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.

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.

[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.

Evolution of sentinel lymph node biopsy in breast cancer, in and out of vogue?

Sentinel lymph node biopsy (SLNB) was introduced 2 decades ago and thereafter validated for routine surgical management of breast cancer, including cases treated with neoadjuvant chemotherapy. As the number of lymph nodes for staging has decreased, pathologists have scrutinized SLN with a combination of standard hematoxylin and eosin, levels, immunohistochemistry (IHC), and molecular methods. An epidemic of small-volume metastases thereby arose, leading to modifications in the American Joint Committee on Cancer staging to accommodate findings such as isolated tumor cells (ITC) and micrometastases. With the goal of determining the significance of these findings, retrospective followed by prospective trials were performed, showing mixed results. The ACOSOG Z10 and NSABP B-32 trials both independently showed that ITC and micrometastases were not significant and thus discouraged the use of levels and IHC for detecting them. However, the Surveillance Epidemiology and End Results database showed that patients with micrometastases had an overall decreased survival. In addition, the MIRROR (Micrometastases and ITC: Relevant and Robust or Rubbish?) trial, showed that patients with ITC and micrometastases treated with adjuvant therapy had lower hazard ratios compared with untreated patients. Subsequently, the ACOSOG Z0011 trial randomized patients with up to 2 positive SLN to axillary lymph node dissection (ALND) or not, all treated with radiation and chemotherapy, showing no difference in survival or recurrence rates between the 2 groups and causing a shift from ALND. As the rate of ALND has declined, the necessity of performing levels, IHC, frozen section, and molecular studies on SLN needs to be revisited.

Updates in diagnostic immunohistochemistry in endometrial carcinoma.

Diagnostic difficulty in the morphologic assessment of endometrial carcinomas may arise in pathology practice. Challenges in tumor classification exist especially in the setting of high-grade carcinomas. These include FIGO grade 3 endometrioid, serous, clear cell, and undifferentiated carcinomas, in addition to carcinomas of mixed cell type and those exhibiting ambiguous morphologic features. This comprehensive review details key morphologic and immunophenotypic features of prototypic endometrial carcinomas, including a description of both well-established and novel immunohistochemical markers in the evaluation of these tumors. It also provides recommendations regarding prudent use of these ancillary techniques in distinguishing between various histologic subtypes of endometrial carcinoma that frequently result in persistent diagnostic problems.

A practical approach to immunohistochemical diagnosis of ovarian germ cell tumours and sex cord-stromal tumours.

Immunohistochemistry can be useful in the diagnosis of ovarian germ cell tumours and sex cord-stromal tumours. A wide variety of markers are available, including many that are novel. The aim of this review is to provide a practical approach to the selection and interpretation of these markers, emphasizing an understanding of their sensitivity and specificity in the particular differential diagnosis in question. The main markers discussed include those for malignant germ cell differentiation (SALL4 and placental alkaline phosphatase), dysgerminoma (OCT4, CD117, and D2-40), yolk sac tumour (α-fetoprotein and glypican-3), embryonal carcinoma (OCT4, CD30, and SOX2), sex cord-stromal differentiation (calretinin, inhibin, SF-1, FOXL2) and steroid cell tumours (melan-A). In addition, the limited role of immunohistochemistry in determining the primary site of origin of an ovarian carcinoid tumour is discussed.

Immunocytochemistry as an adjunct to diagnostic cytology.

Immunocytochemistry as a routine ancillary test remains a distant reality for most diagnostic laboratories. Notable barriers to the mass deployment of ICC include: the large variety of specimen preparations, the small specimen size, lack of validation and lack of control specimens. As clinicians constantly strive to answer questions relating to diagnosis, therapy and prognosis with minimally invasive sampling techniques, the cytopathology community must endeavour to adopt ancillary specimen testing by ICC as a core element of diagnostic cytology.

Immunocytochemistry in Europe: results of the European Federation of Cytology Societies (EFCS) inquiry.

OBJECTIVE: This report describes the results of the first chosen topic of the European Federation of Cytology Societies (EFCS) scientific committee, which concerns the application of immunocytochemistry (ICC) to cytological material. While ICC has become an important ancillary method, not only for diagnosis but also to assess prognostic and predictive factors on cytological material, there are many different methodologies used and the lack of standardization has been criticized. This inquiry aimed first to obtain an overview of the techniques used and second to suggest mechanisms for standardization and quality control. METHODS: We report the results of 28 replies from 13 countries to a web-based inquiry into types of specimens, preparations and technical methods. RESULTS: Conventional smears were the preparations most commonly used, followed by cytospins, cell blocks and liquid-based preparations, in that order. Avidin-biotin complex, labelled streptavidin-biotin or peroxidase anti-peroxidase were used in 61% and enhanced-polymer technology in 39%. Automated staining techniques were used by 64%. More than half used the same antibody dilutions as histopathology although only 20% used cell blocks. CONCLUSION: Reduction of variability by using automation, appropriate controls and customized dilution of antibodies according to different samples could improve the quality of ICC and standardize the techniques, along with quality control and quality assurance measures.

