Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations.

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.

Increased DNA damage in hematopoietic cells of mice with graft-versus-host disease.

Patients who received hematopoietic cell transplants have an increased risk for a new malignancy. In addition to genotoxic regimens such as radiotherapy and chemotherapy, graft-versus-host disease (GVHD) is a risk factor for development of new malignancies in long-term survivors. To understand mechanisms underlying this malignant transformation, we evaluated genomic damage in several murine models of GVHD by enumerating reticulocytes containing micronuclei (MN) in the blood after semi-allogeneic (parent-into-F1) hematopoietic cell transplantation. On day 40 after transplantation, MN frequencies were significantly increased in unirradiated (C57BL6 x DBA/2) F1 (BDF1) and (BALB/c x C57BL6) F1 (CBF1) mice that received cells from C57BL6 (B6) donors. MN frequencies were not significantly increased in F1 mice that received cells from DBA/2 or BALB/c donors. Serum levels of tumor necrosis factor-alpha (TNF-alpha) were higher after transplantation with B6 donors than with DBA/2 or BALB/c donors. The results indicate that GVHD, without irradiation, can induce genomic damage associated with inflammatory reactions manifested by increased TNF-alpha levels.

Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR.

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.

A novel RET rearrangement (ACBD5/RET) by pericentric inversion, inv(10)(p12.1;q11.2), in papillary thyroid cancer from an atomic bomb survivor exposed to high-dose radiation.

During analysis of RET/PTC rearrangements in papillary thyroid cancer (PTC) among atomic bomb survivors, a cDNA fragment of a novel type of RET rearrangement was identified in a PTC patient exposed to a high radiation dose using the improved 5' RACE method. This gene resulted from the fusion of the 3' portion of RET containing tyrosine kinase domain to the 5' portion of the acyl-coenzyme A binding domain containing 5 (ACBD5) gene, by pericentric inversion inv(10)(p12.1;q11.2); expression of the fusion gene was confirmed by RT-PCR. ACBD5 gene is ubiquitously expressed in various human normal tissues including thyroid. Full-length cDNA of the ACBD5-RET gene was constructed and then examined for tumorigenicity. Enhanced phosphorylation of ERK proteins in the MAPK pathway was observed in NIH3T3 cells transfected with expression vector encoding the full-length ACBD5/RET cDNA, while this was not observed in the cells transfected with empty expression vector. Stable NIH3T3 transfectants with ACBD5-RET cDNA induced tumor formation after their injection into nude mice. These findings suggest that the ACBD5-RET rearrangement is causatively involved in the development of PTC.

Improved method for analysis of RNA present in long-term preserved thyroid cancer tissue of atomic bomb survivors.

BACKGROUND: Since many thyroid cancer tissue samples from atomic bomb (A-bomb) survivors have been preserved for several decades as unbuffered formalin-fixed, paraffin-embedded specimens, molecular oncological analysis of such archival specimens is indispensable for clarifying the mechanisms of thyroid carcinogenesis in A-bomb survivors. Although RET gene rearrangements are the most important targets, it is a difficult task to examine all of the 13 known types of RET gene rearrangements with the use of the limited quantity of RNA that has been extracted from invaluable paraffin-embedded tissue specimens of A-bomb survivors. In this study, we established an improved 5' rapid amplification of cDNA ends (RACE) method using a small amount of RNA extracted from archival thyroid cancer tissue specimens. METHODS: Three archival thyroid cancer tissue specimens from three different patients were used as in-house controls to determine the conditions for an improved switching mechanism at 5' end of RNA transcript (SMART) RACE method; one tissue specimen with RET/PTC1 rearrangement and one with RET/PTC3 rearrangement were used as positive samples. One other specimen, used as a negative sample, revealed no detectable expression of the RET gene tyrosine kinase domain. RESULTS: We established a 5' RACE method using an amount of RNA as small as 10 ng extracted from long-term preserved, unbuffered formalin-fixed, paraffin-embedded thyroid cancer tissue by application of SMART technology. This improved SMART RACE method not only identified common RET gene rearrangements, but also isolated a clone containing a 93-bp insert of rare RTE/PTC8 in RNA extracted from formalin-fixed, paraffin-embedded thyroid cancer specimens from one A-bomb survivor who had been exposed to a high radiation dose. In addition, in the papillary thyroid cancer of another high-dose A-bomb survivor, this method detected one novel type of RET gene rearrangement whose partner gene is acyl coenzyme A binding domain 5, located on chromosome 10p. CONCLUSION: We conclude that our improved SMART RACE method is expected to prove useful in molecular analyses using archival formalin-fixed, paraffin-embedded tissue samples of limited quantity.

Expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells: analysis using a monoclonal antibody to CD43 that has a novel lineage specificity.

We have used HSCA-2, an mAb that recognizes a sialic acid-dependent epitope on the low molecular mass (approximately 115-kDa) glycoform of CD43 that is expressed in resting T and NK cells, to examine the expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells. Having previously reported that the memory cells that respond to recall Ags in a CD4+ CD45RO+ T cell population almost all belong to a subset whose surface CD43 expression levels are elevated, we now find that exposing these same memory T cells to HSCA-2 mAb markedly increases their proliferative responsiveness to recall Ags. We think it unlikely that this increase in responsiveness is a result of CD43-mediated monocyte activation, especially given that the HSCA-2 mAb differs from all previously used CD43 mAbs in having no obvious binding specificity for monocyte CD43. Predictably, treatment with HSCA-2 mAb did not lead to significant recall responses in CD4+ CD45RO+ T cells, whose CD43 expression levels were similar to or lower than those of naive cells. Other experiments indicated that the HSCA-2 mAb was capable of enhancing the proliferative responsiveness of CD4+ memory T cells that had been exposed to polyclonal stimulation by monocyte-bound CD3 mAb and could also act in synergy with CD28 mAb to enhance the responsiveness of CD4+ T cells to CD3 stimulation. Taken together, these findings suggest that the CD43 molecules expressed on CD4+ memory T cells may be capable of enhancing the costimulatory signaling and hence providing accessory functions to TCR-mediated activation processes.

Memory functions and death proneness in three CD4+CD45RO+ human T cell subsets.

We propose a classification of human CD4(+)CD45RO(+) memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-gamma and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4(+)CD45RO(+) T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.