Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

Detection of poliovirus-infected macrophages in thymus of patients with myasthenia gravis.

BACKGROUND: Genetic and environmental factors are thought to contribute to the etiology of the autoimmune disease myasthenia gravis (MG). Viral involvement has long been suspected, but direct evidence of involvement has not been found. We recently reported that Toll-like receptor 4 (TLR4)-a key activator of innate immunity-was overexpressed in the thymus of some patients with MG, suggesting that thymic infection by pathogens might be involved in MG pathogenesis. We searched for evidence of intrathymic infection in patients with MG. METHODS: Twenty-seven MG thymuses (6 involuted, 7 hyperplastic, 5 thymitis, and 9 thymoma) previously tested for TLR4 expression, 18 nonpathologic control thymuses, and 10 pathologic control thymuses from patients without MG (8 thymoma and 2 hyperplastic) were analyzed for cytomegalovirus, varicella-zoster virus, herpes simplex virus types 1 and 2, eubacteria, respiratory syncytial virus, and enteroviruses using PCR techniques. Immunohistochemistry and double immunofluorescence were used to detect enterovirus capsid protein VP1 in thymic specimens and analyze TLR4 expression in VP1-positive cells. RESULTS: Poliovirus was detected in 4 MG thymuses (14.8%; 2 thymitis and 2 thymoma). No virus was detected in any control thymus. A linear correlation between plus and minus strand poliovirus RNA levels was observed in all 4 thymuses, suggesting persistent thymic infection. VP1 protein was detected in the cytoplasm of CD68-positive macrophages scattered through thymic medulla in all PV-positive thymuses. VP1 and TLR4 colocalized in infected cells. CONCLUSIONS: Poliovirus-infected macrophages are present in thymus of some patients with myasthenia gravis, suggesting a viral contribution to the intrathymic alterations leading to the disease.

Cervical cancer screening programs in low-income communities. Experiences from Ecuador. Low cost detection of HPV infection in a developing country.

OBJECTIVES: With the support of the independent humanitarian organization "Amici Fundation Terra Nueva" in Quito, Ecuador, we evaluated the feasibility of a cytologic screening program sustained by volunteers on the field and in Italy. METHODS: 250 women underwent a cervical Pap-test. The women with a positive Pap-smear were re-called for visual inspection with acetic acid (VIA), whereas those with a negative smear were invited for a new Pap-test after 3 years. To obtain samples for molecular assays, cytologic material was removed from slides, submitted to DNA extraction and amplified by nested PCR of the L1 region of HPV DNA. PCR-positive samples were sequenced. RESULTS. Six (2.6%) samples showed squamous intra-epithelial lesions (SILs): 4 low grade and 2 high grade SILs were present in women more than 40 years old. The overall rate of successful DNA recovery on a per-slide basis was 96.5%. High grade SILs were characterized by HPV 16 and 18 co-infection. HPV 16 was detected in one low grade SIL. HPV-DNA was detected in 11 smears (4.95%): in all 6 SILS and in 5 of the 216 negative smears. CONCLUSION: Independent humanitarian organizations could play a role in supporting national screening programs offering skilled field professionals and technical support by scientists operating in their countries. Our molecular technique has the potential to provide important epidemiological information in many resource-poor areas of developing countries.

Facial granulomatous diseases: a study of four cases tested for the presence of Mycobacterium tuberculosis DNA using nested polymerase chain reaction.

The histopathologic diagnosis of cutaneous tuberculosis (CTB) is often troublesome, because there are several other entities (tuberculids, demodicidosis, granulomatous rosacea, and acne agminata) that may display granulomatous inflammation with caseation necrosis. The current study describes four cases of granulomatous disease of the face. The final diagnosis (assessed on the basis of the clinical response to therapy) was CTB in three cases and granulomatous rosacea in one case. Histologically, epithelioid granulomas were a constant feature; in one case of CTB, they displayed a palisading (granuloma annulare-like) arrangement. Caseation necrosis was a prominent feature only in the case of granulomatous rosacea. Routinely processed biopsy specimens were evaluated with nested polymerase chain reaction (nPCR) for Mycobacterium tuberculosis (MBT) DNA. The correlation between nPCR results and clinical outcome was less than optimal; in fact, one case showed an excellent clinical response to the antituberculous drug therapy despite the absence of MBT DNA amplification. In granulomatous diseases of the face, the importance of evaluating not only nPCR but the overall clinicopathologic picture so as to avoid diagnostic misinterpretations is emphasized.

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields.

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF-MF). We report that ELF-MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re-established the apoptotic response to ELF-MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF-MF.

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples.

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.

Nested polymerase chain reaction on vaginal smears of tuberculous cervicitis. A case report.

