Cervical cancer affects over half a million people worldwide each year, the majority of whom are in resource-limited settings where cytology screening is not available. As persistent human papilloma ...virus (HPV) infections are a key causative factor, detection of HPV strains now complements cytology where screening services exist. This work demonstrates the efficacy of a handheld Lab-on-Chip (LoC) device, with an external sample extraction process, in detecting cervical cancer from biopsy samples. The device is based on Ion-Sensitive Field-Effect Transistor (ISFET) sensors used in combination with loop-mediated isothermal amplification (LAMP) assays, to amplify HPV DNA and human telomerase reverse transcriptase (hTERT) mRNA. These markers were selected because of their high levels of expression in cervical cancer cells, but low to nil expression in normal cervical tissue. The achieved analytical sensitivity for the molecular targets resolved down to a single copy per reaction for the mRNA markers, achieving a limit of detection of 10
for hTERT. In the tissue samples, HPV-16 DNA was present in 4/5 malignant and 2/5 benign tissues, with HPV-18 DNA being present in 1/5 malignant and 1/5 benign tissues. hTERT mRNA was detected in all malignant and no benign tissues, with the demonstrated pilot data to indicate the potential for using the LoC in cervical cancer screening in resource-limited settings on a large scale.
Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of ...circulating cell-free DNA fragments in the patients' blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy. Next generation sequencing platforms and digital droplet PCR can be used to profile circulating tumour DNA from liquid biopsies; however, they are expensive and time consuming for clinical use. Here, we report a novel strategy with proof-of-concept data that supports the usage of loop-mediated isothermal amplification (LAMP) to detect PIK3CA c.3140 A > G (H1047R), a prevalent BC missense mutation that is attributed to BC tumour growth. Allele-specific primers were designed and optimized to detect the p.H1047R variant following the USS-sbLAMP method. The assay was developed with synthetic DNA templates and validated with DNA from two breast cancer cell-lines and two patient tumour tissue samples through a qPCR instrument and finally piloted on an ISFET enabled microchip. This work sets a foundation for BC mutational profiling on a Lab-on-Chip device, to help the early detection of patient relapse and to monitor efficacy of systemic therapies for personalised cancer patient management.
A growing number of studies has shed light on the role of the NF-κΒ in non-small-cell lung cancer (NSCLC). To address the significance of major effectors of the NF-κΒ alternative pathway, we ...investigated the relationship between NF-κΒ2, RelB, NIK and Bcl3 expression (mRNA and protein) and the clinical outcome of NSCLC patients. NF-κΒ2, RelB, NIK and Bcl3 protein expression levels were assessed by immunohistochemistry in tissue samples from 151 NSCLC patients who had curative resection. mRNA levels were also evaluated in 69 patients using quantitative real-time PCR. Although all studied proteins were overexpressed in NSCLC (P < 0.001 for all), only RelB mRNA levels were strongly increased in cancerous specimens compared to tumor-adjacent non-neoplastic tissues (P = 0.009). Moreover, NF-κB2, RelB and Bcl3 expression was associated with overall survival (OS). In particular, cytoplasmic and mRNA expression of RelB was related to 5-year OS (P = 0.014 and P = 0.006, respectively). Multivariate analysis also showed that Bcl3 expression (nuclear and cytoplasmic) was associated with increased 5-year OS (P = 0.002 and P = 0.036, respectively). In addition, higher Bcl3 mRNA levels were associated with inferior OS in stages I & II and improved OS in stages III and IV after 5-year follow-up (P = 0.004 and P = 0.001, respectively). Furthermore, stage I patients with lower NF-κB2 mRNA levels had better 5-year survival in univariate and multivariate analysis (P = 0.031 and P = 0.028, respectively). Interestingly, RelB expression (cytoplasmic and mRNA) was inversely associated with relapse rates (P = 0.027 and P = 0.015, respectively), while low NIK cytoplasmic expression was associated with lower relapse rates (P = 0.019). Cytoplasmic NIK expression as well as NF-κB2/ Bcl3 detection was associated with lymph node infiltration (P = 0.039 and P = 0.014, respectively). The present study confirms the deregulation of the NF-κB alternative pathway in NSCLC and also demonstrates the importance of this pathway in prognosis, recurrence and infiltration of regional lymph nodes.
