Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis.

Chromogenic immunohistochemistry (immunostaining using an enzyme-labeled probe) is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis in routine pathology services. In neoplastic lesions, immunohistochemistry allows the study of specific clinical and biological features such as histogenesis, behavioral characteristics, therapeutic targets, and prognostic biomarkers. The needs for appropriate and reproducible methods of immunostaining are prompted by technical development and refinement, commercial availability of a variety of antibodies, advanced applicability of immunohistochemical markers, accelerated analysis of clinicopathological correlations, progress in molecular targeted therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. Pathologists should learn from previous mistakes and failures and from results indicating false positivity and false negativity. The present review article describes various devices, technical hints, and trouble-shooting guides to keep in mind when performing immunostaining.

Homologous recombination deficiency real-time clinical assays, ready or not?

Cancers with deficiencies in homologous recombination-mediated DNA repair (HRR) demonstrate improved clinical outcomes and increased survival. Approximately 50% of high-grade serous ovarian cancers (HGSOC) exhibit homologous recombination deficiency (HRD). HRD can be caused by germline or somatic mutations of genes involved in the HR pathway. Given platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPis) are used in HGSOC, double-strand breaks (DSBs) are common. Unrepaired DSBs are toxic to cells as genomic instability ensues and cells eventually die. Thus, tumor cells with DSBs utilize the high-fidelity HRR as one of the central pathways for repair. In tumors that have HRD, an alternate pathway such as non-homologous end-joining (NHEJ) is used and leads to error-prone repair. To date, methods for clinical detection of homologous recombination deficiency (HRD) are limited to genomic changes of HRR genes and genomic mutation patterns resulting from HRD genes involved in HR-mediated DNA repair. However, these tests detect genomic scars that might not always correlate well with PARP inhibitor or platinum sensitivity in the current state. Therefore, a functional HRD assay should be able to more accurately predict tumor response in real-time. RAD51 foci formation has been used as a functional assay to define HRD and closely correlates with chemotherapy and PARPi sensitivity. The inability to form RAD51 foci is a common feature of HRD. DNA damage can also cause transient slowing or stalling of replication forks defined as replication stress. Replication fork stalling can lead to fork degradation and decreased cell viability if forks do not resume DNA synthesis. Fork degradation has been found to lead to chemosensitivity in BRCA-deficient tumors. To determine this fork degradation phenotype, replication fork/DNA fiber assays are utilized. This review will highlight functional assays for HRD in the context of translating these to real-time clinical assays.

Advances in quantitative immunohistochemistry and their contribution to breast cancer.

Introduction: Automated image analysis provides an objective, quantitative, and reproducible method of measurement of biomarkers. Image quantification is particularly well suited for the analysis of tissue microarrays which has played a major pivotal role in the rapid assessment of molecular biomarkers. Data acquired from grinding up bulk tissue samples miss spatial information regarding cellular localization; therefore, methods that allow for spatial cell phenotyping at high resolution have proven to be valuable in many biomarker discovery assays. Here, we focus our attention on breast cancer as an example of a tumor type that has benefited from quantitative biomarker studies using tissue microarray format.Areas covered: The history of immunofluorescence and immunohistochemistry and the current status of these techniques, including multiplexing technologies (spectral and non-spectral) and image analysis software will be addressed. Finally, we will turn our attention to studies that have provided proof-of-principle evidence that have been impacted from the use of these techniques.Expert opinion: Assessment of prognostic and predictive biomarkers on tissue sections and TMA using Quantitative immunohistochemistry is an important advancement in the investigation of biologic markers. The challenges in standardization of quantitative technologies for accurate assessment are required for adoption into routine clinical practice.

"Hey! Whatever happened to hemangiopericytoma and fibrosarcoma?" An update on selected conceptual advances in soft tissue pathology which have occurred over the past 50 years.

Hemangiopericytoma and fibrosarcoma represented at one time two of the most common diagnoses in soft tissue pathology. Both terms are now largely extinct. This article will review the clinicopathologic, immunohistochemical and molecular genetic advances that have led to these changes, and review the pathologic features of a select group of soft tissue tumors previously classified as hemangiopericytoma or fibrosarcoma.

