Crossing the Andes: Challenges and opportunities for digital pathology in Latin America.

The most widely accepted and used type of digital pathology (DP) is whole-slide imaging (WSI). The USFDA granted two WSI system approvals for primary diagnosis, the first in 2017. In Latin America, DP has the potential to reshape healthcare by enhancing diagnostic capabilities through artificial intelligence (AI) and standardizing pathology reports. Yet, we must tackle regulatory hurdles, training, resource availability, and unique challenges to the region. Collectively addressing these hurdles can enable the region to harness DP's advantages-enhancing disease diagnosis, medical research, and healthcare accessibility for its population. Americas Health Foundation assembled a panel of Latin American pathologists who are experts in DP to assess the hurdles to implementing it into pathologists' workflows in the region and provide recommendations for overcoming them. Some key steps recommended include creating a Latin American Society of Digital Pathology to provide continuing education, developing AI models trained on the Latin American population, establishing national regulatory frameworks for protecting the data, and standardizing formats for DP images to ensure that pathologists can collaborate and validate specimens across the various DP platforms.

Cell block specimens applied to diagnostic routine of thyroid fine-needle aspiration biopsy / Aplicação do emblocado em parafina ou cell block na rotina diagnóstica de biópsia aspirativa por agulha fina de tireoide

ABSTRACT Introduction: Various preparations can be used in diagnostic cytology, including conventional smears (CS), liquid-based preparations (LBP) and cell block (CB). Objective: The aim of this study is to evaluate the quality of CB preparations in addition to conventional cytological specimens in cases of fine-needle aspiration biopsy (FNAB) of thyroid nodules in diagnostic routine. Method: One hundred and six consecutive cases of FNAB routine thyroid nodules were independently evaluated by two cytopathologists (Obs1 and Obs2) on the cellularity of SM, LBP and CB. Results: The cellularity was rich/moderate in 56 (52.8%) CBs for both observers. LBP showed rich/moderate cellularity in 86 (81.1%) cases for Obs1 and 91 (85.8%) for Obs2; among these cases, CB showed the same cellularity in 52/86 (60.4%) cases for Obs1 and 54/91 (59.3%) for Obs2. SM showed rich/moderate cellularity in 86 (81.1%) cases for Obs1 and 87 (82%) for Obs2; among these cases, CB showed the same cellularity in 48/86 (55.8%) cases for Obs1 and 54/87 (62%) for Obs2. CB cellularity was higher than that in LBP in only five cases for Obs1 and three for Obs2. LBP was assessed as low/absent in only five (4.7%) and six (5.6%) cases for Obs1 and Obs2, respectively. Conclusion: CB can be routinely used as additional specimen in material obtained from thyroid nodules FNAB, without adversely affecting LBP specimens, enabling the conduction of further immunohistochemical and molecular studies.

RESUMO Introdução: Vários preparados podem ser utilizados na citologia diagnóstica, como esfregaços (SM), preparados do tipo meio líquido (ML) e emblocado em parafina ou cell block (CB). Objetivo: Avaliar a qualidade do CB, além dos espécimes citológicos convencionais, em casos de biópsia aspirativa por agulha fina (BAAF) de nódulos de tireoide na rotina diagnóstica. Método: Cento e seis casos consecutivos de BAAF de nódulos de tireoide foram avaliados independentemente por dois citopatologistas (Obs1 e Obs2) quanto à celularidade dos preparados de SM, ML e CB. Resultados: A celularidade foi rica/moderada em 56 (52,8%) CB para ambos observadores. ML mostrou celularidade rica/moderada em 86 (81,1%) casos para o Obs1 e 91 (85,8%) para o Obs2; desses casos, CB mostrou a mesma celularidade em 52/86 (60,4%) casos para o Obs1 e 54/91 (59,3%) para o Obs2. SM mostrou celularidade rica/moderada em 86 (81,1%) casos para o Obs1 e 87 (82%) para o Obs2; desses casos, CB apresentou a mesma celularidade em 48/86 (55,8%) casos para o Obs1 e 54/87 (62%) para o Obs2. A celularidade do CB foi maior do que a do ML em apenas cinco casos para o Obs1 e três para o Obs2. ML foi avaliado como escasso/ausente em apenas cinco (4,7%) e seis (5,6%) casos para os Obs1 e Obs2, respectivamente. Conclusão: CB pode ser utilizado rotineiramente como espécime adicional no material obtido de BAAF de nódulos de tireoide, sem prejuízo dos espécimes de meio líquido, o que possibilita a realização de estudo complementar, principalmente imuno-histoquímico e molecular.

Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care?

OBJECTIVES: With the development of next-generation sequencing (NGS) technologies, DNA sequencing has been increasingly utilized in clinical practice. Our goal was to investigate the impact of genomic evaluation on treatment decisions for heavily pretreated patients with metastatic cancer. METHODS: We analyzed metastatic cancer patients from a single institution whose cancers had progressed after all available standard-of-care therapies and whose tumors underwent next-generation sequencing analysis. We determined the percentage of patients who received any therapy directed by the test, and its efficacy. RESULTS: From July 2013 to December 2015, 185 consecutive patients were tested using a commercially available next-generation sequencing-based test, and 157 patients were eligible. Sixty-six patients (42.0%) were female, and 91 (58.0%) were male. The mean age at diagnosis was 52.2 years, and the mean number of pre-test lines of systemic treatment was 2.7. One hundred and seventy-seven patients (95.6%) had at least one identified gene alteration. Twenty-four patients (15.2%) underwent systemic treatment directed by the test result. Of these, one patient had a complete response, four (16.7%) had partial responses, two (8.3%) had stable disease, and 17 (70.8%) had disease progression as the best result. The median progression-free survival time with matched therapy was 1.6 months, and the median overall survival was 10 months. CONCLUSION: We identified a high prevalence of gene alterations using an next-generation sequencing test. Although some benefit was associated with the matched therapy, most of the patients had disease progression as the best response, indicating the limited biological potential and unclear clinical relevance of this practice.

Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care?

OBJECTIVES: With the development of next-generation sequencing (NGS) technologies, DNA sequencing has been increasingly utilized in clinical practice. Our goal was to investigate the impact of genomic evaluation on treatment decisions for heavily pretreated patients with metastatic cancer. METHODS: We analyzed metastatic cancer patients from a single institution whose cancers had progressed after all available standard-of-care therapies and whose tumors underwent next-generation sequencing analysis. We determined the percentage of patients who received any therapy directed by the test, and its efficacy. RESULTS: From July 2013 to December 2015, 185 consecutive patients were tested using a commercially available next-generation sequencing-based test, and 157 patients were eligible. Sixty-six patients (42.0%) were female, and 91 (58.0%) were male. The mean age at diagnosis was 52.2 years, and the mean number of pre-test lines of systemic treatment was 2.7. One hundred and seventy-seven patients (95.6%) had at least one identified gene alteration. Twenty-four patients (15.2%) underwent systemic treatment directed by the test result. Of these, one patient had a complete response, four (16.7%) had partial responses, two (8.3%) had stable disease, and 17 (70.8%) had disease progression as the best result. The median progression-free survival time with matched therapy was 1.6 months, and the median overall survival was 10 months. CONCLUSION: We identified a high prevalence of gene alterations using an next-generation sequencing test. Although some benefit was associated with the matched therapy, most of the patients had disease progression as the best response, indicating the limited biological potential and unclear clinical relevance of this practice.

Unraveling molecular pathways of poorly differentiated neuroendocrine carcinomas of the gastroenteropancreatic system: A systematic review.

