Molecular/biomarker testing in lung cytology: A practical approach

The increasing comprehension of molecular mechanisms underlying lung cancer and the discovery of targetable genomic alterations has dramatically change the pathological approach to lung cancer, especially non-small cell lung cancer (NSCLC). This unstoppable knowledge has taken pathologists to the leading front on lung cancer management. This is especially relevant in the world of cytopathology where “doing more with less” is a daily challenge. Nowadays with a growing number of predictive biomarkers needed to manage patients with NSCLC, there has been a paradigm shift in care and handling of diagnostic samples. One of the main emphasis and interest relies on the utilization of cytologic samples and small biopsies for not only diagnostic purposes but also for ancillary testing. Moreover, lung cytopathology is in continuous evolutions with implementation of new diagnostic techniques, new tools, and facing new challenges. The goal of this paper will be to provide the reader with the necessary concepts than can be used to exploit the cytological samples in order to use these samples for comprehensive diagnosis and relevant ancillary testing purposes.     

Advances in cytology of lung cancer

Cytopathology has emerged as a promising platform in precision oncology especially after the revolutionary change in our understanding of the concept of lung cancer etiopathogenesis. With increasing use of minimally invasive techniques for sample acquisition, it becomes almost mandatory to utilize precious cytology samples maximally and judiciously by appropriate triaging of the specimen and timely action of the cytopathology team. Existing patient management protocols require accurate morphologic and molecular diagnosis of the lung cancer specimens which needs knowledge about evolving techniques related to specimen procurement, updates of genomic variants of lung cancer and recently developed molecular testing platforms and algorithms which are capable enough to use even miniscule amount of diagnostic material. This review provides a brief knowledge about advances in cytology of lung cancer which are helpful for developing correct clinical management strategies.     

Molecular testing in cytology

In the era of personalized medicine, molecular testing plays a critical role in patient care. The rapid advance of molecular techniques, especially next-generation sequencing, makes molecular diagnosis feasible in daily practice. Molecular testing can be used as a valuable ancillary test to increase diagnostic sensitivity and specificity, especially in small biopsy or cytology samples. In addition, molecular testing plays an important role in selecting patients for appropriate treatment by detecting therapeutic and predictive biomarkers in tissue or cytology samples. Molecular studies can be applied in all cytology samples, sometimes with better results than histology. As molecular testing has become essential for patient care and is often requested to be performed in cytology samples, it is critical for cytopathologists to understand the basics of molecular diagnostic methods, indications for molecular testing, and how to best utilize different cytologic samples for this purpose. In this special issue, experts in various areas of cytopathology and molecular pathology review the literature and discuss the basics of molecular techniques and the application of molecular testing in various types of cytology samples. It is our hope that after reading the articles in this special issue, the readers can know better about the possibilities of molecular cytology, a very exciting field of pathology.     

Does genomic sequencing early in the diagnostic trajectory make a difference? A follow-up study of clinical outcomes and cost-effectiveness

PurposeTo systematically investigate the longer-term clinical and health economic impacts of genomic sequencing for rare-disease diagnoses.MethodsWe collected information on continuing diagnostic investigation, changes in management, cascade testing, and parental reproductive outcomes in 80 infants who underwent singleton whole-exome sequencing (WES).ResultsThe median duration of follow-up following result disclosure was 473 days. Changes in clinical management due to diagnostic WES results led to a cost saving of AU$1,578 per quality-adjusted life year gained, without increased hospital service use. Uninformative WES results contributed to the diagnosis of non-Mendelian conditions in seven infants. Further usual diagnostic investigations in those with ongoing suspicion of a genetic condition yielded no new diagnoses, while WES data reanalysis yielded four. Reanalysis at 18 months was more cost-effective than every 6 months. The parents of diagnosed children had eight more ongoing pregnancies than those without a diagnosis. Taking the costs and benefits of cascade testing and reproductive service use into account, there was an additional cost of AU$8,118 per quality-adjusted life year gained due to genomic sequencing.ConclusionThese data strengthen the case for the early use of genomic testing in the diagnostic trajectory, and can guide laboratory policy on periodic WES data reanalysis.     

Fluorescence in situ hybridization, comparative genomic hybridization, and other molecular biology techniques in the analysis of effusions

Over the last 20 yr, the introduction of immunocytochemistry as a diagnostic tool has dramatically revolutionized diagnostic pathology. With the introduction of molecular methods as part of the diagnostic armamentarium, the practicing pathologist is facing the new challenge of grasping novel concepts of the molecular cytogenetics era. Herein, we review the diagnostic contribution of ancillary molecular techniques, including fluorescent and chromogenic in situ hybridization, telomerase assays, loss of heterozygosity, comparative genomic hybridization (CGH), and microarray-based CGH, for the practicing cytopathologists and discuss how these techniques will help pathologists in decision-making.     

