary tumor volume was significantly lower after treatment. The baseline DCE parameters of time to peak and wash-out were significantly different between the pathologic complete response group and the residual tumor group (p < 0.05). The developed model, based on parallel random forests and DCE parameters, was able to predict therapy response with a sensitivity of 78.7% (95% CI 71.24-84.93) and a specificity of 78.6% (95% CI 67.13-87.48). The model had an area under the ROC curve of 0.866 (95% CI 0.819-0.914).
DCE parameters indicated treatment response at follow-up, and a random forest machine learning algorithm based on DCE parameters was able to predict treatment response to induction chemo-immunotherapy.
Anilin actin binding protein (ANLN) and transducing-like enhancer protein 2 (TLE2) are associated with cancer patient survival and progression. The impact of their gene expression on progression-free ...survival (PFS) of patients with muscle invasive bladder cancer (MIBC) treated with radical cystectomy (RC) and subtype association has not yet been investigated. qRT-PCR was used to measure the transcript levels of
and
in the Mannheim cohort, and validated in silico by The Cancer Genome Atlas (TCGA) cohort. Uni- and multivariate Cox regression analyses identified predictors for disease-specific survival (DSS) and overall survival (OS). In the Mannheim cohort, tumors with high
expression were associated with lower OS and DSS, while high
expression was associated with a favorable OS. The TCGA cohort confirmed that high
and low
expression was associated with shorter OS and disease-free survival (DFS). In both cohorts, multivariate analyses showed
and
expression as independent outcome predictors. Furthermore,
was more highly expressed in cell lines and patients with the basal subtype, while
expression was higher in cell lines and patients with the luminal subtype.
and
are promising biomarkers for individualized bladder cancer therapy including cancer subclassification and informed MIBC prognosis.
Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder ...cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.
The treatment of oral cancer remains challenging due to its infiltrative nature and a high tendency for tumour relapse leading to an overall poor prognosis. In the case of early recurrence, the ...patient's prognosis deteriorates dramatically, with survival rate dropping to below 30%. Minimal improvements in survival trends in recurrent and advanced stage tumours have been reported in recent decades. Neoadjuvant immunotherapy may represent a new therapeutic approach changing the standard of care in advanced oral cancer therapy.
We describe the case of a woman in her late 30's who presented in mid-2019 with oral squamous cell carcinoma (OSCC) localized to the floor of the mouth. After initial R0 resection, selective neck dissection, and adjuvant brachytherapy, an early recurrence of OSCC located between the hyoid bone and the mandible was diagnosed at the end of 2019. An off-label treatment regimen was performed with neoadjuvant use of Pembrolizumab 19 days prior to salvage surgery. Radiological and histological assessment of T-cell and programmed cell death protein 1 ligand 1 (PD-L1) expression was performed before and after checkpoint inhibitor application. Neoadjuvant immunotherapy resulted in increased T-cell infiltration and PD-L1 expression, as well as a significant tumour necrosis rate. One cycle of Pembrolizumab led to significant regressive tumour changes with increases in immune infiltration, sclerosis, and necrosis of 75% of the tumour mass with only 25% vital tumour cells remaining. By June 2020, the patient remained without recurrence.
The case presented outlines the potential effects of neoadjuvant immunotherapy in recurrent or advanced OSCC prior to definitive surgical tumour treatment. The benefit of additional adjuvant treatment after histologic response will be discussed. The case is also analysed considering ongoing clinical trials of neoadjuvant immunotherapy for head and neck malignancies.
Growing evidence supports the pivotal role of long non-coding RNAs (lncRNAs) in the regulation of cancer development and progression. Their expression patterns and biological function in muscle ...invasive bladder cancer (MIBC) remain elusive.
Transcript levels of lncRNA miR-31 host gene (MIR31HG) and its splice variants were measured in our MIBC cohort (n = 102) by qRT-PCR, and validated in silico by the TCGA cohort (n = 370). Kaplan-Meier and multiple Cox regression analysis were conducted to evaluate the survival significance of MIR31HG and its splice variants. Functional experiments were performed to examine the proliferation and migration abilities of MIR31HG and its splice variants by knockdown approaches.
In this study, a decreased expression of MIR31HG was found in bladder cancer cells and tissues, except in the basal subtype. Survival analysis showed that high expression of MIR31HG was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with MIBC of basal subtype. Two splice variants of MIR31HG lacking exon 1 (MIR31HGΔE1) and exon 3 (MIR31HGΔE3) were identified to have specific expression patterns in different molecular subtypes of our MIBC cohort. MIR31HGΔE3 was highly expressed in basal subtype tumors. A high expression of MIR31HGΔE1 and MIR31HGΔE3 was associated with worse OS and DFS in our cohort. In vitro experiments revealed that knockdown of MIR31HG inhibits cell proliferation, colony formation, and migration in bladder cancer. Cell proliferation and migration assays after knockdown of splice variants of MIR31HG showed corresponding roles for the full-length transcript.
Our study demonstrates that MIR31HG and its splice variants could serve as biomarkers for the classification and prognosis prediction of patients with MIBC.