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.

Immunohistology--past, present, and future.

The rapid development of immunohistochemistry, a morphology-based technique, has come about through refinements in detection systems and an increasing range of sensitive and specific antibodies that have allowed application of the technique to formalin-fixed, paraffin-embedded tissues. The introduction of heat-induced antigen retrieval has been a significant milestone to compliment these developments so that the immunohistochemistry is firmly entrenched as an indispensable adjunct to morphologic diagnosis. Although this ancillary stain was initially used in a qualitative manner, problems surrounding the many variables that influence antigen preservation in formalin-fixed, paraffin-embedded tissues were not a major issue and laboratories strived to optimize their staining protocols to the material they accessioned and processed. The advent of personalized medicine and targeted cancer treatment has imposed the need to quantitate the stain reaction product and has resulted in calls to standardize the process of immunostaining. A closer examination of the variables that influence the ability to show antigens in formalin-fixed, paraffin-embedded tissues revealed many important variables, particularly in the preanalytical phase of the assay, that are beyond the control of the accessioning laboratory. Although analytical factors have the potential to be standardized, the actions of many pivotal procedures including fixation and antigen retrieval are not completely understood. Postanalytical processes including threshold and cut-off values require consensus and standardization and it is clear that some of these goals can be achieved through the direction of national and international organizations associated with cancer diagnosis and treatment. With the ability to serve as a surrogate marker of many genetic abnormalities, immunohistochemistry enters a new era and the need to better understand some of the mechanisms fundamental to the technique become more pressing and the development of true quantitative assays is imperative. There is also an increasing appreciation that the technique highlights patterns of staining that reflect exquisite localization to organelles and tissue structures that are not appreciable in routine stains, adding a further dimension to morphologic diagnosis.

Current concepts of neuropathological diagnostics in practice: neurodegenerative diseases.

Definitive diagnosis of neurodegenerative diseases (NDDs) relies on the neuropathological evaluation. NDDs are defined as disorders with progressive loss of neurons showing distinct anatomical distribution, and accordingly different clinical phenotypes. Recent research has identified a spectrum of immunohistochemically detectable proteins deposited in the central nervous system which serve as a basis for protein-based disease classification. Accordingly, diagnostic criteria and disease staging have been updated. Furthermore, it has become evident that there is considerable overlap between deposited proteins and pathologies. This review summarizes recent achievements in neuropathological diagnosis and classification of NDDs and recommends approaches to be used during the diagnostic procedure in practice, thus to serve as guideline for a common level of diagnostic quality.

[Application of semi-conducting nanocrystals (quantum dots) in immunocytochemistry].

Semi-conducting nanocrystals represent a new class of fluorescent inorganic objects which have a promising perspective for the application in biology and medicine. The aim of the current work was a determination of advantages and shortcimingss of quantum dots (QD) application in immunocytochemistry. It was shown that streptavidin-QD conjugates have more advantages (i.e. high intensity of fluorescence, photostability, wide excitation range, short and symmetric emission range) as compared to streptavidin conjugates with organic fluorochromes. This allows to recommend the use of QD in immunocytochemical studies. However, there are several disadvantages (like lower stability during long-term storage as compared to that one of organic fluorochrome conjugates, poor safety of aliquots, impossibility of long-term preservation of fluorescence of stained sections during their storage, incompatibility with several commercial mounting media) that limit the widespread application of nanocrystals in immunocytochemical studies.

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.

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.

Congenital myopathies--a comprehensive update of recent advancements.

The congenital myopathies are relatively newly discovered compared with other categories of muscle diseases. Current research continues to clarify and classify the congenital myopathies. These pose a diagnostic problem and cannot be diagnosed by routine hematoxylin and eosin stain. A lot of special techniques are required to diagnose them correctly and it's various subtypes. The disease specific structural changes seen in the muscle are detected by enzyme histochemistry, immunohistochemistry and electron microscopy. Through this review we provide an up-to-date analysis of congenital myopathies including clinical and pathologic aspects.

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.

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.

Tissue microarray: a simple technology that has revolutionized research in pathology.

Tissue microarray (TMA) technology is a high-throughput research tool, which has greatly facilitated and accelerated tissue analyses by in-situ technologies. TMAs are amenable to every research method that can be applied on the standard whole sections at enhanced speed. It plays a central role in target verification of results from cDNA arrays, expression profiling of tumors and tissues, and is proving to be a powerful platform for proteomic research. In this review article, primarily meant for students of pathology and oncology, we briefly discuss its basic methodology, applications and merits and limitations.