Immunohistochemical analysis of tumor budding in stage II colon cancer: exploring zero budding as a prognostic marker.

Tumor budding, a biomarker traditionally evaluated using hematoxylin and eosin (H&E) staining, has gained recognition as a prognostic biomarker for stage II colon cancer. Nevertheless, while H&E staining offers valuable insights, its limitations prompt the utilization of pan-cytokeratin immunohistochemistry (IHC). Consequently, this study seeks to evaluate the prognostic significance of tumor budding using IHC in a contemporary cohort of stage II colon cancer patients, aiming to deepen our understanding of this critical facet in cancer prognosis. We conducted a retrospective, population-based cohort study including 493 patients with stage II colon cancer and evaluated tumor budding using IHC, following the H&E-based guidelines proposed by the International Tumor Budding Consensus Conference Group. Correlation between H&E-based and IHC-based tumor budding was assessed using a four-tiered scoring system that included a zero budding (Bd0) category. Survival analyses explored the prognostic significance of tumor budding assessed by IHC and H&E. As expected, IHC-based tumor budding evaluation yielded significantly higher bud counts compared to H&E (p < 0.01). Interestingly, 21 patients were identified with no tumor budding using IHC. This was associated with significantly improved recurrence-free survival (HR = 5.19, p = 0.02) and overall survival (HR = 4.47, p = 0.04) in a multivariate analysis when compared to tumors with budding. The Bd0 category demonstrated a 100% predictive value for the absence of recurrence. In conclusion, IHC-based tumor budding evaluation in stage II colon cancer provides additional prognostic information. The absence of tumor budding is associated with a favorable prognosis and may serve as a potential marker for identifying patients with no risk of recurrence.

The Transcriptional Landscape of Coding and Noncoding RNAs in Recurrent and Nonrecurrent Colon Cancer.

A number of patients with colon cancer with local or local advanced disease suffer from recurrence and there is an urgent need for better prognostic biomarkers in this setting. Here, the transcriptomic landscape of mRNAs, long noncoding RNAs, snRNAs, small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs, pseudogenes, and circular RNAs, as well as RNAs denoted as miscellaneous RNAs, was profiled by total RNA sequencing. In addition to well-known coding and noncoding RNAs, differential expression analysis also uncovered transcripts that have not been implicated previously in colon cancer, such as RNA5SP149, RNU4-2, and SNORD3A. Moreover, there was a profound global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in more advanced tumors. A global down-regulation of circular RNAs in tumors relative to normal tissues was observed, although only a few were expressed differentially between tumor stages. Many previously undescribed transcripts, including RNU6-620P, RNU2-20P, VTRNA1-3, and RNA5SP60, indicated strong prognostic biomarker potential in receiver operating characteristics analyses. In summary, this study unveiled numerous differentially expressed RNAs across various classes between recurrent and nonrecurrent colon cancer. Notably, there was a significant global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in advanced tumors. Many of these newly discovered candidates demonstrate a strong prognostic potential for stage II colon cancer.

The prognostic value of tumor budding in a thoroughly characterized stage II colon cancer population in the context of a national screening program.

Tumor budding as a prognostic marker in colorectal cancer has not previously been investigated in a cohort of screened stage II colon cancer patients. We assessed the prognostic significance of tumor budding in a thoroughly characterized stage II colon cancer population comprising surgically resected patients in the Region of Southern Denmark from 2014 to 2016. Tumors were re-staged according to the 8th edition of UICC TNM Classification, undergoing detailed histopathological evaluation and tumor budding assessment following guidelines from the International Tumor Budding Consensus Conference. Prognostic evaluation utilized Kaplan-Meier curves, log-rank tests, and Cox proportional hazard models for time to recurrence (TTR), recurrence-free survival (RFS), and overall survival (OS). Out of 497 patients, 20% were diagnosed through the national colorectal cancer screening program. High-grade tumor budding (Bd3) was found in 19% of tumors and was associated with glandular subtype, perineural invasion, mismatch repair proficient tumors, and tumor recurrence (p < 0.001, p < 0.001, p = 0.045, and p = 0.007 respectively). In multivariable Cox regression, high-grade budding was a significant prognostic factor for TTR compared to low-grade (Bd3 HR 2.617; p = 0.007). An association between tumor budding groups and RFS was observed, and the difference was significant in univariable analysis for high-grade compared to low-grade tumor budding (Bd3 HR 1.461; p = 0.041). No significant differences were observed between tumor budding groups and OS. High-grade tumor budding is a predictor of recurrence in a screened population of patients with stage II colon cancer and should be considered a high-risk factor in a shared decision-making process when stratifying patients to adjuvant chemotherapy.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

STING activation counters glioblastoma by vascular alteration and immune surveillance.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

Gene Expressions and High Lymphocyte Count May Predict Durable Clinical Benefits in Patients with Advanced Non-Small-Cell Lung Cancer Treated with Immune Checkpoint Inhibitors.

