A Multi-Analyte Blood Test for Cancer Detection and Localization

Cancer, usually referred to uncontrolled growth of abnormal cells, leading to the formation of a tumour. It is strongly believed that the best chance to arrest cancer is its early detection. Nevertheless, it is not possible to detect many tumors until it is grown sufficiently or it spreads across other parts of the body. Numerous efforts are constantly being made by several researchers to develop effective methods for cancer detection. During the uncontrolled growth of cancer cells some of them would die and shed their mutated DNA into the bloodstream. Liquid biopsy test could detect the DNA carrying mutations, which are associated with cancer. However, development of liquid biopsy test that is capable of screening healthy people remains a big challenge. In addition, the inability of the liquid biopsy test to detect the location of the cancer is a major limitation. A team of researchers at Johns Hopkins University School of Medicine, Baltimore led by Nickolas Papadopoulos and Bert Vogelstein have developed a multi-analyte blood test, referred as “CancerSEEK” for early detection of cancer. CancerSEEK is a ‘liquid biopsy test” that examines mutations in cell-free DNA and proteins circulating in the bloodstream (Fig. 1). This research work is funded by National Institute of Health (NIH), USA and the findings of this study is published recently in Science (J. D. Cohen et al., Science 10.1126/science.aar3247 (2018)).

Fig. 1 Schematic of the liquid biopsy test – Tumour cells shed protein and DNA into the blood stream that can be used as biomarkers for early cancer detection

About 1,005 patients diagnosed with ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast cancers (Stage I to III) were used to check the ability of CancerSEEK in which none of them have received chemotherapy prior to blood sample collection and none had evident distant metastasis at the time of study. CancerSEEK evaluates the levels of 8 proteins and the presence of mutations in 2,001 genomic positions in 16 different genes, which helps to identify at least eight common types of cancers. Since the test uses a combination of protein biomarkers along with genetic biomarkers, a better sensitivity is achieved without compromising specificity. CancerSEEK is capable of not only identifying the presence of tumours but also localize the organ at which the cancer cells are grown.

CancerSEEK was found to be 98% accurate for tumours in ovary and liver. The median sensitivity of CancerSEEK was estimated to be 73% and 78% for stage II and stage III cancers, respectively. Unfortunately, the success rate of  CancerSEEK for stage I cancer was limited to 43% (Fig. 2(a)). In spite of its low detection ability for stage I cancer, its ability to narrow down the localization of the cancer in 83% of the patients (Fig. 2(b)) makes CancerSEEK as a most reliable method for cancer detection.

Fig. 2 Performance of CancerSEEK: (a) Sensitivity of CancerSEEK by stage; Bars represent the median sensitivity of the eight cancer types and error bars represent standard errors of the median; and (b) Sensitivity of CancerSEEK by tumor type. Error bars represent 95% confidence intervals.

CancerSEEK is expected to be available in the next few years at an estimated cost of less than US$500. Since cancer-related proteins used by Cancer-SEEK could also appear in people with inflammatory diseases such as arthritis, the applicability of this test for such patients is questioned. As an early detection is the key to surgically remove cancer cells before they metastasise, the detection level of 43% for stage I cancers needs to be improved by a large margin.

T.S.N. Sankara Narayanan

For more information, the reader may kindly refer to: J. D. Cohen et al., Science 10.1126/science.aar3247 (2018)

Non-Endoscopic Balloon-Based Device for Sampling Cancer Detection

Esophageal Adenocarcinoma (EAC) – the cancer that occurs in the lower portion of the esophagus (the food pipe that runs between throat and stomach) is the most common form of cancer in the United States. In spite of a steady increase in the incidence of EAC over the past 3 decades, the prognosis remains poor. Barrett’s esophagus (BE) is the only known precursor for EAC and it is currently diagnosed using an endoscope. Sanford Markowitz at Case Western Reserve University, Ohio, and his colleagues have demonstrated the feasibility of a non-endoscopic molecular cytology screening method for BE and EAC (Moinova et al., Science Translational Medicine, Vol. 10, Issue 424, eaao5848)

The non-endoscopic swallowable balloon-based esophageal sampling device consists of a pill-sized capsule (16 × 9 mm) attached to a thin 2.16 mm silicone catheter (Fig. 1, A and B), which can be easily swallowed. After delivery into the stomach, the balloon is inflated by injecting 5 cm³ of air through the catheter (Fig. 1C). The inflated balloon can be gently moved through the distal esophagus to collect samples from the luminal epithelial surface. Subsequently, the balloon is deflated and inverted back into the capsule (Fig. 1D). After complete retrieval of the capsule through the mouth, DNA is extracted from the balloon surface for molecular analysis. One of the prime advantages of this balloon-based sampling device is its ability to deploy rapidly by inflation unlike the conventional sponge-based devices, which requires sufficient waiting time for the coating to dissolve. The ability of the balloon to retract back into its capsule after sampling protects the sample from dilution or contamination from the proximal esophagus or oral cavity. The swallowable balloon-based device enables a simple and rapid method to collect DNA samples from the distal esophagus of unsedated outpatients. A combination of this balloon-based sampling device with bisulfite sequencing for detecting DNA methylation, provides a highly sensitive and specific yet minimally invasive screening protocol that could be clinically used for the detection and screening of BE.

