Archival FFPE Tumor Subtyping Using Mineral Oil and ddPCR
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Solution Overview
Problem
Existing molecular tests, such as Oncotype Dx, Prosigna, EndoPredict, MamaPrint, and Breast Cancer Index, are ineffective on archival tissue samples that are old and degraded, limiting the ability to perform PCR and molecular subtyping in pathology laboratories.
Innovation Solution
A method involving enhanced solubilization of old and degraded FFPE samples using mineral oil, digestion with proteinase, incubation with DNase and guanidine salt buffer, RNA concentration and isolation, pre-amplification, and digital droplet PCR to perform molecular subtyping of cancer samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional molecular tests (Oncotype Dx, Prosigna, EndoPredict, MamaPrint, Breast Cancer Index) are performed on archival tissue samples, then the samples must be fresh and undegraded, but this limits the ability to perform PCR and molecular subtyping on old archival materials
Solution Approach 1:
The protocol modifies multiple parameters including using mineral oil for enhanced solubilization, optimizing proteinase digestion conditions, adjusting DNase treatment, and using guanidine salt buffer for RNA isolation. These parameter changes enable the extraction of sufficient quality RNA from degraded archival samples to perform molecular subtyping
Solution Approach 2:
The method performs preliminary actions including enhanced solubilization of FFPE samples with mineral oil, extensive proteinase digestion, and DNase treatment before RNA extraction. These preliminary steps prepare the degraded archival samples by removing inhibitors and freeing RNA from protein complexes, making subsequent PCR possible
2Measurement precision
If fresh specimens are used for molecular studies, then diagnostic yield is maximized, but fresh material is not always available for testing
Solution Approach 1:
The protocol uses mineral oil as an intermediary substance to enhance solubilization of FFPE samples. The mineral oil penetrates the paraffin-embedded tissue, facilitates proteinase K penetration, and helps release RNA from the degraded tissue matrix, acting as a mediator between the fixed tissue and the extraction reagents
Solution Approach 2:
The method replaces the need for fresh mechanical tissue processing with chemical/enzymatic processing of archival samples. Instead of requiring fresh tissue sectioning and processing, the protocol uses chemical solubilization (mineral oil), enzymatic digestion (proteinase K, DNase), and chemical extraction (guanidine buffer) to process archived FFPE blocks directly
3Loss of time
If archival material is used for molecular analysis, then retrospective studies become possible, but the probes used in standard assays do not work well on old and degraded samples
Solution Approach 1:
The protocol optimizes multiple parameters including extending proteinase digestion time, adjusting mineral oil to sample ratio, optimizing DNase treatment conditions, and using specific guanidine buffer concentrations. These parameter optimizations ensure sufficient RNA quality and quantity from degraded archival samples for reliable molecular subtyping
Solution Approach 2:
The method creates a copy of the fresh tissue molecular state by extracting and amplifying RNA from archival samples. Through pre-amplification and digital droplet PCR, the protocol generates sufficient signal from the limited degraded RNA to accurately determine molecular subtype, effectively copying the diagnostic information that would be present in fresh tissue
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables accurate molecular subtyping of cancer samples from archival tissues, even those that are 5 years old or older, by enhancing RNA extraction and amplification, allowing for retrospective clinical trials and therapeutic response prediction.
Implementation Method 1
enhanced solubilization of the old and degraded FFPE sample material through the non-discretionary use of mineral oil
Implementation Method 2
digesting the sample with a proteinase
Implementation Method 3
digesting the sample with a proteinase; the sample is digested at about 65° C. to about 70° C. for about 75 minutes
Implementation Method 4
incubating the digested sample from step a) with a DNase
Implementation Method 5
incubating the mixture from step b) with a guanidine salt based buffer, concentrating and isolating the RNA
Implementation Method 6
performing digital droplet PCR; the target nucleic acid or fragment thereof encodes a Estrogen receptor 1 (ESR1), Progesterone receptor (PGR)
Data Source
AI summary
The present disclosure encompasses methods for molecularly subtyping formalin-fixed paraffin-embedded tumor samples. The disclosure works particularly well for old and degraded (archival) samples for which standard methods are unfeasible. Further, the methods disclosed allow for the correlation of patient outcome data with the molecular subtype of the tumor and provides a wealth of information which will guide treatment decisions and/or selection of therapeutic agents.