BACKGROUND: Not all patients with advanced non-small cell lung cancer (NSCLC) benefit from immune checkpoint inhibitors (ICIs). Therefore, we aimed to assess the predictive potential of gene expression profiling (GEP), peripheral immune cell counts, and clinical characteristics. METHODS: The primary endpoint of this prospective, observational study was a durable clinical benefit (DCB) defined as progression-free survival >6 months. In a subgroup with histological biopsies of sufficient quality (n = 25), GEP was performed using the nCounter® PanCancer IO 360 panel. RESULTS: DCB was observed in 49% of 123 included patients. High absolute lymphocyte count (ALC) and absence of liver metastases were associated with DCB (OR = 1.95, p = 0.038 and OR = 0.36, p = 0.046, respectively). GEP showed clustering of differentially expressed genes according to DCB, and a strong association between PD-L1 assessed by GEP (CD274) and immunohistochemistry (IHC) was observed (p = 0.00013). The TGF-ß, dendritic cell, and myeloid signature scores were higher for patients without DCB, whereas the JAK/STAT loss signature scores were higher for patients with DCB (unadjusted p-values < 0.05). CONCLUSIONS: ALC above 1.01 × 109/L and absence of liver metastases were significantly associated with DCB in ICI-treated patients with NSCLC. GEP was only feasible in 20% of the patients. GEP-derived signatures may be associated with clinical outcomes, and PD-L1 could be assessed by GEP rather than IHC.

Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells.

Circular RNAs (circRNA) are covalently closed molecules that can play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA sequencing or microarrays, emphasizing a strong translational potential. Most previous studies have been performed using RNA from bulk tissues and lack information on the spatial expression patterns of circRNAs. Here, we showed that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues were surprisingly not differentially expressed when comparing cancer cells directly with normal epithelial cells. Manipulating the proliferation rates of cells grown in culture revealed that these discrepancies were explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability; on the contrary, circRNAs were diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, different subcompartments of colon tumors and adjacent normal tissues exhibited striking differences in circRNA expression patterns. Likewise, the high circRNA content in muscle cells was also a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancers. Together, these findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. SIGNIFICANCE: The abundance of circRNAs varies systematically between subcompartments of solid tumors and adjacent tissues, implying that differentially expressed circRNAs discovered in bulk tissue analyses may reflect differences in cell type composition between samples.

Global circRNA expression changes predate clinical and histological improvements of psoriasis patients upon secukinumab treatment.

Psoriasis is a common chronic inflammatory skin disease accompanied by heterogenous clinical and histological features, including a characteristic keratinocyte hyperproliferation and dermal immunogenic profile. In addition, psoriasis is associated with widespread transcriptomic alterations including changes in microRNA (miRNA) and circular RNA (circRNA) abundance, which constitute non-coding RNA (ncRNA) classes with specific regulatory capacities in diverse physiological and pathological processes. However, the knowledge about the expression dynamics of ncRNA during psoriasis treatment is sparse. To elucidate the dynamics of miRNA and circRNA abundance during secukinumab (anti-IL-17A) treatment, we studied their expression patterns in skin biopsies from 14 patients with severe plaque-type psoriasis before and during an 84-day secukinumab therapy at day 0, 4, 14, 42, and 84 using NanoString nCounter technology. We found a comprehensive downregulation of the majority of investigated circRNAs and specific alterations in the miRNA profile, including an upregulation of miR-203a-3p, miR-93-5p, and miR-378i in lesional compared to non-lesional skin before treatment. During treatment, the circRNAs progressively returned to the expression levels observed in non-lesional skin and already four days after treatment initiation most circRNAs were significantly upregulated. In comparison, for miRNAs, the normalization to baseline during treatment was delayed and limited to a subset of miRNAs. Moreover, we observed a strong correlation between multiple circRNAs, including ciRS-7 and circPTPRA, and the psoriasis area and severity index (PASI). Similar pronounced correlations could, however, not be found for miRNAs. Finally, we did not observe any significant changes in circRNA expression in peripheral blood mononuclear cells during treatment. In conclusion, we uncovered a rapid shift in global circRNA abundance upon anti-IL-17A treatment, which predated clinical and histological improvements, and a strong correlation with PASI, indicating a biomarker potential of individual circRNAs.

