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Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

Nassiri M, Ramos S, Zohourian H, Vincek V, Morales AR, Nadji M - BMC Clin Pathol (2008)

Bottom Line: Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05).The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative.Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida, USA. mnassiri@med.miami.edu

ABSTRACT

Background: The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer - estrogen receptor and HER2 - at the RNA and protein levels.

Methods: Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.

Results: The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.

Conclusion: The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

No MeSH data available.


Related in: MedlinePlus

Scatter plot (logarithmic scale) of transcript copy number data for ER (A), HER2 (B), Cyclophilin A (PPIA) (C) and RN7SL1 (D).
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Figure 4: Scatter plot (logarithmic scale) of transcript copy number data for ER (A), HER2 (B), Cyclophilin A (PPIA) (C) and RN7SL1 (D).

Mentions: While the DNA yield was similar in formalin and UMFIX samples (Figure 3A), the RNA yield was significantly higher in the latter (p < 0.05 t-test, Figure 3B). Using the same amount of RNA template (2 μg), the cDNA yield was similar between two groups. Real-time PCR for HER2- and ER-RNA showed at least a ten fold difference in transcript copy number between UMFIX and formalin samples (Figure 4). Two control transcripts, PPIA and RN7sL had different dynamic ranges. PPIA was, on average, twofold higher in UMFIX samples. However, RN7SL1 copies were 50 times higher in UMFIX samples. These results indicate greater reverse-transcription efficiency and amplification in the UMFIX samples.


Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

Nassiri M, Ramos S, Zohourian H, Vincek V, Morales AR, Nadji M - BMC Clin Pathol (2008)

Scatter plot (logarithmic scale) of transcript copy number data for ER (A), HER2 (B), Cyclophilin A (PPIA) (C) and RN7SL1 (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267798&req=5

Figure 4: Scatter plot (logarithmic scale) of transcript copy number data for ER (A), HER2 (B), Cyclophilin A (PPIA) (C) and RN7SL1 (D).
Mentions: While the DNA yield was similar in formalin and UMFIX samples (Figure 3A), the RNA yield was significantly higher in the latter (p < 0.05 t-test, Figure 3B). Using the same amount of RNA template (2 μg), the cDNA yield was similar between two groups. Real-time PCR for HER2- and ER-RNA showed at least a ten fold difference in transcript copy number between UMFIX and formalin samples (Figure 4). Two control transcripts, PPIA and RN7sL had different dynamic ranges. PPIA was, on average, twofold higher in UMFIX samples. However, RN7SL1 copies were 50 times higher in UMFIX samples. These results indicate greater reverse-transcription efficiency and amplification in the UMFIX samples.

Bottom Line: Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05).The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative.Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida, USA. mnassiri@med.miami.edu

ABSTRACT

Background: The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer - estrogen receptor and HER2 - at the RNA and protein levels.

Methods: Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.

Results: The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.

Conclusion: The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

No MeSH data available.


Related in: MedlinePlus