Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples.

Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.

The story of poorly differentiated thyroid carcinoma: From Langhans' description to the Turin proposal via Juan Rosai.

Rosai, reinterpreting Langhans' "proliferating goiter," adopted the term "poorly differentiated carcinoma" for a specific thyroid tumor with insular features 30 years ago. This tumor type is only one of those approached by Dr. Rosai in the thyroid field (a PubMed search as of August 31, 2015 on "Rosai & thyroid" disclosed 73 articles), but seems the most innovative and representative of his heavy contribution to thyroid tumor classification. The diagnostic problems associated with PDTC recognition date back a long time, with a still ongoing debate on the nature of PDTC, its morphological diagnostic features, its clinical significance, and its optimal therapeutic approach. In 2004, PDTC was at last incorporated in the WHO classification of thyroid tumors, but the proposed diagnostic criteria were heterogeneous, controversial, and hardly applicable in the diagnostic practice. A consensus conference held in Turin in 2006 was lead by the authors and Dr. Rosai and confirmed the presence of geographical differences among claimed classical PDTC forms, which were responsible for a poor interobserver reproducibility of the diagnostic criteria. A diagnostic algorithm was therefore designed to define the crucial parameters to categorize PDTC and better stratify these distinctly aggressive tumors.

Towards a formalin-free hospital. Levels of 15-F2t-isoprostane and malondialdehyde to monitor exposure to formaldehyde in nurses from operating theatres.

Purpose: nurses are exposed to formaldehyde when managing surgical samples that are to be later transferred to histopathology. We evaluated the conditions favouring the risk of exposure to this toxic reagent and the effect of measures to prevent it. Methods: we conducted a cross-sectional study where 94 female workers were enrolled as being potentially exposed to formaldehyde. From each nurse were collected: (1) personal air-formaldehyde by a personal dosimeter (8 hours), (2) a standardized questionnaire, (3) a urine sample to test 15-F2t-isoprostane, malondialdehyde, cotinine. Results: the results indicate a marked difference related to the adoption of the under vacuum sealing procedure, as an alternative to formaldehyde for preserving tissues. Nurses using the under vacuum sealing system in the operating rooms are exposed to levels of formaldehyde 75% lower than those who do not use that system. Oxidative stress biomarkers (15-F2t-isoprostane, malondialdehyde) are significantly higher in nurses using formaldehyde (p < 0.001) and in the absence of the under vacuum sealing system (p = 0.027), in particular in those workers who use liquid formaldehyde in the operating theatre (p = 0.012). Conclusions: analysis of the biological biomarkers confirms a direct responsibility of air formaldehyde on the onset of oxidative stress while the use of the under vacuum sealing technique is associated with a significant reduction of the exposure to air-formaldehyde and redox status. Our findings can be useful to characterize the environmental health risk in operating theatres and to plan preventive measures such as the under vacuum sealing procedure.

A synonymous EGFR polymorphism predicting responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients.

Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild-type patients benefit from that treatment. In this study, we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11 % of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG + AA genotypes. Taken together, our findings could be used to better define CRC populations responding to anti-EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness.

Critical roles of specimen type and temperature before and during fixation in the detection of phosphoproteins in breast cancer tissues.

The most efficient approach for therapy selection to inhibit the deregulated kinases in cancer tissues is to measure their phosphorylation status prior to the treatment. The aim of our study was to evaluate the influence of pre-analytical parameters (cold ischemia time, temperature before and during tissue fixation, and sample type) on the levels of proteins and phosphoproteins in breast cancer tissues, focusing on the PI3 kinase/AKT pathway. The BALB-neuT mouse breast cancer model expressing HER2 and pAKT proteins and human biopsy and resection specimens were analyzed. By using quantitative reverse phase protein arrays (RPPA), 9 proteins and 16 phosphoproteins relevant to breast cancer biology were assessed. Cold temperatures before and during fixation resulted in a marked improvement in the preservation of the reactivity of biological markers (eg, ER, HER2) in general and, specifically, pHER2 and pAKT. Some phosphoproteins, eg, pHER2 and pAKT, were more sensitive to prolonged cold ischemia times than others (eg, pS6RP and pSTAT5). By comparing the phosphoprotein levels in core needle biopsies with those in resection specimens, we found a marked decrease in many phosphoproteins in the latter. Cold conditions can improve the preservation of proteins and phosphoproteins in breast cancer tissues. Biopsies ≤ 1 mm in size are the preferred sample type for assessing the activity of deregulated kinases for personalized cancer treatments because the phosphoprotein levels are better preserved compared with resection specimens. Each potential new (phospho)protein biomarker should be tested for its sensitivity to pre-analytical processing prior to the development of a diagnostic assay.