Spatial analysis of photoplethysmography in cutaneous squamous cell carcinoma.

The primary treatment of the common malignancy squamous cell carcinoma is surgical removal. In this process, sufficient tissue removal is balanced against unnecessary mutilation. We recently presented a remote photoplethysmography algorithm, which revealed significant differences between processed video recordings of cancer biopsy areas and surrounding tissue. The aim of this study was to investigate whether spatial analyses of photoplethysmography data correlate with post-excision pathological analyses and thus have potential to assist in tumour delineation. Based on high speed video recordings of 11 patients with squamous cell carcinoma, we examined different parameters derived from temporal remote photoplethysmography variations. Signal characteristics values in sites matching histological sections were compared with pathological measures. Values were ranked and statistically tested with a Kendall correlation analysis. A moderate, negative correlation was found between signal oscillations and the width and transversal area of squamous cell carcinoma in the frequencies below 1 Hz and specifically from 0.02 to 0.15 Hz. We have presented a correlation between frequency content and prevalence of cancer based on regular video recordings of squamous cell carcinoma. We believe this is supported by published findings on malignant melanoma. Our findings indicate that photoplethysmography can be used to distinguish SCC from healthy skin.

The emerging roles of circRNAs in cancer and oncology.

Over the past decade, circular RNAs (circRNAs) have emerged as a large class of primarily non-coding RNA molecules, many of which have key roles in cancer development and progression through diverse mechanisms of action. CircRNAs often have tissue-restricted and cancer-specific expression patterns, and accumulating data suggest that these molecules are of potential clinical relevance and utility. In particular, circRNAs have strong potential as diagnostic, prognostic and predictive biomarkers, which is underscored by their detectability in liquid biopsy samples such as in plasma, saliva and urine. However, technical issues in the detection and assessment of circRNAs as well as biological knowledge gaps need to be addressed to move this relatively young field of research forward and bring circRNAs to the forefront of clinical practice. Herein, we review the current knowledge regarding circRNA biogenesis, regulation and functions in cancer as well as their clinical potential as biomarkers, therapeutic agents and drug targets.

Photoplethysmography for demarcation of cutaneous squamous cell carcinoma.

A video processing algorithm designed to identify cancer suspicious skin areas is presented here. It is based on video recordings of squamous cell carcinoma in the skin. Squamous cell carcinoma is a common malignancy, normally treated by surgical removal. The surgeon should always balance sufficient tissue removal against unnecessary mutilation, and therefore methods for distinction of cancer boundaries are wanted. Squamous cell carcinoma has angiogenesis and increased blood supply. Remote photoplethysmography is an evolving technique for analysis of signal variations in video recordings in order to extract vital signs such as pulsation. We hypothesize that the remote photoplethysmography signal inside the area of a squamous cell carcinoma is significantly different from the surrounding healthy skin. Based on high speed video recordings of 13 patients with squamous cell carcinoma, we have examined temporal signal differences in cancer areas versus healthy skin areas. A significant difference in temporal signal changes between cancer areas and healthy areas was found. Our video processing algorithm showed promising results encouraging further investigation to clarify how detailed distinctions can be made.

Validating Methylated HOXA9 in Bronchial Lavage as a Diagnostic Tool in Patients Suspected of Lung Cancer.

Diagnosing lung cancer requires invasive procedures with high risk of complications. Methylated tumor DNA in bronchial lavage has previously shown potential as a diagnostic biomarker. We aimed to develop and validate methylated HOXA9 in bronchial lavage as a diagnostic biomarker of lung cancer. Participants were referred on suspicion of lung cancer. Ten mL lavage fluid was collected at bronchoscopy for analysis of methylated HOXA9 based on droplet digital PCR according to our previously published method. HOXA9 status was compared with the final diagnosis. The Discovery and Validation cohorts consisted of 101 and 95 consecutively enrolled participants, respectively. In the discovery cohort, the sensitivity and specificity were 73.1% (95% CI 60.9-83.2%) and 85.3% (95% CI 68.9-95.0%), respectively. In the validation cohort, the values were 80.0% (95% CI 66.3-90.0%) and 75.6% (95% CI 60.5-87.1%), respectively. A multiple logistic regression model including age, smoking status, and methylated HOXA9 status resulted in an AUC of 84.9% (95% CI 77.3-92.4%) and 85.9% (95% CI 78.4-93.4%) for the Discovery and Validation cohorts, respectively. Methylated HOXA9 in bronchial lavage holds potential as a supplementary tool in the diagnosis of lung cancer with a clinically relevant sensitivity and specificity. It remained significant when adjusting for age and smoking status.

