Adenocarcinoma predominant pattern subtyping and nuclear grading in cytology: Is there a role in prognostication of advanced pulmonary adenocarcinomas?

INTRODUCTION: Primary lung adenocarcinomas (ADs) show varied architectural patterns, and pattern-based subtyping of ADs is currently recommended due to prognostic implications. Predicting AD patterns on cytology is challenging; however, cytological nuclear features appear to correlate with histological grade and survival in early stage lung ADs. The feasibility and value of AD pattern prediction and nuclear grading on cytology in advanced lung ADs is not known. We aimed to predict patterns and analyse nuclear features on cytology and evaluate their role in prognostication. METHODS: One-hundred patients of Stage III/IV lung AD with available matched cytology and histology samples were included. Cyto-patterns based on cell arrangement patterns (flat sheets vs three-dimensional clusters vs papillae) and cyto-nuclear score based on nuclear features (size, shape, contour), nucleoli (macronucleoli vs prominent vs inconspicuous), and nuclear chromatin were determined, and correlated with predominant histological-pattern observed on the matched small biopsy and outcome. RESULTS: Higher cyto-nuclear scores were observed with high-grade histo-patterns (solid, micropapillary and cribriform), while the predicted cyto-patterns did not correspond to the predominant pattern on histology in 77% cases. Highest cyto-histo agreement was observed for solid pattern (72%). High grade histo-patterns and cyto-nuclear scores > 3 showed a trend towards inferior survival (not significant). CONCLUSIONS: Nuclear grade scoring on cytology is simple to perform, and is predictive of high grade patterns. Its inclusion in routine reporting of cytology samples of lung ADs may be valuable.

Doing more with less: integrating small biopsies in cytology practice.

Cytopathologists are at the forefront of specimen acquisition during many different procedures while providing rapid on site evaluation (ROSE). This has added pressure to cytopathologists as more and more ancillary testing is being requested on smaller amounts of tissue. By focusing on the most common organ sites: lung, head and neck, and pancreas, there is a discussion of what the cytopathologist needs to know to triage tissue successfully. Finally, there is a discussion of the logistical aspects of integrating small biopsies into everyday practice.

Pulmonary Cytopathology: Current and Future Impact on Patient Care.

Small biopsies of lung are routinely obtained by many methods, including several that result in cytologic specimens. Because lung cancer is often diagnosed at a stage for which primary resection is not an option, it is critical that all diagnostic, predictive, and prognostic information be derived from such small biopsy specimens. As the number of available diagnostic and predictive markers expands, cytopathologists must familiarize themselves with current requirements for specimen acquisition, handling, results reporting, and molecular and other ancillary testing, all of which are reviewed here.

Pulmonary Cytopathology: Current and Future Impact of Microscopy and Immunohistochemistry.

With the advancement of tissue procurement techniques, in-depth knowledge of morphology is crucial for cytopathologists to diagnose neoplastic and nonneoplastic lung diseases optimally. Cytopathologists must also be well versed in immunohistochemistry/immunocytochemistry markers and their interpretation for an accurate diagnosis.

One procedure-one report: the Re-Imagine Cytopathology Task Force position paper on small tissue biopsy triage in anatomic pathology.

INTRODUCTION: Endoscopic biopsy procedures increasingly generate multiple tissue samples from multiple sites, and frequently retrieve concurrent cytologic specimens and small core needle biopsies. There is currently lack of consensus in subspecialized practices as to whether cytopathologists or surgical pathologists should review such samples, and whether the pathology findings should be reported together or separately. MATERIALS AND METHODS: In December 2021, the American Society of Cytopathology convened the Re-Imagine Cytopathology Task Force to examine various workflows that would facilitate unified pathology reporting of concurrently obtained biopsies and improve clinical care. RESULTS AND CONCLUSIONS: This position paper summarizes the key points and highlights the advantages, challenges, and resources available to support the implementation of such workflows that result in "one procedure-one report".

Chromatin Immunoprecipitation Sequencing (ChIP-seq) Protocol for Small Amounts of Frozen Biobanked Cardiac Tissue.

Chromatin immunoprecipitation and sequencing (ChIP-seq) is a well-established method to study the epigenetic profile at the genome-wide scale, including histone modifications and DNA-protein interactions. It provides valuable insights to better understand disease mechanisms. Here we present an optimized ChIP-seq protocol suitable for human cardiac tissues, especially the frozen biobanked small biopsy samples.

Maximizing Small Biopsy Patient Samples: Unified RNA-Seq Platform Assessment of over 120,000 Patient Biopsies.