The pre-analytical phase in surgical pathology.

Several sequential passages are involved in the pre-analytical handling of surgical specimens from resection in the surgical theater to paraffin-embedding and storage. Each passage is highly critical and can significantly affect the preservation of morphology, antigens, and nucleic acids. Some key points in this process are still undefined and are subject to high variability among hospitals. High quality and standardization are demanded and pathologists should therefore work to comply with all novel clinical requests (such as genomic and antigenic testing for targeted molecular therapies). Under-vacuum sealing of surgical pieces can be a safe and reliable alternative to storage in large formalin-filled boxes; it prevents dehydration and favors cooling by removing air. Moreover, it implements tissue banking and preservation of nucleic acids. After transport of specimens to pathological anatomy laboratories, the next passage, fixation, has been the object of several attempt to find alternatives to formalin. However, none of the substitutes proved successful, and formalin fixation is still considered the gold standard for preservation of morphology and antigens. RNA has instead been found to be heavily affected by degradation and fragmentation in formalin-fixed tissues. Based on the hypothesis that RNA degradation would be inhibited by maintaining a low temperature, a protocol based on processing tissues with formalin at low temperature (cold fixation) was evaluated and proved useful in obtaining a reduction in RNA fragmentation. Finally, the problem of storage is discussed, in order to find ways to guarantee feasibility of molecular analyses even years after the original diagnosis.

[DRY PREPARATIONS OF ANATOMICAL LESIONS IN PATHOLOGICAL ANATOMY MUSEUMS]. / PREPARAZIONI ANATOMICHE A SECCO NELLE COLLEZIONI DEI MUSEI DI ANATOMIA PATOLOGICA.

Collection of pathological specimens began soon after the seminal description of autopsy by Giovan Battista Morgagni in Padoa in the second half of the 18th Century. Pathologists soon realized difficulties of preserving the form and to prevent decay caused by autolysis and attack by bacteria and parasites. The ancient procedures devoted to mummification were applied to the purpose, and a number of personal experiences were reported in the first half of the 19th century, mainly in Northern Italy and France, testifying a dedicated interest of the time in those areas. A combination of chemical fixation (with corrosive sublimate/mercuric chloride and/or tannic acid) and careful drying allowed to produce dry preparations, once very numerous in the Pathological Anatomy's Museums so much popular in the 19th and early 20th Century. In fact, it was the sole way to give visual evidence of disease and pathological processes. Only a limited number of these dry preparations are still present and visible in Pathology Museums, mainly in Universities of Northern Italy, while a few examples can be traced in the other European Country.

Presence of osteoinductive factors in bovine colostrum.

New approaches in the treatment of skeletal defects may benefit from the use of soluble biological factors. We previously standardized a derivative of bovine colostrum (SBCD), deprived of casein and fat and rich in cytokines. In the present study, we tested its possible use as an adjuvant in bone healing. SBCD contained factors involved in stromal cell stimulation and differentiation and induced cytokine production from stimulated mesenchymal stem cells (MSCs). In vitro, SBCD promoted proliferation, migration and, in association with osteogenic factors, osteogenic differentiation of osteoblastic and MSCs. In in vivo experiments of subcutaneous Matrigel injection in mice, SBCD plus hydroxyapatite, but not hydroxyapatite nor SBCD alone, induced recruitment of macrophages and stromal cells. After 60 days, plugs containing SBCD and hydroxyapatite were densely calcified and diffusely positive for osteocalcin, supporting the occurrence of an early osteogenic process. These results indicate that SBCD is a rich source of factors with osteoinductive properties.

Cell size as a prognostic factor in oncocytic poorly differentiated carcinomas of the thyroid.