The CRISPR/Cas9 Minipig-A Transgenic Minipig to Produce Specific Mutations in Designated Tissues.

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

The prognostic impact of circulating homeobox A9 methylated DNA in advanced non-small cell lung cancer.

BACKGROUND: The homeobox A9 gene encodes a transcription factor, and aberrantly methylated homeobox A9 in the circulation has been suggested as a prognostic marker in early stage non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the prognostic impact of methylated homeobox A9 in plasma from patients with advanced NSCLC. METHODS: Blood samples were prospectively collected from patients with NSCLC stage III and IV receiving standard first line chemotherapy. Sampling took place before treatment initiation and subsequently before each treatment cycle. Plasma was stored at -80 °C until analysis. DNA was extracted, and following bisulfite conversion methylated homeobox A9 was analyzed by methylation specific droplet digital polymerase chain reaction. Detection of methylated homeobox A9 was assessed as a binary variable. The primary endpoint was overall survival (OS). RESULTS: A total of 231 patients were included. At baseline methylated homeobox A9 was detected in 78.5% of the patients with a clear correlation to survival. The median OS for patients with and without detectable methylated homeobox A9 was 7.4 and 11.1 months, respectively [hazard ratio (HR) 1.79, 95% confidence interval (CI): 1.35-2.38, P<0.001]. The difference increased after the first cycle of treatment. At this time point the median OS was 6.2 and 15.6 months for patients with and without detectable methylated homeobox A9, respectively (HR 2.07, 95% CI: 1.58-2.73, P<0.001). The independent prognostic impact of detectable methylated homeobox A9 after one treatment cycle assessed by multiple Cox regression including known prognostic factors resulted in a HR of 3.79 (2.19-6.54, P<0.001) compared to undetectable methylated homeobox A9. CONCLUSIONS: Measurable methylated homeobox A9 after the first treatment cycle may serve as a valuable prognostic marker in patients with advanced NSCLC. Routine clinical application with treatment reconsideration calls for further studies, preferably in prospective clinical trials.

Comparative Analysis of Stk11/Lkb1 versus Pten Deficiency in Lung Adenocarcinoma Induced by CRISPR/Cas9.

This study focused on STK11, PTEN, KRAS, and TP53, which are often found to be mutated in lung cancer. We compared Stk11 and Pten implication in lung cancer in combination with loss of Trp53 and gain of function of Kras in a CRISPR/Cas9 mouse model. Mice with loss of Stk11, Trp53, and KrasG12D mutation (SKT) reached human endpoint at around four months post-initiation. In comparison, mice with loss of Pten, Trp53, and KrasG12D mutation (PKT) survived six months or longer post-initiation. Pathological examination revealed an increase in proliferation in SKT deficient lung epithelia compared to PKT. This difference was independent of Pten loss, indicating that loss of Pten is dispensable for cell proliferation in lung adenocarcinoma. Furthermore, tumors with loss of Stk11, Trp53, and KrasG12D mutation had a significantly higher progression rate, monitored by PET/MRI scanning, compared to mice with loss of Pten, Trp53, and KrasG12D mutation, revealing that mutations in Stk11 are essential for adenocarcinoma progression. Overall, by using the CRISPR/Cas9 mouse model of lung adenocarcinoma, we showed that mutations in Stk11 are a key driver, whereas loss of Pten is dispensable for adenocarcinoma progression.

In vivo CRISPR inactivation of Fos promotes prostate cancer progression by altering the associated AP-1 subunit Jun.

Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.

Characterization of circular RNA transcriptomes in psoriasis and atopic dermatitis reveals disease-specific expression profiles.