Despite its wide-ranging benefits, whole-transcriptome or RNA exome profiling is challenging to implement in a clinical diagnostic setting. The Unified Assay is a comprehensive workflow wherein exome-enriched RNA-sequencing (RNA-Seq) assays are performed on clinical samples and analyzed by a series of advanced machine learning-based classifiers. Gene expression signatures and rare and/or novel genomic events, including fusions, mitochondrial variants, and loss of heterozygosity were assessed using RNA-Seq data generated from 120,313 clinical samples across three clinical indications (thyroid cancer, lung cancer, and interstitial lung disease). Since its implementation, the data derived from the Unified Assay have allowed significantly more patients to avoid unnecessary diagnostic surgery and have played an important role in guiding follow-up decisions regarding treatment. Collectively, data from the Unified Assay show the utility of RNA-Seq and RNA expression signatures in the clinical laboratory, and their importance to the future of precision medicine.

WT1 and Cyclin D1 Immunohistochemistry: A Useful Adjunct for Diagnosis of Pediatric Small Round Blue Cell Tumors on Small Biopsies.

Pediatric small round blue cell tumors (SRBCTs) are a heterogeneous group of neoplasms with overlapping morphological appearance. Accordingly, their diagnosis is one of the most difficult in the field of surgical pathology. The most common tumors include rhabdomyosarcoma, Ewing's sarcoma, neuroblastoma, lymphoblastic lymphoma and Wilms' tumor (the blastemal component). Over time their diagnosis has become more difficult due to the increasing use of small biopsies. However, the advent of immunohistochemistry has improved the quality of diagnosis in most cases by the application of an adequate panel of immunomarkers. Recently, WT1 and Cyclin D1 have been shown to be useful in the differential diagnosis of SRBCTs on surgically-resected specimens, showing a diffuse cytoplasmic positivity of the former in all RMSs and a diffuse nuclear staining of the latter in both EWS and NB. The aim of the present study was to investigate the expression of WT1 and Cyclin D1 on small biopsies from a series of 105 pediatric SRBCTs to evaluate their diagnostic utility. Both immunomarkers were differentially expressed, with a diffuse and strong cytoplasmic staining for WT1 limited to all cases of RMS, and a diffuse nuclear staining for cyclin D1 restricted to all cases of EWS and NB. Notably, the expression of WT1 and cyclin D1 was also retained in those cases in which the conventional tumor markers (myogenin, desmin and MyoD1 for RMS; CD99 for EWS; NB84 for NB) were focally expressed or more rarely absent. The present study shows that WT1 and Cyclin D1 are helpful immunomarkers exploitable in the differential diagnosis of pediatric SRBCTs on small biopsies, suggesting their applicability in routine practice.

Architectural aspects of cell-blocks as small biopsies.

Cell-block preparations have become an essential part of integrated cytology diagnosis. They are essentially microbiopsies that are formalin fixed and embedded in paraffin. This has become more prevalent with greater sample procurement due to the advent of newer biopsy techniques and needles. Cell-blocks allow retrieval of small tissue fragments from cytology specimens that sometimes cannot be processed by alternate cytologic techniques. They represent concentrated, cell-enriched preparations that provide cytologists with the opportunity to evaluate cellular architecture, as well as to perform ancillary testing. A cell-block compatible sample may thus obviate the need for a more invasive procedure such as a tissue biopsy. Microscopic examination of cell-blocks is quick, avoids obscuring material, permits cells to be evaluated in one focal plane, and allows the histologic architecture such as glandular differentiation, papillary formations, and sometimes invasion to be easily identified. This new era of "cytohistology" accordingly requires practicing cytologists to become more familiar with histopathology. This review article discusses the benefit of various architectural patterns identifiable in cell-blocks employed as an adjunct to Pap tests, exfoliative fluid specimens, and fine-needle aspirations.

Lung cancer cytology and small biopsy specimens: diagnosis, predictive biomarker testing, acquisition, triage, and management.

In the 21st century, there has been a dramatic shift in the management of advanced-stage lung carcinoma, and this has coincided with an increasing use of minimally invasive tissue acquisition methods. Both have had significant downstream effects on cytology and small biopsy specimens. Current treatments require morphologic, immunohistochemical, and/or genotypical subtyping of non-small cell lung carcinoma. To meet these objectives, standardized classification of cytology and small specimen diagnoses, immunohistochemical algorithms, and predictive biomarker testing guidelines have been developed. This review provides an overview of current classification, biomarker testing, methods of small specimen acquisition and triage, clinical management strategies, and emerging technologies.

An update on touch preparations of small biopsies.