Reporting Practices and Resource Utilization in the Era of Intraductal Carcinoma of the Prostate: A Survey of Genitourinary Subspecialists.

Intraductal carcinoma of the prostate (IDC-P) has been recently recognized by the World Health Organization classification of prostatic tumors as a distinct entity, most often occurring concurrently with invasive prostatic adenocarcinoma (PCa). Whether documented admixed with PCa or in its rare pure form, numerous studies associate this entity with clinical aggressiveness. Despite increasing clinical experience and requirement of IDC-P documentation in protocols for synoptic reporting, the specifics of its potential contribution to assessment of grade group (GG) and cancer quantitation of PCa in both needle biopsies (NBx) and radical prostatectomy (RP) specimens remain unclear. Moreover, there are no standard guidelines for incorporating basal cell marker immunohistochemistry (IHC) in the diagnosis of IDC-P, either alone or as part of a cocktail with AMACR/racemase. An online survey containing 26 questions regarding diagnosis, reporting practices, and IHC resource utilization, focusing on IDC-P, was undertaken by 42 genitourinary subspecialists from 9 countries. The degree of agreement or disagreement regarding approaches to individual questions was classified as significant majority (>75%), majority (51% to 75%), minority (26% to 50%) and significant minority (≤25%). IDC-P with or without invasive cancer is considered a contraindication for active surveillance by the significant majority (95%) of respondents, although a majority (66%) also agreed that the clinical significance/behavior of IDC-P on NBx or RP with PCa required further study. The majority do not upgrade PCa based on comedonecrosis seen only in the intraductal component in NBx (62%) or RP (69%) specimens. Similarly, recognizable IDC-P with GG1 PCa was not a factor in upgrading in NBx (78%) or RP (71%) specimens. The majority (60%) of respondents include readily recognizable IDC-P in assessment of linear extent of PCa at NBx. A significant majority (78%) would use IHC to confirm or exclude intraductal carcinoma if other biopsies showed no PCa, while 60% would use it to confirm IDC-P with invasive PCa in NBx if it would change the overall GG assignment. Nearly half (48%, a minority) would use IHC to confirm IDC-P for accurate Gleason pattern 4 quantitation. A majority (57%) report the percentage of IDC-P when present, in RP specimens. When obvious Gleason pattern 4 or 5 PCa is present in RP or NBx, IHC is rarely to almost never used to confirm the presence of IDC-P by the significant majority (88% and 90%, respectively). Most genitourinary pathologists consider IDC-P to be an adverse prognostic feature independent of the PCa grade, although recommendations for standardization are needed to guide reporting of IDC-P vis a vis tumor quantitation and final GG assessment. The use of IHC varies widely and is performed for a multitude of indications, although it is used most frequently in scenarios where confirmation of IDC-P would impact the GG assigned. Further study and best practices recommendations are needed to provide guidance with regards to the most appropriate indications for IHC use in scenarios regarding IDC-P.

Companion diagnostic tests for treatment of lung cancer patients: what are the current and future challenges?

INTRODUCTION: Companion diagnostic tests (CDXs) are considered mandatory for decision-making for treatment with targeted therapies in thoracic oncology. The emergence of immunotherapy has also given rise to the development of CDXs. Some CDXs, in particular PD-L1 immunohistochemistry tests, have been questioned and re-examined for use with new combination therapies that are being evaluated in clinical trials. Current questions include: Can we establish therapeutic indications in thoracis oncology without CDXs? Would the addition of new tests benefit patient outcome? Areas covered: This review covers the use of CDXs for decision-making in the treatment of lung cancer but also covers the limits of certain tests. It discusses the major challenges for present and future development of CDXs in daily practice. Expert opinion: CDXs can predict the efficacy of drugs if crucial steps in development and validation are fully controlled. Future development of CDXs must consider the detection of biomarkers of resistance and toxicity that are complementary to CDXs predicting therapeutic drug efficacy. Certain CDXs that have already been developed may be of interest for new indications in the field of thoracic oncology.

Immunohisto(cyto)chemistry: an old time classic tool driving modern oncological therapies.