Atopic dermatitis (AD) and psoriasis are two common chronic inflammatory skin diseases that are associated with various comorbidities. Circular RNA (circRNA) constitutes a major class of non-coding RNAs that have been implicated in many human diseases, although their potential involvement in inflammatory skin diseases remains elusive. Here, we compare and contrast the circRNA expression landscapes in paired lesional and non-lesional skin from psoriasis and AD patients relative to skin from unaffected individuals using high-depth RNA-seq data. CircRNAs and their cognate linear transcripts were quantified using the circRNA detection algorithm, CIRI2, and in situ hybridization and Sanger sequencing was used for validation purposes. We identified 39,286 circRNAs among all samples and found that psoriasis and AD lesional skin could be distinguished from non-lesional and healthy skin based on circRNA expression landscapes. In general, circRNAs were less abundant in lesional relative to non-lesional and healthy skin. Differential expression analyses revealed many significantly downregulated circRNAs, mainly in psoriasis lesional skin, and a strong correlation between psoriasis and AD-related circRNA expression changes was observed. Two individual circRNAs, ciRS-7 (also known as CDR1as) and circZRANB1, were specifically dysregulated in psoriasis and show promise as biomarkers for discriminating AD from psoriasis. In conclusion, the circRNA transcriptomes of psoriasis and AD share expression features, including a global downregulation relative to healthy skin, but this is most pronounced in psoriasis, and only psoriasis is characterized by several circRNAs being dysregulated independently of their cognate linear transcripts. Finally, specific circRNAs could potentially be used to distinguish AD from psoriasis.

Comparison of Mutated KRAS and Methylated HOXA9 Tumor-Specific DNA in Advanced Lung Adenocarcinoma.

Circulating tumor DNA (ctDNA) has been suggested as a biomarker in non-small cell lung cancer. The optimal target for measuring ctDNA has not yet been established. This study aimed to investigate methylated Homeobox A9 (meth-HOXA9) as an approach to detect ctDNA in advanced lung adenocarcinoma and compare it with mutated Kirsten rat sarcoma viral oncogene homolog (mut-KRAS) in order to determine the mutual agreement. DNA was purified from formalin-fixed, paraffin-embedded non-malignant lung tissue and lung adenocarcinoma tissue, and plasma from healthy donors and lung adenocarcinoma patients, respectively. KRAS mutations in tumor tissue were identified by next-generation sequencing and quantified in tumor and plasma by droplet digital polymerase chain reaction (ddPCR). The meth-HOXA9 analysis was based on bisulfite-converted DNA from tumor and plasma and quantified by ddPCR. Samples consisted of 20 archival non-malignant lung tissues, 48 advanced lung adenocarcinomas with matched plasma samples, and 100 plasma samples from healthy donors. A KRAS mutation was found in the tumor in 34/48 (70.8%) adenocarcinoma patients. All tumors were positive for meth-HOXA9, while none of the non-malignant lung tissues were. Meth-HOXA9 was detected in 36/48 (75%) of plasma samples, and the median level was 0.7% (range of 0-46.6%, n = 48). Mut-KRAS was detected in 29/34 (85.3%) of the plasma samples, and the median level was 1.2% (range of 0-46.1%, n = 34). There was a good correlation between meth-HOXA9 and mut-KRAS in plasma (Spearman's rho 0.83, p < 0.001). Meth-HOXA9 is present in tissue from incurable lung adenocarcinoma but not in non-malignant lung tissue. It may be used as an approach for detecting ctDNA. The results demonstrated a high agreement between meth-HOXA9 and mut-KRAS in patients with advanced lung adenocarcinoma.

Spatial expression analyses of the putative oncogene ciRS-7 in cancer reshape the microRNA sponge theory.

Circular RNAs (circRNAs) have recently gained substantial attention in the cancer research field where most, including the putative oncogene ciRS-7 (CDR1as), have been proposed to function as competitive endogenous RNAs (ceRNAs) by sponging specific microRNAs. Here, we report the first spatially resolved cellular expression patterns of ciRS-7 in colon cancer and show that ciRS-7 is completely absent in the cancer cells, but highly expressed in stromal cells within the tumor microenvironment. Additionally, our data suggest that this generally apply to classical oncogene-driven adenocarcinomas, but not to other cancers, including malignant melanoma. Moreover, we find that correlations between circRNA and mRNA expression, which are commonly interpreted as evidence of a ceRNA function, can be explained by different cancer-to-stromal cell ratios among the studied tumor specimens. Together, these results have wide implications for future circRNA studies and highlight the importance of spatially resolving expression patterns of circRNAs proposed to function as ceRNAs.