Investigative pathology: leading the post-genomic revolution

The completion of the Human Genome Project and the development of genome-based technologies over the past decade have set the stage for a new era of personalized medicine. By all rights, molecularly trained investigative pathologists should be leading this revolution. Singularly well suited for this work, molecular pathologists have the rare ability to wed genomic tools with unique diagnostic skills and tissue-based pathology techniques for integrated diagnosis of human disease. However, the number of pathologists with expertise in genome-based research has remained relatively low due to outdated training methods and a reluctance among some traditional pathologists to embrace new technologies. Moreover, because budding pathologists may not appreciate the vast selection of jobs available to them, they often end up choosing jobs that focus almost entirely on routine diagnosis rather than new frontiers in molecular pathology. This review calls for changes aimed at rectifying these troubling trends to ensure that pathology continues to guide patient care in a post-genomic era.     

Variant call concordance between two laboratory-developed,solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n = 17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.     

Targeted RNA sequencing: A routine ancillary technique in the diagnosis of bone and soft tissue neoplasms

The past decade has witnessed remarkable progress in delineating the molecular pathogenesis of many mesenchymal neoplasms. This, in large part, is attributable to the application of next‐generation sequencing. As these techniques decrease in cost, and increasingly support the use of routine clinical specimens—such as formalin‐fixed paraffin‐embedded tissue and cytology samples—they are beginning to be routinely implemented in diagnostic pathology laboratories. The breadth of testing possible by next‐generation sequencing makes this a useful adjunct for pathologists, particularly with the emergence of targeted therapies. The intent of this article is to share our experience, over 2 years, as an early adopter of targeted RNA sequencing as an ancillary diagnostic technique for fusion gene detection in bone and soft tissue neoplasms.     

Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a novel cause of Lynch syndrome

Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders.     

Cytopathology in the diagnosis,treatment and management of malignant respiratory disease

Lung carcinomas are almost always diagnosed using small samples of cellular material. Diagnostic cytopathology has a major role and, with efficient and thoughtful processing and examination of fine needle aspiration or fluid specimens, it is possible to accurately diagnose and perform a full range of predictive testing. This short piece begins by placing cytopathology within an overall umbrella of cellular pathology and briefly describes recent developments in lung cancer diagnosis and treatment. Cytopathological features of the main relevant tumour types and their differential diagnoses are discussed together with their appearance in commonly encountered specimen types such as those from endobronchial ultrasound. There are suggestions for efficient workflow and illustrations of diagnostic pitfalls. Although some trainees are wary of cytopathology, there is no magic (good or bad) involved and any time spent acquiring a facility with interpretation of these specimens as well as their formalin-fixed counterparts will enhance diagnostic skill and, ultimately, patient care.     

Advances in DNA sequencing technologies for high resolution HLA typing

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of bacterial and fungal isolates

Identification of bacterial and fungal isolates from clinical specimens has traditionally been performed by examination of colony morphology and by biochemical characterization using classic tube sets. While these methods remain the gold standard for identification, they can be laborious to perform and subjective to interpret. These drawbacks have largely been alleviated with the advent of automated biochemical testing platforms and with the development of DNA sequencing and polymerase chain reaction techniques. Though reliable and rapid, molecular-based platforms are associated with significantly higher cost and require advanced user expertise for assay development and performance. These factors limit the routine implementation of molecular diagnostic methods to large hospitals and reference laboratories. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is an automated, molecular platform recently adopted by many clinical laboratories worldwide, which offers a rapid, straightforward, and inexpensive method for the identification of bacterial and fungal organisms.     

Molecular pathology in anatomic pathology practice: a review of basic principles

Molecular testing in pathology emerged shortly after polymerase chain reaction became a standard molecular biology assay. Testing efforts began in the clinical laboratories primarily with assays for genetically inherited diseases and assays for clonality in hematologic malignancies. Today, the field has evolved into "molecular diagnostics," which encompasses testing in almost every area of anatomic pathology. Molecular testing is now even making its way definitively into both surgical pathology and cytopathology, although molecular anatomic pathology is still young with few standard tissue-based molecular assays. As more clinically valuable information is gained from molecular pathology testing of tissues, unique challenges are also becoming apparent at the intersection between tissue diagnosis and DNA diagnosis. This review focuses on basic molecular pathology concepts, with particular emphasis on the challenge of tissue-based testing in anatomic pathology.     

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.     

Molecular techniques in cytopathology practice

In the last decade, new molecular techniques were introduced into pathology laboratories. Cytology also benefited from the innovations emerging from this new era. Molecular cytopathology (MCP) can be defined as molecular studies applied on all types of cytological specimens, namely gynaecology cytology, exfoliative non- gynaecology cytology and fine needle aspirates. The development of many new ancillary techniques has paralleled the emergence of clinical cytology as a major diagnostic specialty. Clinical applications of these techniques have been growing in the last decade. The widespread acceptance of liquid-based systems in gynaecological cytology emphasises the relation between cells and molecules. The increased use of morphology and molecular biology in human papillomavirus-induced lesions for example, showed the potential to optimise, in one single brushed sample, diagnosis and research. Cytology samples from serous effusions, the pulmonary tree, urine, and aspirations, among others, are now likely to be studied by different molecular techniques for diagnosis, prognosis, or even assessment of therapeutic targets. In this review, the main published results concerning the application of molecular techniques in different fields of cytopathology are highlighted, and their applications discussed.     