Cancer remains a leading cause of death worldwide, despite many advances in diagnosis and treatment. Precision medicine has been a key area of focus, with research providing insights and progress in ...helping to lower cancer mortality through better patient stratification for therapies and more precise diagnostic techniques. However, unequal access to cancer care is still a global concern, with many patients having limited access to diagnostic tests and treatment regimens. Noninvasive liquid biopsy (LB) technology can determine tumour-specific molecular alterations in peripheral samples. This allows clinicians to infer knowledge at a DNA or cellular level, which can be used to screen individuals with high cancer risk, personalize treatments, monitor treatment response, and detect metastasis early. As scientific understanding of cancer pathology increases, LB technologies that utilize circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) have evolved over the course of research. These technologies incorporate tumour-specific markers into molecular testing platforms. For clinical translation and maximum patient benefit at a wider scale, the accuracy, accessibility, and affordability of LB tests need to be prioritized and compared with gold standard methodologies in current use. In this review, we highlight the range of technologies in LB diagnostics and discuss the future prospects of LB through the anticipated evolution of current technologies and the integration of emerging and novel ones. This could potentially allow a more cost-effective model of cancer care to be widely adopted.
This paper presents the concept of using a novel ISFET-based readout circuit for ratiometric detection of DNA methylation, which gives a discrete answer once a certain percentage of methylation is ...reached. This allows the development of a tool for the early detection of multiple cancer types using a minimally invasive approach, by determining the differences between a pathogenic gene through cancer development and a normally methylated one. Analysis of such ratiometric markers will be based upon the proportion of methylated sites presented above a pre-defined, empirically set threshold value, using ISFETs in standard CMOS. The designed circuit utilizes the weak-inversion region of operation to allow analogue computation at low complexity and power coming from methylated and unmethylated DNA concentrations, by following the translinear principle of current division of biochemical signals.
Immune system-related receptors CD40 (tumor necrosis factor receptor superfamily member 5), BAFFR (tumor necrosis factor receptor superfamily member 13C), and LTβR (tumor necrosis factor receptor ...superfamily member 3) play a pivotal role in non-small-cell lung cancer (NSCLC). To further evaluate their role in NSCLC,
rs1883832 (T>C),
rs7290134 (A>G), and
rs10849448 (A>G) single-nucleotide polymorphisms (SNPs) were investigated regarding their impact in risk and clinical outcome of NSCLC patients.
The three selected SNPs were evaluated in 229 NSCLC patients and 299 healthy controls, while CD40, BAFFR, and LTβR protein expression was assessed by immunohistochemistry in 96 tumor specimens from NSCLC patients.
In total,
rs1883832 was associated with NSCLC risk, with the T allele, after adjusting for cofactors, being related to increased risk (p = 0.007; OR 1.701). Moreover, the CT genotype was associated with increased risk (p = 0.024; OR 1.606) and poorer 5-year overall survival (OS) after adjusting for cofactors (p = 0.001, HR 1.829), while CC was associated with higher CD40 expression in tumorous cells (p = 0.040) and in stromal cells (p = 0.036). In addition, AA homozygotes for the
rs10849448 had increased risk for NSCLC in multivariate analysis (p = 0.008; OR, 2.106) and higher LTβR membranous expression (p = 0.035). Although
rs7290134 was associated with BAFFR membranous expression (p = 0.039),
rs7290134 was not associated with neither the disease risk nor the prognosis of NSCLC patients.
In conclusion,
rs1883832 and
rs10849448 seem to be associated with increased risk for NSCLC, while
rs1883832 is also associated with OS of patients with NSCLC.