In the era of precision medicine immunohistochemistry (IHC) and immunocytochemistry (ICC) share some of the highlights in personalized treatment. Survival data obtained from clinical trials shape the cut-offs and IHC scoring that serve as recommendations for patient selection both for targeted and conventional therapies. Assessment of Estrogen and Progesterone Receptors along with HER2 status has been among the first approved immunostaining assays revolutionizing breast cancer treatment. Similarly, ALK positivity predicts the efficacy of ALK inhibitors in patients with non-small cell lung cancer (NSCLC). In recent years, Programmed Death Ligand 1 (PD-L1) IHC assays have been approved as companion or complimentary diagnostic tools predicting the response to checkpoint inhibitors. Anti-PD-L1 and anti-PD-1 monoclonal antibodies have inaugurated a new period in the treatment of advanced cancers, but the path to approval of these biomarkers is filled with immunohistochemical challenges. The latter brings to the fore the significance of molecular pathology as a hub between basic and clinical research. Besides, novel markers are translated into routine practice, suggesting that we are at the beginning of a new exciting period. Unraveling the molecular mechanisms involved in cellular homeostasis unfolds biomarkers with greater specificity and sensitivity. The introduction of GL13 (SenTraGor®) for the detection of senescent cells in archival material, the implementation of key players of stress response pathways and the development of compounds detecting common mutant P53 isoforms in dictating oncological treatments are paradigms for precision oncology.

Advances in Molecular Testing Techniques in Cytologic Specimens.

There has been a paradigm shift in the practice of cytopathology with the advent of highly sensitive molecular tests using small amounts of tissue that can provide diagnostic, prognostic, and predictive information for clinical management. The cytopathologist plays a key role in providing a timely and accurate diagnosis as well as ensuring appropriate processing and handling of the specimen and judicious triaging of the tissue for molecular testing that guide therapeutic decisions. As the era of "precision medicine" continues to evolve and expand, cytopathology remains a dynamic field with advances in the practice of molecular cytopathology providing new paradigms in clinical care.

Estudio de la evolución en la actividad en un Departamento de Anatomía Patológica de un hospital de tercer nivel en la última década (2007-2016) / Study of the evolution in the activity of a Department of Pathology from a third level hospital in the last decade (2007-2016)

Objetivo: Estudiar la evolución de variables de interés de un Departamento de Anatomía Patológica de un hospital de tercer nivel durante la última década. Valorar el impacto del traslado hospitalario en la actividad (año 2014). Material y método: Estudio retrospectivo observacional en el que se analizaron las muestras registradas (biopsias, citologías, PAAF, autopsias, intraoperatorias) así como las técnicas complementarias (IHQ, histoquímica, IF y FISH) y la cartera de servicios, durante la década 2007-2016. Para el análisis estadístico se compararon los quinquenios 2007-2011 y 2012-2016. Resultados: Las siguientes variables han sido estadísticamente significativas: citologías (34055,8±1994,0 vs. 26590,4±2938,3; p=0,002), autopsias (156,2±27,3 vs. 122,0±14,78; p=0,039), inmunohistoquímica (17855,4±3424,2 vs. 28559,2±4734,7; p=0,003), histoquímica (11117,8±2300,9 vs. 6225,0±1330,5; p=0,003) e inmunofluorescencia (610,2±185,3 vs. 1205,4±154,0; p=0,001). Se han identificado correlaciones estadísticas de interés entre las variables. En el año 2014 se observó que las variables de mayor peso específico (biopsias, citologías, IHQ e histoquímica) en la carga asistencial del Departamento mostraron un descenso medio del 12,5%. Se ha identificado un incremento generalizado en el catálogo de muestras disponibles. El número de anticuerpos (78,7%), la histoquímica (38,7%) y FISH (400%) fueron los que mayor aumento mostraron. Conclusión: Se han identificado variaciones relevantes del volumen de actividad, así como en la cartera de servicios, especialmente en las técnicas orientadas a mejorar la precisión diagnóstica (IHQ e IF); y un descenso importante en el número de citologías, autopsias e histoquímica. En el año 2014 se ha observado un descenso de más del 12% en las principales variables del estudio