A sensitive and specific diagnostic test for hearing loss using a microdroplet PCR‐based approach and next generation sequencing

Implementing DNA diagnostics in clinical practice for extremely heterogeneous diseases such as hearing loss is challenging, especially when attempting to reach high sensitivity and specificity in a cost‐effective fashion. Next generation sequencing has enabled the development of such a test, but the most commonly used genomic target enrichment methods such as hybridization‐based capture suffer from restrictions. In this study, we have adopted a new flexible approach using microdroplet PCR‐based technology for target enrichment, in combination with massive parallel sequencing to develop a DNA diagnostic test for autosomal recessive hereditary hearing loss. This approach enabled us to identify the genetic basis of hearing loss in 9 of 24 patients, a success rate of 37.5%. Our method also proved to have high sensitivity and specificity. Currently, routine molecular genetic diagnostic testing for deafness is in most cases only performed for the GJB2 gene and a positive result is typically only obtained in 10–20% of deaf children. Individuals with mutations in GJB2 had already been excluded in our selected set of 24 patients. Therefore, we anticipate that our deafness test may lead to a genetic diagnosis in roughly 50% of unscreened autosomal recessive deafness cases. We propose that this diagnostic testing approach represents a significant improvement in clinical practice as a standard diagnostic tool for children with hearing loss. © 2012 Wiley Periodicals, Inc.     

The use of electron microscopy to refine diagnoses in the daily practice of cytopathology

Conceived as a screening tool, cytology is a field that since the 1980s has become more diagnostic in its scope. The advent of the fine-needle aspiration biopsy (FNAB) is responsible for cytology's new place in pathology. In the everyday practice of cytopathology, about 85-90% of the nongynecologic cases can be diagnosed with the use of routine stains (i.e., Papanicolaou and Diff Quik). The other 10-15% of the cases require the use of ancillary diagnostic techniques for a precise diagnosis. Immunohistochemistry helps solve approximately 50% of these cases, and the other half of these challenging cases are best approached and diagnosed by using electron microscopy (EM). In their practice, the authors obtain cytologic samples for EM routinely in difficult cases. Unfortunately, a percentage of these cases collected for ultrastructural evaluation do not have enough cells after processing, and others only have a few diagnostic cells available. In the cases in which at least a handful of cells are available, EM is almost invariably helpful in one way or another, either making a definitive diagnosis or refining the diagnosis. A sampling of FNAB cases from the authors' everyday practice is prevented to illustrate the use of EM in the practice of cytopathology. The cases have been selected from among the most common diagnostic challenges to highlight the important role that ultrastructural evaluation plays in a busy cytology practice. In our practice ultrastructural evaluation is a piece of the puzzle, which, along with the clinical history, clinical impression, light microscopic/cytologic features, and other ancillary techniques (IHC, flow cytometry, and molecular pathology), help compile an accurate diagnosis. Many times EM is the most important component of the diagnostic algorithm.     

An FBN1 Deep Intronic Mutation in a Familial Case of Marfan Syndrome: An Explanation for Genetically Unsolved Cases?

Marfan syndrome (MFS) is caused by mutations in the FBN1 (fibrillin‐1) gene, but approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS family that had remained negative after extensive molecular genomic DNA FBN1 testing, including denaturing high‐performance liquid chromatography, Sanger sequencing, and multiplex ligation‐dependent probe amplification. Linkage analysis in the family and cDNA sequencing of the proband revealed a deep intronic point mutation in intron 56 generating a new splice donor site. This mutation results in the integration of a 90‐bp pseudo‐exon between exons 56 and 57 containing a stop codon, causing nonsense‐mediated mRNA decay. Although more than 90% of FBN1 mutations can be identified with regular molecular testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole‐genome sequencing.     

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene,gene panel,or exome/genome sequencing

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing–based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.Genet Med17 6, 444–451.     

Molecular cytogenetics: recent developments and applications in cancer

Aneuploidy or alteration in chromosome numbers is a characteristic feature in cancer that is generally a consequence of defective chromosome segregation during cell division. Molecular cytogenetic analyses have conferred substantial evidence with regards to the chromosomal architectures in cancer. Most importantly, the fluorescence in situ hybridization (FISH) technique that plays a leading role in diagnostic pathology for its single‐cell analysis has provided crucial information regarding genomic variations in malignant cells. Further development of molecular cytogenetic methodologies such as chromosome specific FISH karyotyping and comparative genomic hybridization have also helped in the detection of cryptic genetic changes in cancer. But, the recent advancement of high throughput sequencing technologies have provided a more comprehensive genomic analyses resulting in novel chromosome rearrangements, somatic mutations as well as identification of fusion genes leading to new therapeutic targets. This review highlights the application of early molecular cytogenetics and the recent high throughput genomic approaches in characterizing various cancers and their invaluable support in cancer therapeutics.