Understanding our health shouldn’t be a daunting process. Researchers, like Alex Rolland and his team at Cancer Treatment Options and Management Inc., are working to provide clear answers to critical oncology questions. They focus on determining if treatments are effective and if patients are in remission. Discover how their research aims to improve cancer diagnostics and treatment, bringing greater certainty to patient care.

Alex Rolland, co-founder and chief scientist of Cancer Treatment Options and Management Inc. (CTOAM) and Liquid Biopsy Labs, embarked on a search for effective ct-DNA extraction kits for their digital PCR blood-based liquid biopsy lab in late 2018. Despite testing various kits, none provided the required ct-DNA yield for their Taqman-ddPCR platform until they discovered Norgen Biotek.

Attracted by Norgen’s centrifuge-based extraction technology, Rolland’s team initially obtained a small but usable amount of ct-DNA. Following a conversation with a Senior Research Scientist at Norgen, they were introduced to a new procedure involving two different-sized columns. This innovation, along with Norgen’s cf-DNA/cf-RNA Preservative Tubes (Cat. 63950), significantly improved their yield to 7-10 mL of plasma/serum from two tubes, enhancing their real-life testing capabilities.

Figure 1. Blood samples from six donors were tested in duplicate, with one set remaining in the lab and the other shipped (72 hours total). Norgen’s cf-DNA/cf-RNA Preservative Tubes maintained consistent plasma volumes (6-7 mL) both before and after shipping. In contrast, Competitor and EDTA tubes showed a significant volume decrease from ~4 mL to ~2.5 mL.

Testing Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit (Cat. 55600) and Plasma/Serum Exosome Purification and RNA Isolation Midi Kit (Cat. 58500), Rolland’s team achieved outstanding results. By focusing on ct-DNA from active tumors and excluding DNA from apoptosis, they identified cancer-specific ct-DNA fragments of 146bps and smaller. Norgen’s kits proved instrumental in enabling the team to concentrate on these critical cancer-associated ct-DNA fragments, advancing their research and diagnostic capabilities significantly.

Summary of Benefits from Norgen Products

• High yield of plasma/serum from blood tubes.
• High yield of cancer/exosome-associated ct-DNA from their extraction kits.

Examples of ct-DNA Results

Alex Rolland and his team have developed hundreds of custom TaqMan probes to monitor cancer patients, targeting mutations in oncogenes, tumor suppressor genes, and Variants of Unknown Significance (VUSs). This approach enhances their understanding of disease progression and treatment effectiveness.

–          Case 1: In a CRC patient, a VUS in the PRKDC tumor suppressor gene, identified through tumor DNA sequencing, was predicted pathogenic via in-silico analysis. This informed early recurrence detection and treatment efficacy assessment.

Figure 2: Amount of PRKDC A358T exosomal DNA obtained from patient’s whole blood. The error bars indicate the 95% Confidence Intervals (CI).

–          Case 2: In a late-stage breast cancer patient, a PIK3CA mutation was detected. Although Alpelisib was accessed through compassionate means despite lacking Canadian approval, the patient could not tolerate it for long, leading to treatment cessation. Monitoring with ct-DNA showed increased levels without benefit.

Figure 3: PIK3CA P1047L exosomal DNA was obtained from the patient’s whole blood. The error bars indicate the 95% Confidence Intervals (CI).

–          Case 3: A woman with lung cancer undergoing immunotherapy was thought to have disease progression based on traditional imaging, leading to consideration of stopping treatment. Using ct-DNA, it was determined she was experiencing pseudoprogression, allowing her to continue with immunotherapy and achieve a positive response.

Figure 4: Amount of KRAS G12D exosomal DNA obtained from patient’s whole blood. The error bars indicate the 95% Confidence Intervals (CI).

–          Case 4: Following a significant response to chemotherapy, the patient initiated immunotherapy. However, ct-DNA analysis later revealed significant progression, indicating ineffective treatment. This real-time data enabled the patient’s doctor to promptly adjust their treatment plan.

Figure 5: Amount of KRAS G12D exosomal DNA obtained from patient’s whole blood. The error bars indicate the 95% Confidence Intervals (CI).

Norgen Biotek’s Role

Recently, Norgen partnered with Alex Rolland and his team to isolate blood-based exosomes and exosomal-RNA, achieving RNA-seq-based expression analysis of 20,000 genes from tumor-associated exosomes of cancer patients in various states—remission and recurrence. However, attempts to apply this method to individuals without cancer didn’t yield adequate sequencing reads, highlighting the specificity of Norgen’s exosome isolation for tumors.

In their research, Alex Rolland’s team developed a panel of RNAs and genes exclusive to cancers, meticulously comparing these with normal tissue profiles. This approach enables:

1. Early detection of cancer activity in non-diagnosed individuals.
2. Evaluation of cancer remission or early recurrence.
3. Assessment of treatment effectiveness.
4. Mapping widespread gene expression in cancer.
5. Identification of potential drivers of cancer progression, guiding tissue-specific targeted therapies.
6. Evaluation of VUS roles in cancer based on their expression levels from tumor DNA sequencing.

Globally, these tools significantly enhance cancer treatment accuracy, ensuring appropriate therapies and improving outcomes for patients worldwide.

Looking ahead, scientists such as Alex Rolland and his team are revolutionizing oncological research, and Norgen Biotek is proud to support their efforts. Improved testing promises more precise treatments, aiming to reduce cancer-related deaths globally.

We extend our gratitude to Alex Rolland and the CTOAM Inc. team for their unwavering dedication and hard work.

Source: Improving cancer diagnostics with exosomal ct-DNA