Objective: To study the evolution of variables of interest in a department of pathology from a third level hospital during the last decade and to evaluate the impact on these of the hospital relocation in 2014. Material and method: Retrospective observational study in which the recorded samples (biopsies, cytology specimens, FNA, autopsies, intraoperative) as well as the complementary techniques (IHC, Histochemistry, IF and FISH) and portfolio of services were analyzed during the years 2007-2016 inclusive. For the statistical analysis, the five-year periods 2007-2011 and 2012-2016 were compared. Results: The following variables were statistically significant: cytology (34055.8±1994.0 vs 26590.4±2938.3, p=0.002), autopsies (156.2±27.3 vs 122.0±14.78, p=0.039), immunohistochemistry (17855.4±3424.2 vs 28559.2±4734.7, p=0.003), histochemistry (11117.8±2300.9 vs 6225.0±1330.5, p=0.003) and immunofluorescence (610.2±185.3 vs. 1205.4±154.0, p=0.001). Statistical correlations of interest among variables have been identified. In 2014, it was observed that the variables of greater specific weight (biopsies, cytology, IHQ and histochemistry) in the work load of the Department showed an average decrease of 12.5%. A generalized increase in the panel of available samples has been identified, the largest increase being seen in the number of antibodies (78.7%), histochemistry (38.7%) and FISH (400%). Conclusion: Relevant variations in work volume, as well as the service portfolio, have been identified, especially in the techniques aimed at improving diagnostic accuracy (IHQ and FI), and a significant decrease in the number of cytology specimens, autopsies and histochemistry. In the year 2014 a decrease of more than 12% in the main variables of the study was observed

Tumor biomarker testing in non-small-cell lung cancer: A decade of change.

INTRODUCTION: Although a growing list of essential genomic/immune-based biomarkers are linked to approved non-small-cell lung cancer (NSCLC) therapies worldwide, few reports have detailed the evolution of NSCLC predictive biomarker assessment in routine clinical practice. METHODS: We retrospectively reviewed the first one thousand plus NSCLC patient specimens from our institution analyzed for predictive biomarkers from 2004 to 2017 and evaluated patterns of testing as well as correlation with clinical-pathologic characteristics. RESULTS: The majority of 1009 NSCLC patients had advanced stages of adenocarcinoma with most tissues obtained from the lung, mediastinal/hilar nodes, or pleura. The majority of testing was performed on cytology or small biopsy specimens. All were tested for EGFR mutations, 895 for ALK rearrangement, 841 for KRAS mutation, 537 for ROS1 rearrangement, and 179 using comprehensive genomic profiling. Implementation of near-universal genomic biomarker testing at our institution for EGFR, ALK, ROS1 and PD-L1 all occurred within the first year following evidence of clinical activity or regulatory body approval of an associated inhibitor. The overall testing failure rate after use of the best specimen for the most common tests was ≤5.5%. A quarter of tumors had a driver oncogene identified (EGFR/ALK/ROS1/BRAF V600E) with an approved oral targeted therapy, with the highest prevalence in those patients with no or light (≤15 pack-years) history of tobacco use. CONCLUSIONS: Tumor biomarker testing using clinical NSCLC specimens in routine oncologic care evolves rapidly following approval of targeted therapies linked to diagnostic assays. Our practice's decade plus experience highlights the rapid evolution of biomarker testing and confirms the therapeutic relevance of such testing in all patients-particularly those patients with light/no history of tobacco use.

Histologic Mimics of Basal Cell Carcinoma.

CONTEXT: - Basal cell carcinoma (BCC) is the most common human malignant neoplasm and is a frequently encountered diagnosis in dermatopathology. Although BCC may be locally destructive, it rarely metastasizes. Many diagnostic entities display morphologic and immunophenotypic overlap with BCC, including nonneoplastic processes, such as follicular induction over dermatofibroma; benign follicular tumors, such as trichoblastoma, trichoepithelioma, or basaloid follicular hamartoma; and malignant tumors, such as sebaceous carcinoma or Merkel cell carcinoma. Thus, misdiagnosis has significant potential to result in overtreatment or undertreatment. OBJECTIVE: - To review key features distinguishing BCC from histologic mimics, including current evidence regarding immunohistochemical markers useful for that distinction. DATA SOURCES: - Review of pertinent literature on BCC immunohistochemistry and differential diagnosis. CONCLUSIONS: - In most cases, BCC can be reliably diagnosed by histopathologic features. Immunohistochemistry may provide useful ancillary data in certain cases. Awareness of potential mimics is critical to avoid misdiagnosis and resulting inappropriate management.