Histological and cytological criteria in predicting clinical outcomes in patients with oncocytic poorly differentiated carcinoma (PDC) of the thyroid were investigated. In a set of 102 PDC patients, we performed a computer-assisted evaluation of cell size based on two different methods. Univariate analysis showed that cell size was a discriminant prognostic parameter in oncocytic PDC (30 cases) but not in the non-oncocytic carcinoma cases (72 cases). Patients with oncocytic PDC with small-medium cell size had a significantly increased risk of death (P = .029) and a decrease of disease-free survival (P = .014). This correlation was absent in cases of non-oncocytic PDC, where age and extensive vascular invasion were significant indicators of progression. The proposed morphological signature shows a robust discriminatory ability when tested on the oncocytic PDC group, and cell size assessment could thus be proposed as an inexpensive and readily evaluable parameter for predicting prognosis and planning therapy in this tumor type.

C and APUD cells and endocrine tumours. Pearse's laboratory in the years 1965-1969: a personal recollection.

This paper is a personal recollection of the studies, conducted in Prof. Pearse's laboratory in London in the years 1965-1969, which led to the discovery of production of calcitonin by parafollicular C cells and medullary carcinomas of the thyroid. The author's intention is to underline the combination of technical excellence, brilliant intuition, dedication and serendipity which led to a series of major discoveries and, historically, established the pivotal role to be played by immunohistochemistry in endocrine research and diagnosis. The formulation of Pearse's APUD cell theory gave a formal credence to the existence of common endocrine mechanisms, molecular markers and structural features in dispersed cells, all belonging to a diffuse endocrine system. This represented a major breakthrough which primed, in the following years, the studies on polypeptide hormone-producing cells and tumours, thus paving the way to the endocrine histology and pathology as we know, and practice them today.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue.

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

"To be or not to be in a good shape": diagnostic and clinical value of nuclear shape irregularities in thyroid and breast cancer.

Variation in both nuclear shape and size ("pleomorphism"), coupled with changes in chromatin amount and distribution, remains the basic criteria for microscopy in a cytologic diagnosis of cancer. The biological determinants of nuclear shape irregularities are not clarified, so, rather than on the genesis of nuclear irregularities, we here focus our attention on a descriptive analysis of nuclear pleomorphism. We keep in mind that evaluation of nuclear shape as currently practiced in routine preparations is improper because it is indirectly based on the distribution of DNA as revealed by the affinity for basic dyes. Therefore, over the last years we have been using as criteria morphological features of nuclei of thyroid and breast carcinomas as determined by immunofluorescence, in situ hybridization, and 3D reconstruction. We have translated this approach to routine diagnostic pathology on tissue sections by employing immunoperoxidase staining for emerin. Direct detection of nuclear envelope irregularities by tagging nuclear membrane proteins such as lamin B and emerin has resulted in a more objective definition of the shape of the nucleus. In this review we discuss in detail methodological issues as well as diagnostic and prognostic implications provided by decoration/staining of the nuclear envelope in both thyroid and breast cancer, thus demonstrating how much it matters "to be in the right shape" when dealing with pathological diagnosis of cancer.

Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis.

BACKGROUND: The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. RESULTS: A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. CONCLUSIONS: Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.

Search for neuro-endocrine markers (chromogranin A, synaptophysin and VGF) in breast cancers. An integrated approach using immunohistochemistry and gene expression profiling.

Discordant data are reported in the literature on the definition, incidence and clinical features of neuroendocrine (NE) carcinomas of the breast. This tumour entity is currently assessed by immunohistochemistry (IHC) detecting "general" NE markers such as chromogranin A (CHGA) and synaptophysin (SYP), but other markers have been considered as well. In the present study, in addition to CHGA and SYP, we investigated the expression of VGF, a neurotrophin-inducible gene, which is emerging as a new specific NE marker. In order to evaluate the differential expression of these neuro-endocrine markers in breast cancers, we conducted parallel immunohistochemical and gene expression analyses, using PCR, gene array and real-time quantitative PCR procedures. Data obtained in 28 cases were further validated with a meta-analysis of published datasets of 103 breast cancer cases. The value of IHC positivity (irrespective of the percentage of positive cells) was confirmed by over-expression of the related gene. However, the genetic approach emerged as more sensitive, showing over-expression of NE markers in a subset of IHC-negative carcinomas. In conclusion, the present study confirms, by a novel approach, the occurrence of NE differentiation in breast cancers. Over-expression of one or more NE marker (CHGA and/or SYP and/or VGF) characterizes a significant fraction (approximately 10 %) of infiltrative breast cancers.