Bereaved relatives often refuse to give consent for post-mortem investigation of deceased cancer patients, mainly because of the mutilation due to conventional autopsy (CA). Minimally invasive ...autopsy (MIA) may be a more acceptable alternative and, if implemented in clinical practice, creates an opportunity to more often obtain post-mortem tissue samples of (recurred) primary tumors and metastases for molecular research. As a measure for tissue quality for molecular studies, we hereby present a feasibility study, comparing the RNA quality of MIA and CA samples, and fresh frozen samples as reference.
Tissue samples of heart, liver and kidney were prospectively collected from 24 MIAs followed by CA, and compared to corresponding archival fresh frozen tissue. After RNA isolation and RT-qPCR, RNA integrity numbers (RIN) and GAPDH expression (six amplicon sizes ranging from 71 to 530 base pairs) were measured. RIN values and GAPDH Cq values were analyzed and compared between all sample groups and post-mortem intervals (PMI).
RIN values in MIA samples were significantly higher than those in CA samples. GAPDH was expressed significantly higher in MIA samples than in CA samples and 530 bp PCR products could be measured in all cases. GAPDH expression was significantly lower in samples with PMI >15 hours. As expected, the samples of the fresh frozen reference standard performed best in all analyses.
MIA samples showed better RNA quality than CA samples, probably due to shorter PMI. Both had lower RNA quality and expression levels than fresh frozen tissue, however, remaining GAPDH RNA was still sufficiently intact. Therefore, other highly expressed genes are most likely also detectable. Gene array analysis should be performed to gain insight into the quality of entire post-mortem genomes. Reducing PMI will further improve the feasibility of demanding molecular research on post-mortem tissues, this is most likely more feasible with MIA than CA.
Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological ...analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.
Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the ...molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.
About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity ...Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.
The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to ...standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (
k
w
) = 0.73 (95 % confidence interval (CI), 0.41–0.94)), with a higher agreement between the reference evaluation and the PFPE samples (
k
w
= 0.86 (95 % CI, 0.67–1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (
k
w
= 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.
Proteasome inhibitors are effective in Waldenström's macroglobulinemia (WM) but require parenteral administration and are associated with polyneuropathy. We investigated efficacy and toxicity of the ...less neurotoxic oral proteasome inhibitor ixazomib combined with rituximab, in patients with relapsed WM.
We conducted a multicenter phase I/II trial with ixazomib, rituximab, and dexamethasone (IRD). Induction consisted of eight cycles IRD wherein rituximab was started in cycle 3, followed by rituximab maintenance. Phase I showed feasibility of 4 mg ixazomib. Primary end point for phase II was overall response rate (ORR ≥ minimal response) after induction.
A total of 59 patients were enrolled (median age, 69 years; range, 46-91 years). Median number of prior treatments was 2 (range, 1-7); 70% had an intermediate or high WM-IPSS (International Prognostic Scoring System for WM) score. After eight cycles, ORR was 71% (42 out of 59) (14% very good partial response PR, 37% PR, and 20% minor response). Depth of response improved until month 12 (best ORR 85% 50 out of 59: 15% very good PR, 46% PR, and 24% minor response). Median duration of response was 36 months. The average hematocrit level increased significantly (0.33-0.38 L/L) after induction (
< .001). After two cycles of ixazomib and dexamethasone, immunoglobulin M levels decreased significantly (median 3,700-2,700 mg/dL,
< .0001). Median time to first response was 4 months. Median progression-free survival and overall survival were not reached. After median follow-up of 24 months (range, 7.4-54.3 months), progression-free survival and overall survival were 56% and 88%, respectively. Toxicity included mostly grade 2 or 3 cytopenias, grade 1 or 2 neurotoxicity, and grade 2 or 3 infections. No infusion-related reactions or immunoglobulin M flare occurred with use of subcutaneous rituximab. Quality of life improved significantly after induction. In total, 48 patients (81%) completed at least six cycles of IRD.
Combination of IRD shows promising efficacy with manageable toxicity in patients with relapsed or refractory WM.
Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles and complicate molecular diagnostics. To investigate this effect, we ...characterized such pre-analytical effects in 143 non-malignant liver samples obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts treated either with the Pringle manoeuvre or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-PCR. We found 179 mutually deregulated genes which point to elevated cytokine signaling with NFκB as a dominant pathway in ischemia responses. In contrast to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFκB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were present in non-tumor tissue. The reported inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a set of 230 pre-analytically highly robust genes identified from histologically normal livers (<2% covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications.
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•Inter-patient variability represents key pre-analytical difference in responses to stress.•IRI induces core genes independent of the surgical technique.•TNF, MAPK and NFκB are dominant pathways in IRI in humans.•Down-regulation of FOS/JUN may hinder AP-1 activation in steatotic livers in IRI.•230 genes unaffected by IRI encourage further exploration in research or diagnostics.
The quality of human tissue specimens can have a significant impact on analytical data sets for biomarker research. The aim of this study was to characterize fluctuations of protein and ...phosphoprotein levels in human tissue samples during the preanalytical phase. Eleven intestine and 17 liver specimens were surgically resected, aliquoted, and either snap-frozen or fixed in formalin immediately or exposed to different ischemic conditions before preservation. Protein levels in the resultant samples were investigated by reverse phase protein array, Western blot analysis, and liquid chromatography–tandem mass spectrometry. Our data revealed that the degree of sensitivity of proteins and phosphoproteins to delayed preservation varied between different patients and tissue types. For example, up-regulation of phospho-p42/44 MAPK in intestine samples was seen in some patients but not in others. General trends toward up- or down-regulation of most proteins were not evident due to pronounced interpatient variability but signal intensities of only a few proteins, such as cytokeratin 18, were altered from baseline in postresection samples. In contrast, glyceraldehyde 3-phosphate dehydrogenase was found to be stable during periods of cold ischemia. Our study represents a proper approach for studying potential protein fluctuations in tissue specimens for future biomarker development programs.
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