Primary Cutaneous Acral CD8+ T-Cell Lymphoma.

Primary cutaneous acral CD8+ T-cell lymphoma is a new provisional entity in the 2016 revision of the World Health Organization classification of lymphoid neoplasms. This is a challenging diagnosis because of its rarity, as well as its morphologic and immunophenotypic overlap with other CD8+ cytotoxic lymphoid proliferations. Appropriate classification of this entity is crucial because of its indolent clinical behavior compared with other CD8+ T-cell lymphomas. Knowledge of the clinical setting, sites of involvement, and morphologic features can aid in correct diagnosis. Here, we review the clinical and pathologic features of primary cutaneous acral CD8+ T-cell lymphoma with an emphasis on the differential diagnosis among other C8+ T-cell lymphomas.

Mixed-Phenotype Acute Leukemia: Diagnostic Criteria and Pitfalls.

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous category in the World Health Organization classification that comprises acute leukemias with discrete admixed populations of myeloid and lymphoid blasts ("bilineal") or with extensive coexpression of lymphoid and myeloid markers in a single blast population ("biphenotypic"). Flow cytometric findings suggestive of MPAL are often met with consternation by pathologists and oncologists alike, owing to unfamiliarity with the disease and uncertainty about how MPAL fits into established paradigms for treatment of acute leukemia. The purpose of this review is to explain the diagnostic criteria for MPAL, summarize its biological and clinical features, and address common diagnostic pitfalls of these unusual leukemias.

Colorectal cancer molecular profiling: from IHC to NGS in search of optimal algorithm.

Advances in defining the mutational landscape of colorectal cancer (CRC) over the past decades have revolutionized the molecular understanding and clinical testing algorithms for this disease. Mutation testing is standard of care for the work-up of CRCs. This review focuses on the current indications and strategies for molecular testing in CRC and discusses the potential changes in CRC testing approach associated with the emerging clinical application of genomic-based technologies.

Recent advances in diagnostic testing for gastroesophageal reflux disease.

INTRODUCTION: Gastroesophageal reflux disease (GERD) has a large economic burden with important complications that include esophagitis, Barrett's esophagus, and adenocarcinoma. Despite endoscopy, validated patient questionnaires, and traditional ambulatory pH monitoring, the diagnosis of GERD continues to be challenging. Areas covered: This review will explore the difficulties in diagnosing GERD with a focus on new developments, ranging from basic fundamental changes (histology and immunohistochemistry) to direct patient care (narrow-band imaging, impedance, and response to anti-reflux surgery). We searched PubMed using the noted keywords. We included data from full-text articles published in English. Further relevant articles were identified from the reference lists of review articles. Expert commentary: Important advances in novel parameters in intraluminal impedance monitoring such as baseline impedance monitoring has created some insight into alternative diagnostic strategies in GERD. Recent advances in endoscopic assessment of esophageal epithelial integrity via mucosal impedance measurement is questioning the paradigm of prolonged ambulatory testing for GERD. The future of reflux diagnosis may very well be without the need for currently employed technologies and could be as simple as assessing changes in epithelia integrity as a surrogate marker for GERD. However, future studies must validate such an approach.

Aptahistochemistry in diagnostic pathology: technical scrutiny and feasibility.

Antibodies have been the workhorse for diagnostic immunohistochemistry to specifically interrogate the expression of certain protein to aid in histopathological diagnosis. This review introduces another dimension of histochemistry that employs aptamers as the core tool, the so-called aptahistochemistry. Aptamers are an emerging class of molecular recognition elements that could recapitulate the roles of antibodies. The many advantageous properties of aptamers suited for this diagnostic platform are scrutinized. An in-depth discussion on the technical aspects of aptahistochemistry is provided with close step-by-step comparison to the more familiarized immunohistochemical procedures, namely functionalization of the aptamer as a probe, antigen retrieval, optimization with emphasis on incubation parameters and visualization methods. This review offers rationales to overcome the anticipated challenges in transition from immunohistochemistry to aptahistochemistry, which is deemed feasible for an average diagnostic pathology laboratory.