Touch preparations (TPs) are being increasingly utilized in the era of personalized medicine. They fill a gap in cytopathology practice by providing a method to perform rapid onsite evaluation of small tissue samples such as core needle biopsies. However, there is a paucity of literature about how best to perform and interpret a TP. A high-quality TP can provide excellent diagnostic accuracy and good concordance with core needle biopsy histopathology findings. Although many of the cytomorphologic features of TPs overlap with fine needle aspirate smears, TP cytology is unique and differs from conventional smears in many aspects. It is important for cytologists to recognize these features, as well as potential pitfalls and artifacts in order to avoid misinterpretation. Core depletion of tumor cells is a notable drawback if TPs are performed too aggressively. TP slides are also valuable for ancillary testing because they often contain a cellular and pure population of whole tumor cells. This paper reviews all of the aspects of TPs including their clinical utility, proper slide preparation techniques, distinctive cytomorphologic characteristics, limitations, and potential pitfalls.

Optimizing small liver biopsy specimens: a combined cytopathology and surgical pathology perspective.

Both fine-needle aspiration (FNA) and core needle biopsy (CNB) are widely used to obtain liver biopsy specimens, particularly from mass lesions. However, the advantages and disadvantages of FNA versus CNB in terms of appropriate use, diagnostic yield, complications, and whether or not specimens should be handled by cytopathologists, surgical pathologists, or both remain subjects of controversy. This review addresses the issues of sample adequacy, appropriate use of each technique and complications, and challenges regarding the diagnosis of both hepatic tumors and non-neoplastic liver disease.

Lung cancer and molecular testing in small biopsies versus cytology: The Logics of Worlds.

The 8th Annual National Molecular Cytopathology Meeting, held in Naples, Italy, on December 2 to 3, 2019, addressed updates in diagnostic cytopathology and molecular classifications and specifically focused on lung cancer biomarker testing in cytology samples. Lung cancer continues to be the most commonly diagnosed noncutaneous malignancy in the world. In the majority of patients, lung cancers are frequently identified when they cannot be surgically accessed, and this leads to the use of cytology for a diagnosis and theragnostic testing. The meeting was an international forum for discussing new roles and updates for cytopathology in molecular testing as the basis for provoking new trends and novel approaches. The relevant literature is referenced. The significance of these updates for the practice of pathology in general is discussed.

Navigating small biopsies of salivary gland tumors: a pattern-based approach.

Diagnosis of salivary gland tumors on small biopsy can be difficult because of overlapping morphology, limited tissue availability, and technical artifact. Although a specific diagnosis is not feasible in all cases, a cautious and thoughtful approach to the differential diagnosis and a keen awareness of clinical consequences can facilitate the most complete and useful classification possible. In this review, we present a general strategy for the evaluation of small salivary biopsies, including consideration of clinical and radiographic information, systematic assessment of histologic patterns, and judicious use of immunohistochemistry and molecular studies. We then focus on the distinctive differential diagnoses raised by 6 specific histologic patterns: tubular and cribriform architecture, squamous differentiation, mucin and other secretions, high-grade cytology, epithelial and lymphoid elements, and oncocytic features. Throughout this systematic and pattern-based approach, we focus on practical and cost-effective strategies to overcome the most common diagnostic challenges in limited material.

Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies Guideline from the College of American Pathologists (CAP): implications for the cytology community.

The methods of collecting and handling thoracic small biopsy and cytology specimens can greatly impact downstream ancillary testing success, especially for non-small cell lung cancer (NSCLC). The College of American Pathologists (CAP) in collaboration with multiple other professional societies has recently developed an evidence-based laboratory practice guideline to provide clarity on how to best optimize these pre-analytic variables for small thoracic specimens. A total of 16 guideline statements were developed based on systematic literature review and expert panel consensus, covering topics ranging from optimal materials and techniques for fine-needle aspiration and small biopsy sampling to acquire sufficient amounts of material, to the use of rapid on-site evaluation to help appropriately triage, handle, and process these specimens. These guideline statements hold many implications for the practicing cytologist, though perhaps none more important than the recognition that the variety of cytology specimens we work with on a daily basis (including smears, cell blocks, and liquid-based cytology) can certainly be used for ancillary studies if supported by adequate validation studies. This commentary provides a brief overview of the newly developed CAP thoracic small biopsy and cytology specimen collection and handling guideline, as well as a discussion of how it may specifically impact the cytology community.

Concordance of tumor infiltrating lymphocytes, PD-L1 and p16 expression in small biopsies, resection and lymph node metastases of oropharyngeal squamous cell carcinoma.