The electrical detection of DNA methylation based biomarkers using semiconductor technology shows great promise for early cancer screening. Presented is the very first proof-of-concept example of ...using CMOS-based technology for real-time DNA methylation detection using Ion-Sensitive Field-Effect Transistors (ISFETs). An electrochemical label-free approach was applied in two gene assays, each one of which incorporated the sequences of DAPK1 and CDKN2A/p16-INK4 (p16) gene promoters at a both methylated and unmethylated state, performing isothermal methylation-specific amplification and detection both in-tube and on-chip (real-time). Good discrimination was shown between the two states, achieving a very good average pH signal change for the methylated state of 1.91 for DAPK1 assay and of 1.58 for p16 assay in the tube test. The real-time on-chip test showed similarly good real-time differential signal change in favour of methylated DNA, reaching 37mV for DAPK1 assay and 23mV for p16 assay, validating the results from the proof-of-concept test of pH-LAMP in-tube while confirming the sensitivity of real-time methylation-specific pH-LAMP on-chip.
An increasing number of studies implicates the NF-κB (Nuclear Factor of kappa light chain gene enhancer in B cells) alternative pathway in non-small-cell lung cancer (NSCLC). We assessed the clinical ...significance of CD40 (Tumor necrosis factor receptor superfamily member 5, TNFRSF5), BAFFR (B-cell activating factor receptor), RANK (Receptor activator of NF-κB) and LTβR (lymphotoxin β receptor) receptors, which activate the alternative pathway of NF-κB, in NSCLC. Evaluation of CD40, BAFFR, RANK and LTβR expression was performed based on the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets, while protein expression was assessed by immunohistochemistry in specimens from 119 operated NSCLC patients.
gene overexpression was correlated with improved five-year overall survival (OS) (
< 0.001), while increased
and
mRNA levels were associated with worse OS in patients with adenocarcinomas (
< 0.001 and
< 0.001, respectively). Similarly, patients with adenocarcinomas exhibited a negative correlation between membranous BAFFR protein expression in carcinoma cells and three- and five-year survival (
= 0.021; HR, 4.977 and
= 0.030; HR, 3.358, respectively) as well as between BAFFR protein overexpression in cancer-associated fibroblasts (CAFs) and two-year survival (
= 0.036; HR, 1.983). Patients with increased LTβR nuclear protein staining or stage II patients with lower cytoplasmic LTβR protein expression had worse five-year OS (
= 0.039 and
= 0.008, respectively). Moreover, CD40 protein expression in tumor infiltrating lymphocytes (TILs) and CAFs was positively associated with metastatic spread while BAFFR protein expression in CAFs was negatively associated with bone metastasis (
= 0.041). Our data suggests that CD40, BAFFR, RANK and LTβR play an important role in NSCLC and further supports the role of NF-κB alternative pathway in NSCLC.
This paper presents a low power current-mode method for monitoring differentially derived changes in pH from ion-sensitive field-effect transistor (ISFET) sensors, by adopting the Chemical Gilbert ...Cell. The fabricated system, with only a few transistors, achieves differential measurements and therefore drift minimisation of continuously recorded pH signals obtained from biochemical reactions such as DNA amplification in addition to combined gain tunability using only a single current. Experimental results are presented, demonstrating the capabilities of the front-end at a microscopic level through integration in a lab-on-chip (LoC) setup combining a microfluidic assembly, suitable for applications that require differential monitoring in small volumes, such as DNA detection where more than one gene needs to be studied. The system was designed and fabricated in a typical 0.35 μ m CMOS process with the resulting topology achieving good differential pH sensitivity with a measured low power consumption of only 165 nW due to weak inversion operation. A tunable gain is demonstrated with results confirming 15.56 dB gain at 20 nA of ISFET bias current and drift reduction of up to 100 times compared to a single-ended measurement is also reported due to the differential current output, making it ideal for robust, low-power chemical measurement.
Thirty-four years since its discovery, NF-κB remains a transcription factor with great potential for cancer therapy. However, NF-κB-targeted therapies have yet to find a way to be clinically ...translatable. Here, we focus exclusively on the role of NF-κB in non-small cell lung cancer (NSCLC) and discuss its contributing effect on cancer hallmarks such as inflammation, proliferation, survival, apoptosis, angiogenesis, epithelial-mesenchymal transition, metastasis, stemness, metabolism, and therapy resistance. In addition, we present our current knowledge of the clinical significance of NF-κB and its involvement in the treatment of patients with NSCLC with chemotherapy, targeted therapies, and immunotherapy.
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