BACKGROUND: Poorly differentiated neuroendocrine carcinomas (NECs) are rare and aggressive tumors. Their molecular pathogenesis is still largely unknown, and consequently, the best therapeutic management also remains to be determined. We conducted a systematic review on molecular alterations found in gastroenteropancreatic NECs (GEP-NECs) and discuss potential applications of targeted therapies in setting. MATERIALS AND METHODS: Systematic review of studies about molecular features in tumor tissues of patients with GEP-NECs. The Medline, Lilacs, Embase, Cochrane, Scopus and Opengrey databases were sought, without time, study design or language restrictions. RESULTS: Of the 1.564 studies retrieved, 41 were eligible: 33 were retrospective studies and eight were case reports. The studies spanned the years 1997-2017 and involved mostly colorectal, stomach and pancreas primary tumors. Molecular alterations in the TP53 gene and the p53 protein expression were the most commonly observed, regardless of the primary site. Other consistently found molecular alterations were microsatellite instability (MSI) in approximately 10% of gastric and colorectal NEC, and altered signaling cascades of p16/Rb/cyclin D1, Hedgehog and Notch pathways, and somatic mutations in KRAS, BRAF, RB1 and Bcl2. In studies of mixed adeno-neuroendocrine carcinomas (MANECs) the molecular features of GEP-NEC largely resemble their carcinoma/adenocarcinomas tumor counterparts. CONCLUSIONS: Despite the paucity of data about the molecular drivers associated with GEP-NEC, some alterations may be potentially targeted with new cancer-directed therapies. Collaborative clinical trials for patients with advanced GEP-NEC are urgently needed.

HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity.

Trastuzumab-containing therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17 (CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity.

Experts' opinion: Recommendations for retesting breast cancer metastases for HER2 and hormone receptor status.

The human epidermal growth factor receptor 2 (HER2) and hormone receptor status of recurrent breast cancer may change between the tumor and metastases from negative to positive and vice versa, potentially affecting the treatment regimen. Retesting of metastases may therefore be crucial to allow appropriate selection of patients for whom targeted therapy is indicated; however, retesting is not routinely performed. This article recommends that metastases be retested for HER2 and hormone receptor status and provides practical guidance on when and how to retest, as agreed by a panel of expert pathologists with extensive experience of HER2 and hormone receptor testing.

Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells.

BACKGROUND: Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear. METHODS: High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry. RESULTS: Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases. CONCLUSIONS: The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.

APC +/- alters colonic fibroblast proteome in FAP.

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.

ErbB family immunohistochemical expression in colorectal cancer patients with higher risk of recurrence after radical surgery.

PURPOSE: Investigate ErbB family expression in colorectal cancer patients with higher risk of recurrence after surgical treatment. METHODS: We studied 109 individuals with high risk stage II and stage III patients submitted to radical surgery. ErbB expression was assessed by tissue microarray technique. RESULTS: The immunohistochemical expression was considered positive for EGFR, ErbB2, ErbB3, ErbB4 membrane, and ErbB4 cytoplasmic in respectively 57.8%, 8.3%, 69.7%, 11%, and 19.3% of patients. ErbB3 negative expression was associated with lymphovascular invasion. EGFR, ErbB2, and cytoplasmic ErbB4 expression was not associated with prognosis. Membranous positive ErbB4 expression was an independent prognostic factor for recurrence. ErbB3 negative expression was an independent prognostic factor for recurrence and survival in the multivariate analysis. CONCLUSIONS: The immunohistochemical expression of ErbB3 and ErbB4 may identify a subgroup with stage II and III colorectal cancer at higher risk of recurrence.

One-hit effects in cancer: altered proteome of morphologically normal colon crypts in familial adenomatous polyposis.

We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

Absence of TMPRSS2:ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.