OBJECTIVE: The incidence of oropharyngeal squamous cell carcinoma (OPSCC), especially human papillomavirus (HPV)-associated, is increasing worldwide. Immunotherapy become available for patients with carcinomas in the head and neck region, however without ideal biomarker. Markers like PD-L1 vary in the clone of the antibody used, and the method of evaluation. Adequate and reliable immune cells characterization and evaluation is still not found. Furthermore, studies analyzing representativeness of different tissue samples are scarce. We analyzed small biopsy, lymph node (LN) metastasis and resected OPSCC, in regards of tumor infiltrating lymphocyte (TIL) density, PD-L1 and p16 expression. MATERIAL AND METHODS: Patients with OPSCC diagnosed from 2000 to 2016, with small biopsy, resection specimen and LN metastasis samples were selected. We analyzed TILs on hematoxylin-eosin stain, and PD-L1 and p16 expression in tumor cells. Concordance between different tumor locations was evaluated. RESULTS: 93 patients, with 65 small biopsies, 72 resection specimens, and 70 LN metastases were included. TILs, p16 and PD-L1 demonstrated very high concordance. Additionally, PD-L1 expression in the small biopsies was more representative of the PD-L1 expression in the resection specimens, than the LN samples. CONCLUSION: TILs density can be reliably assessed using hematoxylin-eosin stain with high concordance between the small biopsy, resection specimen and LN metastasis. Evaluation of concordance of p16 expression is very high, nevertheless some cases might be misdiagnosed on a small biopsy or lymph node metastasis. Evaluation of PD-L1 expression is very reliable on the biopsy specimen. Different PD-L1 clones and methods of evaluation still remain to be addressed.

The importance of EGFR mutation testing in squamous cell carcinoma or non-small cell carcinoma favor squamous cell carcinoma diagnosed from small lung biopsies.

BACKGROUND: Adenosquamous carcinoma (ADSC) of the lung, a rare but aggressive subtype of non-small cell lung cancer (NSCLC), is defined as a carcinoma containing components of adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). Mutations of epidermal growth factor receptor (EGFR) are found at a frequency of 15 to 44% in Asian ADSC, and EGFR tyrosine kinase inhibitors (EGFR-TKIs) are a more effective treatment for EGFR-mutated ADSC compared to chemotherapy. However, ADSC in small lung biopsies could be misdiagnosed as SqCC or non-small cell carcinoma (NSCC) favor SqCC due to undersampling, which may result in neglecting of EGFR mutation testing and affecting patients' clinical management, particularly in Asian patients that relatively have high prevalence of EGFR mutation. METHODS: A total of 148 small lung biopsy cases with pathological diagnosis of SqCC or NSCC favor SqCC were retrospectively enrolled. The frequency of EGFR mutations and the correlation between patients' EGFR mutation status and clinicopathological characteristics were evaluated. RESULTS: EGFR mutations were found in 8.8% (13 /148) of all cases with 5.2% (7/135) in SqCC and 46.2% (6/13) in NSCC favor SqCC. There were 7 (53.8%) L858R mutation, 4 (30.8%) exon 19 deletions, and 2 (15.4%) cases with coexistent L858R and T790 M mutations. Multivariate analysis showed that EGFR mutations were more prevalent in never-smokers (83.3% versus 16.7%, p = 0.006) and patients diagnosed as NSCC favor SqCC (46.2% versus 5.2%, p = 0.001). Moreover, 75% (3/4) of EGFR mutation-positive cases with subsequent surgical resection or rebiopsy were further diagnosed as ADSC. CONCLUSIONS: EGFR mutation testing should be performed in Asian patients with SqCC diagnosed from small lung biopsies, especially in never-smokers and patients with diagnosis of NSCC favor SqCC, which have a high probability of being ADSC.

Insulinoma-associated protein 1 is a robust nuclear immunostain for the diagnosis of small cell lung carcinoma in cytology smears.

BACKGROUND: In a significant percentage of patients with small cell lung carcinoma (SCLC), cytology samples represent the only source of tumor tissue. Ancillary immunocytochemistry (ICC) for neuroendocrine markers is an important adjunct for the diagnosis of SCLC. Insulinoma-associated protein 1 (INSM1) is a novel neuroendocrine marker proposed as an economical single-marker alternative to the traditional 3-marker panel of chromogranin, synaptophysin, and CD56. To the authors' knowledge, limited studies have evaluated INSM1 immunohistochemistry (IHC) for the diagnosis of SCLC and reported high sensitivities and specificities. The objective of the current study was to evaluate the sensitivity and specificity of INSM1 ICC on direct smears (DS) from patients with SCLC in comparison with IHC on small biopsies (SBs). METHODS: All available DS and SBs from patients with SCLC who were diagnosed over the previous year were retrieved. Immunostaining for INSM1 was performed on alcohol-fixed DS and formalin-fixed SBs wherever available. A total of 10 DS and SBs from patients with non-small cell lung carcinoma were included for comparison. Nuclear staining for INSM1 in ≥1% tumor cells was interpreted as positive. RESULTS: Among a total of 60 patients with SCLC who were included in the current study, a total of 37 underwent INSM1 IHC on SBs and 36 underwent INSM1 ICC on DS. ICC was noninterpretable in 3 DS due to necrosis. The sensitivity of INSM1 IHC was 97% (36 of 37 cases) whereas the sensitivity of INSM1 ICC was 91% (30 of 33 cases) for the diagnosis of SCLC. Among matched IHC and ICC results available for 11 patients, 91% of cases (10 of 11 patients) demonstrated concordant IHC-ICC staining. All cases of non-small cell lung carcinoma were negative for INSM1 (100% specificity). CONCLUSIONS: INSM1 appears to be a robust and reliable ICC marker for the confirmation of SCLC diagnosis on cytology smears.

Molecular Screening of Small Biopsy Samples Using Next-Generation Sequencing in Korean Patients with Advanced Non-small Cell Lung Cancer: Korean Lung Cancer Consortium (KLCC-13-01).

BACKGROUND: Non-small cell lung cancer (NSCLC) is a common type of cancer with poor prognosis. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in NSCLC might help predict patient outcomes and guide treatment. The aim of this study was to evaluate the clinical adequacy of molecular testing using next-generation sequencing (NGS) for small biopsy samples and characterize the mutational landscape of Korean patients with advanced NSCLC. METHODS: DNA was extracted from small biopsy samples of 162 patients with advanced NSCLC. Targeted NGS of genomic alterations was conducted using Ion AmpliSeq Cancer Hotspot Panel v2. RESULTS: The median age of patients was 64 years (range, 32 to 83 years) and the majority had stage IV NSCLC at the time of cancer diagnosis (90%). Among the 162 patients, 161 patients (99.4%) had novel or hotspot mutations (range, 1 to 21 mutated genes). Mutations were found in 41 genes. Three of the most frequently mutated genes were TP53 (151, 93.2%), KDR (104, 64.2%), and epidermal growth factor receptor (EGFR; 69, 42.6%). We also observed coexistence of EGFR and other oncogene (such as KRAS, PIC3CA, PTEN, and STK11) mutations. Given that 69.6% (48/69) of EGFR mutant patients were treated with EGFR tyrosine kinase inhibitors, EGFR mutant status had higher prognostic ability in this study. CONCLUSIONS: These results suggest that targeted NGS using small biopsy samples is feasible and allows for the detection of both common and rare mutations in NSCLC.

Accuracy of cytology in sub typing non small cell lung carcinomas.

BACKGROUND: Sub typing of non small cell lung carcinoma (NSCLC) has an important task in the era of molecular and targeted therapies. Differentiating between squamous cell carcinoma (SQCC) and adenocarcinoma (ADC) is challenging when limited material is available in lung carcinoma. We investigated the accuracy and feasibility of sub typing NSCLCs in cytology and small biopsy material. METHODS: Concurrent cytology and biopsy material obtained in a single CT- guided procedure in lung carcinoma over a year period retrospectively. Both materials were individually sub typed and analyzed. Immunohistochemistry (IHC) was performed. Accuracy was determined by comparing the results with IHC. RESULTS: Total 107 of 126 cases of NSCLCs were included for analysis, where both cytology and biopsy material were adequate for interpretation. FNAC allowed tumor typing in 83 (77.6%) cases; 36 (33.6%) were ADC, 47 (43.9%) cases were SQCC and 24 (22.4%) cases diagnosed as Non-small cell carcinoma not otherwise specified (NSCLC-NOS). In biopsy, 86 cases (80.4%) were typed, among which 34 (31.8%) were ADC, 52 (48.6%) were SQCC and 21 (19.6%) were of NSCLC-NOS type. The result of Chi-square index was significant. With the aid of IHC, NSCLC-NOS reduced from 14 (13%) cases to 2 (1.9%) cases. CONCLUSION: Cytology and small biopsy specimens achieved comparable specificity and accuracy in sub-typing NSCLC and optimal results were obtain when findings from both modalities combine. The advantage of paired specimens is to maximize overall diagnostic yield and the remaining material will be available for ancillary technique like IHC or for molecular testing. Diagn. Cytopathol. 2017;45:598-603. © 2017 Wiley Periodicals, Inc.