Pathologic diagnosis of breast cancer patients: evolution of the traditional clinical-pathologic paradigm toward "precision" cancer therapy.

We present an updated account of breast cancer treatment and of progress toward "precision" cancer therapy; we focus on new developments in diagnostic molecular pathology and breast cancer that have emerged during the past 2 years. Increasing awareness of new prognostic and predictive methodologies, and introduction of next generation sequencing has increased understanding of both tumor biology and clinical behavior, which offers the possibility of more appropriate therapeutic choices. It remains unclear which of these testing methodologies provides the most informative and cost-effective actionable results for predictive and prognostic pathology. It is likely, however, that an integrated "step-wise" approach that uses the traditional clinical-pathologic paradigms coordinated with molecular characterization of breast tumor tissue, will offer the most comprehensive and cost-effective options for individualized, "precision" therapy for patients with breast cancer.

Comentario a «Metástasis cutáneas a distancia de cáncer de próstata: 2 casos» / Comment on «Distant cutaneous metastases of prostate cancer: a report of 2 cases»

No disponible

[PD1/PD-L1 immunohistochemistry in thoracic oncology: Where are we?] / Immunohistochimie PD-1/PD-L1 en oncologie thoracique : où en sommes-nous ?

The assays for the assessment of the PD-L1 status by immunohistochemistry are available in clinical studies in thoracic oncology to predict response to immunotherapies targeting the PD-1/PD-L1 pathway. With the arrival of this new class of molecules in second line and very soon in first line of treatment for patients with advanced or metastatic non-small cell lung cancer, these tests will certainly be required in routine once these new drugs will be granted marketing authorization. The rapid introduction of these "companion" or "complementary" tests seems essential to select patients to benefit from these effective but also expensive and sometimes toxic therapies. Although challenged by some oncologists (as some patients not expressing PD-L1 may sometimes respond to PD-1/PD-L1 blockade), the anti-PD-L1 immunohistochemically approach seems inevitable in 2017. This new activity developed in the pathology laboratories raises several questions: which anti-PD-L1 clone should be used? On which device? What threshold of positivity should be considered? Should PD-L1 expression be assessed on tumor cells as well as on the immune cells? What controls should be used? Comparative studies are underway or have been already implemented in order to answer some of these questions. This review addresses the different evaluation criteria for immunohistochemistry using the main anti-PD-L1 antibodies used to date as well the recently published studies using these antibodies in thoracic oncology.

Proteomic modulation in breast tumors after metformin exposure: results from a 'window of opportunity' trial

Purpose. Reverse Phase Protein Array (RPPA) is a high-throughput antibody-based technique to assess cellular protein activity. The goal of this study was to assess protein marker changes by RPPA in tumor tissue from a pre-surgical metformin trial in women with operable breast cancer (BC). Methods. In an open-label trial, metformin 1500-mg PO daily was administered prior to resection in 35 non-diabetic patients with stage 0-III BC, body mass index ≥25 kg/m2. For RPPA, formalin-fixed paraffin-embedded (FFPE) samples were probed with 160 antibodies. Paired and two-sample t-tests were performed (p ≤ 0.05). Multiple comparisons were adjusted for by fixing the false discovery rate at 25 %. We evaluated whether pre- and post-metformin changes of select markers by RPPA were identified by immunohistochemistry (IHC) in these samples. We also assessed for these changes by western blot in metformin-treated BC cell lines. Results. After adjusting for multiple comparisons in the 32 tumors from metformin-treated patients vs. 34 untreated historical controls, 11 proteins were significantly different between cases vs. controls: increases in Raptor, C-Raf, Cyclin B1, Cyclin D1, TRFC, and Syk; and reductions in pMAPKpT202,Y204, JNKpT183,pT185, BadpS112, PKC.alphapS657, and SrcpY416. Cyclin D1 change after metformin by IHC was not observed. In cell lines, reductions in JNKpT183 and BadpS112 were seen, with no change in Cyclin D1 or Raptor. Conclusions. These results suggest that metformin modulates apoptosis/cell cycle, cell signaling, and invasion/motility. These findings should be assessed in larger metformin trials. If confirmed, associations between these changes and BC clinical outcome should be evaluated (AU)

No disponible