Since 2016, large nested urothelial carcinoma (LNUC) has been included within the WHO classification of urothelial tumors. Limited reports with mainly small case series have confirmed the malignant ...behavior of LNUC despite its bland morphological appearance. We evaluated, for the first time, markers for new immunooncological or targeted therapies including
mutational status and PD-L1 status, the frequency of
-promoter mutations and the molecular subtype in a cohort of 25 LNUC using SNaPshot analysis and immunohistochemistry. Of the 25 cases, 17 were pure LNUC, with 13 showing an additional exophytic papillary/papillary-like component. Seven mixed LNUCs presented areas of classical nested variant urothelial carcinoma (NVUC) and one showed a component of conventional urothelial carcinoma. Of the 17 evaluable pure LNUCs, 16 were
-mutated with identical mutations in their concomitant papillary/papillary-like components. An
mutation was found in 1/7 evaluable mixed LNUCs combined with NVUC.
-promoter mutations were detected in 86.7% pure and 83.3% mixed tumors. Immunohistochemistry revealed a luminal phenotype; PD-L1 was negative in the majority of tumor cells and tumor-associated immune cells. Pure LNUC is a prime example of a luminal,
-mutated, mostly PD-L1-negative tumor. In contrast,
mutations seem to be rare in mixed LNUC, which may indicate a different pathway of tumor development.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, ...both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed.
In this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy.
We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects.
Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer.
Few-shot learning addresses the problem of classification when little data or few labels are available. This is especially relevant in histopathology, where labeling must be carried out by highly ...trained medical experts. Prototypical Networks promise transferability to new domains by using a pre-trained encoder and classifying by way of a prototypical representation of each class learned with few samples. We examine the applicability of this approach by attempting domain transfer from colon tissue (for training the encoder) to urothelial tissue. Furthermore, we address the problems arising from representing a class via a small amount of representatives (prototypes) by testing two different prototype calculation strategies. We compare the original “Prototype per Class” (PPC) approach to our “Prototype per Annotation” (PPA) method, which calculates one prototype for each example annotation made by the pathologist. We test the domain transfer capability of our approach on a dataset of 55 whole slide images (WSIs) containing six subtypes of urothelial carcinoma in two granularities: “Superclasses”, which combines the tumorous subtypes into a single “tumor” class on top of a aggregated “healthy” and additional “necrosis” class, and “subtypes”, which considers all eleven classes separately. We evaluate the classic PPC approach as well as our PPA approach on this data set. Our results show that the adaptation of the Prototypical Network from colon tissue to urothelial tissue was successful, yielding an F1 score of 0.91 for the “superclasses”. Furthermore, the PPA approach performs very comparably to the PPC strategy. This makes it a viable alternative that places more value on the intent of the pathologist during annotation.
The involvement of immune cell infiltration and immune regulation in the progression of oral squamous cell carcinoma (OSCC) is shown. Anti-PD-1 therapy is approved for the treatment of advanced OSCC ...cases, but not all patients respond to immune checkpoint inhibitors. Hence, further targets for therapeutic approaches are needed. The number of identified cellular receptors with immune checkpoint function is constantly increasing. This study aimed to perform a comparative analysis of a large number of immune checkpoints in OSCC in order to identify possible targets for therapeutic application.
A NanoString mRNA analysis was performed to assess the expression levels of 21 immune regulatory checkpoint molecules in OSCC tissue (
= 98) and healthy oral mucosa (NOM;
= 41). The expression rates were compared between the two groups, and their association with prognostic parameters was determined. Additionally, relevant correlations between the expression levels of different checkpoints were examined.
In OSCC tissue, significantly increased expression of CD115, CD163, CD68, CD86, CD96, GITRL, CD28 and PD-L1 was detected. Additionally, a marginally significant increase in CD8 expression was observed. BTLA and PD-1 levels were substantially increased, but the differential expression was not statistically significant. The expression of CD137L was significantly downregulated in OSCC compared to NOM. Correlations between immune checkpoint expression levels were demonstrated, and some occurred specifically in OSCC tissue.
The upregulation of inhibitory receptors and ligands and the downregulation of activators could contribute to reduced effector T-cell function and could induce local immunosuppression in OSCC. Increased expression of activating actors of the immune system could be explained by the increased infiltration of myeloid cells and T-cells in OSCC tissue. The analysis contributes to the understanding of immune escape in OSCC and reveals potential targets for oral cancer immunotherapy.
This pilot study aimed to assess the feasibility of intraoperative assessment of safe margins with Confocal Laser Endomicroscopy (CLE) during planned partial or total laryngectomy.
Eight patients ...with confirmed larynx squamous cell carcinoma (SCC) and planned partial or total laryngectomy were included in this study in March 2020. Two head and neck surgeons and one pathologist were asked to classify carcinoma or healthy epithelium in a sample of 94 representative sequences (5.640 images), blinded to the histological results (H&E staining).
Healthy mucosa areas showed epithelium with cells of uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture. CLE optical biopsy of SCC demonstrated a disorganized arrangement of variable cellular morphology. We calculated an accuracy, sensitivity, specificity, PPV, and NPV of 80.1%, 72.3%, 87.9%, 85.7%, and 76.1%, respectively. A distinct transition between healthy appearing tissue and suspicious lesions could also be detected.
CLE can be easily integrated into the intraoperative setting, generate real-time, in-vivo microscopic images of the larynx for evaluation and demarcation of cancer. If validated in further studies, CLE could eventually contribute to a less radical approach by enabling a more precise evaluation of the cancer margin.
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