TMPRSS2:ERG gene fusions and PTEN deletions are the most common genomic aberrations in prostate cancer. Recent work has suggested that the TMPRSS2:ERG fusion is associated with a more aggressive phenotype. Similarly, PTEN deletion has been associated with biochemical recurrence and lymph node metastasis. To date, there has been no systematic analysis of the combined influence of genomic PTEN deletion with TMPRSS2:ERG gene fusions on clinical parameters of prostate cancer progression. We carried out a retrospective analysis of 125 prostate cancers with known clinical outcome using interphase fluorescence in situ hybridization to detect the relative prevalence of TMPRSS2:ERG rearrangements and/or PTEN genomic deletions. TMPRSS2:ERG rearrangement was found in 60 of 125 (48%) prostate cancers. Duplication of TMPRSS2:ERG fusion was observed in seven (6%) tumors. Gleason grade (P=0.0002)/score (P=0.001), median tumor volume (P=0.0024), preoperative PSA (P=0.001) and perineural invasion (P=0.0304) were significantly associated with biochemical recurrence by univariate analysis with TMPRSS2:ERG approaching significance (P=0.0523). By multivariate analysis, relevant factors associated with recurrence were Gleason scores 7 (P=0.001) and 8-10 (P=0.015), PTEN homozygous deletion (P=0.013) and concurrent TMPRSS2:ERG fusion and PTEN deletion (P=0.036). Kaplan-Meier analysis indicated that the presence of TMPRSS2:ERG fusion was marginally less favorable in comparison to no fusion. Duplication of fusion gene showed worse prognosis. It was possible to determine the relative frequencies of PTEN deletion and/or TMPRSS2:ERG fusions in 82 of 125 prostate cancers. With biochemical recurrence as an endpoint, the genomic biomarkers identified three patient groups: (1) 'poor genomic grade' characterized by both PTEN deletion and TMPRSS2:ERG fusions (23/82, 28%); (2) 'intermediate genomic grade' with either PTEN deletion or TMPRSS2:ERG fusion (35/82, 43%) and (3) 'favorable genomic grade' in which neither rearrangement was present (24/82, 29%). Kaplan-Meier and multivariate analysis indicate that TMPRSS2:ERG fusion and PTEN loss together are a predictor of earlier biochemical recurrence of disease.

5-aminosalicylic acid inhibits colitis-associated colorectal dysplasias in the mouse model of azoxymethane/dextran sulfate sodium-induced colitis.

BACKGROUND: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. METHODS: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. RESULTS: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7, respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. CONCLUSIONS: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.

Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium.

Loss of p53 function is an early event in colitis-associated neoplasia in humans. We assessed the role of p53 in a mouse model of colitis-associated neoplasia. Colitis was induced in p53-/-, p53+/- and p53+/+ mice using three or four cycles of dextran sulfate sodium (DSS) followed by 120 days of water. Mice were examined for incidence, multiplicity and types of neoplastic lesions. Lesions were examined for mutations in beta-catenin (exon 3), K-ras (codons 12/13) and p53 (exons 5-8) by sequencing and for cellular localization of beta-catenin by immunohistochemistry. The incidence of neoplastic lesions was 57, 20 and 20% in p53-/-, p53+/- and p53+/+ mice, respectively (P = 0.013). p53-/- mice had a greater number of total lesions (P < 0.0001), cancers (P = 0.001) and dysplasias (P = 0.009) per mouse than either p53+/- or p53+/+ mice. Flat lesions were associated with the p53-/- genotype, whereas polypoid lesions were associated with the p53+/- and p53+/+ genotypes (P < 0.0001). beta-Catenin mutations were present in 75% of lesions of p53+/+ mice and absent in lesions from p53-/- mice (P = 0.055). Nuclear expression of beta-catenin was seen only in polypoid lesions (91%). No K-ras or p53 mutations were detected. These data indicate that loss of p53 enhances the induction of colitis-associated neoplasia, particularly flat lesions, and dysregulation of beta-catenin signaling plays an important role in the formation of polypoid lesions in this mouse model. As observed in humans, p53 plays a protective role in colitis-associated neoplasia in the DSS model.

Successful application of microarray technology to microdissected formalin-fixed, paraffin-embedded tissue.

The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson's correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported.