A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

Quantitative and qualitative analysis of stain color using RGB computer color specification

OBJECTIVE: To establish standardized Papanicolaou stain for cytology using RGB color specification. This new method was formerly used in DTP software application for computer color specification. STUDY DESIGN: RGB color specification was taken from a color film, optical constituents of which were made into computer software. Cell samples used in this study were from 100 sputum specimens stained with Papanicolaou stain. We analyzed the color tone of the cytoplasm of squamous cells in the smear. RESULTS: The R and B value of eosinophilic cells were demonstrated statistically by different values between normal, borderline atypical and malignant squamous cells. G and B values of light green-philic cells demonstrated a statistical difference between normal, borderline atypical and malignant squamous cells. No significant differences were found in RGB value between normal, borderline atypical and malignant squamous orangeophilic cells. CONCLUSION: Using our own method of analyzing Papanicolaou-stained sputum, a new quantitative and qualitative analysis of stain color for standardized Papanicolaou stain was introduced.     

Evaluation of applying feulgen stain for DNA analysis on destained hematoxylin-eosin-stained cytologic smears

OBJECTIVE: To evaluate the feasibility and reliability of DNA analysis performed on the original hematoxylin-eosin (HE)-stained cytologic specimens by destaining the slides and restaining with the Feulgen method. STUDY DESIGN: Image cytometric analysis of DNA ploidy status was performed in a comparative study on 20 cytologic preparations from 10 nasopharyngeal carcinomas. Ten smears were stained directly by the Feulgen method, and the others were Feulgen stained after HE destaining. RESULTS: There was 90% overall concordance in DNA determination and a good correlation (r = .97, P < .001) between the DNA indices determined by the 2 methods. Discordance was probably due to tumor heterogeneity. CONCLUSION: This study demonstrated that image cytometric DNA analysis on previously routinely HE-stained cytologic preparations is feasible and reliable. This method permits retrospective studies on archival cytologic material from patients with long term follow-up data.     

Computerized analysis of cellular features and biomarkers for cytologic diagnosis of early lung cancer

OBJECTIVE: To present a set of novel computerized analysis algorithms to construct a computer-aided cytologic diagnosis (CACD) system to differentiate lung cancer biomarkers and identify cancer cells in the tissue-based specimen images. STUDY DESIGN: Molecular methods, including application of cancer-specific markers, may prove to be complementary to cytology diagnosis, especially when they are combined with CACD system for biomarker assessment. We trained a novel CACD system to recognize expression of the cancer biomarkers histone H2AX in lung cancer cells and then tested the accuracy of this system to distinguish resected lung cancer from preneoplastic and normal tissues. The major characteristics of CACD algorithms is to adapt detection parameters according to cellular image contents. Our newly developed wavelet transform is able to adaptively select different resolution and orientation features based on image content requirements. RESULTS: Visual, statistical and quantitative results as CACD performance evaluation are presented in this paper. CONCLUSION: The presented algorithms and CACD system for cellular feature enhancement, segmentation and classification are very important in distinguishing benign and malignant lesions.     

Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique

The use of immunoperoxidase techniques was investigated in 21 fine needle aspiration (FNA) cytology smears that had been previously stained by the Papanicolaou technique. The retrospectively selected slides were destained before applying the immunostain, utilizing antisera to calcitonin, prostatic acid phosphatase (PrAP), prostate-specific antigen (PSA), alpha-lactalbumin (AL), S-100 protein (S-100), carcinoembryonic antigen (CEA), common leukocyte antigen (LA), epithelial membrane antigen (EMA) and alpha-fetoprotein (AFP). Positive results were obtained with six of nine small-cell carcinomas of the lung stained with EMA, all three colonic carcinomas stained with CEA, one of two prostatic carcinomas stained with PSA and PrAP, one of two lymphomas stained with LA and the one medullary thyroid carcinoma stained with calcitonin. Negative staining results were observed in the one melanoma stained with S-100, the two breast carcinomas stained with AL and the one hepatocellular carcinoma stained with AFP. These results indicate that immunostaining can be a helpful diagnostic tool in diagnosing some fine needle aspirates using smears previously stained with the Papanicolaou stain.     

Reliability of cytologic typing of lung cancer.

In a prospective study of 111 cases of lung cancer, cytopathologic diagnoses were compared with histologic diagnoses. In 77.5 per cent of the cases, histopathologic diagnoses were in concurrence with cytopathologic diagnoses. The discrepancies occurred mainly in poorly differentiated adeno and epidermoid carcinoma. Well differentiated adeno and epidermoid carcinoma were cell typed with 100 per cent accuracy and small cell carcinoma with 90 per cent accuracy.     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

A novel method of sputum processing for cytologic diagnosis of lung cancer

OBJECTIVE: To compare the diagnostic sensitivity and cytologic findings of the sputum-processing method with those of the traditional sputum smear method. STUDY DESIGN: From May to December 2001, 300 consecutive sputum samples were collected from 168 patients suspected of having lung cancer in the Chest Department, Taipei Veterans General Hospital. After the sputum smear method, the remaining sputum material was processed by homogenization, filtration and fixation. All the slides were stained with Papanicolaou stain and reviewed by 2 cytologists. RESULTS: Of the 300 sputum samples, 141 from 79 patients were finally diagnosed as lung malignancies. The mean number of sputum samples was 1.78 per patient. Among the 79 patients, 46 had peripheral lung malignancies (58.2%). The overall diagnostic sensitivities of sputum smear and sputum-processing methods were 29% and 31%, respectively (P = .776). Tumor location and cell types did not change the difference significantly; however, among patients with small cell lung cancer, there was a higher detection rate with the sputum-processing method (50% vs. 20%, P = .350). Eight patients with negative results with the sputum smear had positive results with the sputum-processing method (8 of 79 = 10.1%). Microscopic morphologic differences between the 2 methods were described. CONCLUSION: There was no significant difference in diagnostic sensitivity between the sputum smear and sputum-processing methods. However, the sputum-processing method may play a role in patients with small cell lung carcinoma.     

Quantitative monitoring of mouse lung tumors by magnetic resonance imaging

Primary lung cancer remains the leading cause of cancer-related death in the Western world, and the lung is a common site for recurrence of extrathoracic malignancies. Small-animal (rodent) models of cancer can have a very valuable role in the development of improved therapeutic strategies. However, detection of mouse pulmonary tumors and their subsequent response to therapy in situ is challenging. We have recently described MRI as a reliable, reproducible and nondestructive modality for the detection and serial monitoring of pulmonary tumors. By combining respiratory-gated data acquisition methods with manual and automated segmentation algorithms described by our laboratory, pulmonary tumor burden can be quantitatively measured in approximately 1 h (data acquisition plus analysis) per mouse. Quantitative, analytical methods are described for measuring tumor burden in both primary (discrete tumors) and metastatic (diffuse tumors) disease. Thus, small-animal MRI represents a novel and unique research tool for preclinical investigation of therapeutic strategies for treatment of pulmonary malignancies, and it may be valuable in evaluating new compounds targeting lung cancer in vivo.     

Targeted therapy for LIMD1-deficient non-small cell lung cancer subtypes

An early event in lung oncogenesis is loss of the tumour suppressor gene LIMD1 (LIM domains containing 1); this encodes a scaffold protein, which suppresses tumorigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD1, yet this subtype of NSCLC has been overlooked in preclinical and clinical investigations. Defining therapeutic targets in these LIMD1 loss-of-function patients is difficult due to a lack of ‘druggable’ targets, thus alternative approaches are required. To this end, we performed the first drug repurposing screen to identify compounds that confer synthetic lethality with LIMD1 loss in NSCLC cells. PF-477736 was shown to selectively target LIMD1-deficient cells in vitro through inhibition of multiple kinases, inducing cell death via apoptosis. Furthermore, PF-477736 was effective in treating LIMD1^−/− tumours in subcutaneous xenograft models, with no significant effect in LIMD1^+/+ cells. We have identified a novel drug tool with significant preclinical characterisation that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need.Subject terms: Targeted therapies, Non-small-cell lung cancer     

A novel classification of lung cancer into molecular subtypes

The remarkably heterogeneous nature of lung cancer has become more apparent over the last decade. In general, advanced lung cancer is an aggressive malignancy with a poor prognosis. The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer, however, has created new opportunities for targeted therapy and improved outcome. In this paper, we define "molecular subtypes" of lung cancer based on specific actionable genetic aberrations. Each subtype is associated with molecular tests that define the subtype and drugs that may potentially treat it. We hope this paper will be a useful guide to clinicians and researchers alike by assisting in therapy decision making and acting as a platform for further study. In this new era of cancer treatment, the 'one-size-fits-all' paradigm is being forcibly pushed aside-allowing for more effective, personalized oncologic care to emerge.     

Heterogeneity of lipidomic profiles among lung cancer subtypes of patients

Lung cancer is a leading cause of cancer‐related deaths with an increasing incidence and poor prognoses. To further understand the regulatory mechanisms of lipidomic profiles in lung cancer subtypes, we measure the profiles of plasma lipidome between health and patients with lung cancer or among patients with squamous cell carcinomas, adenocarcinoma or small cell lung cancer and to correct lipidomic and genomic profiles of lipid‐associated enzymes and proteins by integrating the data of large‐scale genome screening. Our studies demonstrated that circulating levels of PS and lysoPS significantly increased, while lysoPE and PE decreased in patients with lung cancer. Our data indicate that lung cancer‐specific and subtype‐specific lipidomics in the circulation are important to understand mechanisms of systemic metabolisms and identify diagnostic biomarkers and therapeutic targets. The carbon atoms, dual bonds or isomerism in the lipid molecule may play important roles in lung cancer cell differentiations and development. This is the first try to integrate lipidomic data with lipid protein‐associated genomic expression among lung cancer subtypes as the part of clinical trans‐omics. We found that a large number of lipid protein‐associated genes significantly change among cancer subtypes, with correlations with altered species and spatial structures of lipid metabolites.     

Relationship between patient characteristics and the sputum cytologic diagnosis of lung cancer

In 421 patients with a malignant lung process, from whom samples of sputum of satisfactory quality were received, patient characteristics relevant to the cytologic diagnosis of malignancy were investigated. In patients with primary lung cancer, the presence of blood in the sputum was highly significant from the point of view of its association with a correct positive cytologic diagnosis on sputum. The same relationship was noted in patients with metastatic lung cancer. In patients producing bloody sputum, the examination of at least three sputum samples gave a proportion of correct positive diagnoses of 0.88 in primary lung cancer patients and of 0.77 in patients with metastatic lung disease. Furthermore, a high sensitivity of the sputum cytology diagnosis of malignancy was found in primary lung cancer patients with low forced expiratory volume values (less than 50% of the vital capacity), with large tumors (greater than 24 mm in diameter) and with squamous-cell cancers. A central location of the tumor correlated with significantly better cytodiagnostic results in patients with both primary and metastatic cancers.     

Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections

The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitative immunoprofiles of breast cancer performed by image analysis

OBJECTIVE: To determine the biopathologic profiles of breast cancer for greater knowledge of tumor natural history and clinical outcome. STUDY DESIGN: In 99 in situ (ISC) and 2718 infiltrating breast carcinomas (IC), biologic markers (estrogen receptor [ER], progesterone receptor [PR] proliferation index, cerbB-2/NEU, p53, bcl-2 and DNA ploidy) were evaluated with an image analysis system (CAS 200/486). In 105 mixed invasive cancers with size < or = 1 cm, a separate analysis of in situ (ISCm) and invasive component (ICm) was obtained. A clinical study of 836 invasive breast cancers was performed. RESULTS: Different biophenotypes were obtained: among ISCs, cribriform type exhibited biologic behavior similar to that of normal breast tissue (ER+, PR+, proliferation index [PI] low, NEU-, p53-, bcl-2+) the opposite profile was displayed by comedo type, and intermediate phenotypes were observed in noncomedo and lobular types. Comparing ISC and ISCm, PI and p53 expression had the highest levels in ISCm with respect to other groups. NEU overexpression exhibited a decreasing value from ICm to IC. Younger women (< or = 40 years) with IC demonstrated a worse biologic profile (high PI, p53+, ER- and size > 2 cm). In multivariate analysis, PI and NEU in node-negative patients, and NEU, PR and size in node-positive ones emerged as prognostic parameters. CONCLUSION: The results underline the importance of the quantitative biologic profile for defining tumor behavior and patient management.     

Quantitative computer analysis of signal sequence homologies in DNA

Homologies to prokaryotic recognition sites for RNA polymerase,ribosomes, and cyclic-AMP receptor protein (CRP), are analyzedby a new computer program using weighting factors to accountfor the statistical variation at each position of the consensus.Known signal sequence sites are easily detected by this algorithm,and other sites with equally strong homology are found whosebiological function is still unknown. Some sites are biologicallyactive even though they have very weak homology. No arbitrary‘cutoff score’ can distinguish active recognitionsites from inactive homologies; experiments must determine whycertain weak homologies are able to function while others arenot. Received on May 12, 1986; accepted on August 8, 1986