Fig. 1 Non-endoscopic balloon-based device: (A) Device capsule and catheter (a vitamin pill and a dime are included for size comparison); (B) Capsule containing inverted balloon for swallowing; (C) Capsule with inflated balloon for esophageal sampling; and (D) Capsule containing inverted balloon for device and biospecimen retrieval.

T.S.N. Sankara Narayanan

 

Detecting cancer from urine sample – Will the development of a nanowire based device open the gates for early diagnoses and timely medical checkups for cancer?

MicroRNAs (miRNAs) encapsulated by extracellular vesicles (EVs) are found in body fluids of patients with malignant diseases as well as with those having a better health. The difference in the EV-encapsulated miRNAs between these two groups of people could be used as a signature to identify various diseases. The miRNAs present in urine could serve as biomarkers for detecting cancer. Unfortunately, the concentration of EVs in urine is extremely low (<0.01 volume %) and hence the most commonly used method of extraction – ultracentrifugation is not capable of extracting nearly 90% of the miRNA species, due to their low abundance. Recently, a nanowire-based device anchored to a microfluidic substrate is fabricated for the efficient collection of EVs and in situ extraction of various miRNAs of different sequences, which is believed to open the gates for urine-based early diagnoses and medical checkups for cancer (Yasui et al., Sci. Adv. 2017;3: e1701133).

Si (100) served as the base substrate (Fig. 1(a)), which was coated with a positive photoresist followed by channel patterning using photo lithography (Fig. 1(b)). A 140 nm thick Cr layer was sputter deposited (Fig. 1(c)), followed by removal of the photoresist layer and thermal oxidation of the Cr layer at 400 °C for 2 h (Fig. 1(d)). The thermally oxidized Cr layer served as the seed layer for subsequent growth of ZnO nanowires using a solution mixture of 15 mM hexamethylenetetramine (HMTA) and 15 mM zinc nitrate hexahydrate at 95 °C for 3 h (Fig. 1(e)). PDMS was poured over the ZnO nanowire grown substrate and cured (Fig. 1(f)). Subsequently, the PDMS was removed from the Si substrate (Fig. 1(g)) and the ZnO nanowires in the PDMS were transferred to another PDMS substrate. The transferred nanowires were uniformly and deeply buried into PDMS while their slightly emerged heads served as growth points for the second nanowire growth (Fig. 1(h)), which was carried out by immersing the PDMS in a solution mixture of 15 mM HMTA and 15 mM zinc nitrate hexahydrate at 95 °C for 3 h (Fig. 1(i)). To enhance contact events between the ZnO nanowires and the EVs as well as to avoid any pressure drop, the ZnO nanowire embedded PDMS substrate was anchored to a herringbone-structured PDMS substrate (Fig. 1(j)).

Fig. 1 (a-j) Various stages involved in the fabrication of nanowire-based device; and (k) schematic of the collection and extraction of EV–encapsulated miRNAs.

The nanowire-based device is capable of detecting around 1000 types of species of miRNAs when compared to the conventional ultracentrifugation method. Using this device, it is possible to extract EV–encapsulated miRNAs within 40 min (collection, 20 min; extraction, 20 min) by introducing just 1 ml of urine sample followed by 1 ml of lysis buffer into the device (Fig. 1(k)). In contrast, the ultracentrifugation method requires 20 ml of urine sample and more than 5 h for collection and extraction. The device enables a four-fold increase in the miRNA expression level with a larger variety of extracted species of miRNAs. The ZnO nanowire-based device is found to be superior to the commonly used ultracentrifugation method in terms of treatment time and RNA extraction efficiency. This attribute is due to its large surface area of ZnO nanowires and their positively charged surface (isoelectric point of 9.50 at pH 6 to 8), which electrostatically attracts the negatively charged EVs in urine sample at pH 6-8. In addition, the mechanical stability of ZnO nanowires, which are firmly anchored to the PDMS substrate helps to retain their strength during buffer flow and enhances the extraction efficiency. The positively charged surface of ZnO nanowires offers benefit in collecting negatively charged objects in urine samples, including exosomes, microvesicles, and EV-free miRNAs.

The ZnO nanowire-based device is believed to help in the early diagnoses and timely medical checkups based on urine miRNA analysis. The method is capable of identifying urinary miRNAs that could potentially serve as biomarkers for detecting bladder, prostate lung, pancreas, and liver cancer.

T.S.N. Sankara Narayanan