BACKGROUND: Tuberculous cervicitis (TC) is a rare disease the diagnosis of which is based on the microscopic and/or cultural recognition of mycobacteria. In recent years, the polymerase chain reaction (PCR), especially with double-round amplification ("nested" PCR [nPCR]), has been increasingly used for rapid detection of mycobacteria in clinical samples. CASE: The present case is the first example of tuberculosis diagnosed with the aid of nPCR amplification of mycobacterial DNA fragments on smeared and Papanicolaou-stained cytologic material. First detected on vaginal smears, the amplicon IS6110 was subsequently identified also on paraffin-embedded tissue sections. CONCLUSION: The technique described here could also be applied to aspiration cytology smears to give rapid and accurate information on mycobacterial infections.

Human Immunodeficiency Virus Localization in Human Papillomavirus-Related, High-Grade Squamous Intraepithelial Lesions of the Cervix in Women with HIV Infection: Microdissection and Molecular Analysis on Formalin-Fixed and Paraffin-Embedded Specimens.

OBJECTIVES: To evaluate a possible mechanism of human immunodeficiency virus (HIV) and human papillomavirus (HPV) interaction, we have identified the cervical tissue compartments that harbor HIV. MATERIALS AND METHODS: We studied 39 paraffin-embedded, cervical conization specimens with high-grade cervical intraepithelial neoplasia (CIN3) occurring in HIV-infected women. From selected intraepithelial HPV-positive nonulcerated specimens (confirmed by in situ hybridization), we obtained serial 4- to 5-µm-thick sections that were stained with hematoxylin and eosin, anti-S100 protein, and anti-CD4. The presence of intramucosal Langerhans' cells or dendritic cells or CD4-positive cells was recorded. Three consecutive, nonmicrodissected, full-thickness sections of the same specimens were used for polymerase chain reaction (PCR) analysis (group A). Three other uncovered, consecutive sections from the same blocks were examined with an inverted microscope, and full-thickness specimens of mucosa were dissected from the underlying cervical stroma, were gently removed, and were used for PCR (group B). The quality of DNA was checked by HLA-DQa amplification; then a nested PCR for HIV proviral DNA was performed. RESULTS: Of group A, 5 of 39 cases (12.8%) were positive, whereas HIV was not detected in the microdissected sections of group B, with or without intraepithelial Langerhans' or CD4 cells. CONCLUSIONS: HIV does not affect cervical epithelium. The absence of infected Langerhans' or dendritic cells (or both) indicates a migration to the proximal lymph nodes of the in ….

Local impairment of immunoreactivity in HIV-infected women with HPV-related squamous intraepithelial lesions of the cervix.

AIMS AND BACKGROUND: The aim of this study was to compare the local immune response in two groups of patients with high-grade cervical intraepithelial squamous lesions (SIL): one with HIV infection and the other with HPV infection alone. MATERIALS AND METHODS: 16 conization specimens (8 from HIV-infected and 8 from non-HIV-infected patients) of HPV-related, high-grade SIL were selected. The specimens from non-HIV patients were considered as controls. The total number of Langerhans cells, CD4 and CD8 cells present in 10 field areas (3.120 mm2) was recorded in each case. In HIV patients CD4 and CD8 peripheral counts were performed immediately before surgery. RESULTS: The CD4/CD8 ratio never exceeded 0.71, whereas the lowest ratio in controls was 0.81: this difference was statistically significant (P = 0.0009). The mean number of Langerhans cells was markedly reduced in the high-grade SILs in the HIV patients in comparison with controls (P = 0.001). The number of CD4 cells and the CD4/CD8 ratio correlated with the peripheral CD4 count (P = 0.001 and 0.02). CONCLUSIONS: In our study a marked local impairment of cervical immunoreactivity was observed, which may play a major role in the progression of these lesions in HIV-infected women.

Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissues with granulomatous diseases for rapid diagnosis of tuberculosis.

We evaluated the sensitivity and specificity of a nested polymerase chain reaction (PCR) to the Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue samples from patients with tubercular and other granulomatous lesions. Five groups of patients and samples were studied: (1) 28 samples from HIV-positive patients with tuberculosis, (2) 8 samples from HIV-negative patients with histologically suspected tuberculosis (confirmed by culture in 5 cases), (3) lymph nodes from 5 HIV-positive patients with Mycobacterium avium-intracellulare infection, (4) lymph nodes from 30 patients with sarcoidosis, and (5) specimens from 17 patients with other granulomatous diseases. The DNA was extracted from sections with a total thickness of 60 microm, and PCR amplified an internal fragment of 123 base pairs. All of the cases with M. tuberculosis infection were PCR-positive, although this sensitivity was partially related to the initial concentration of the DNA used for amplification. Two of the group 4 samples also were repeatedly positive, thus reducing the specificity of the method. All of the cases with granulomatous diseases other than sarcoidosis were negative. We propose a simplified and highly sensitive nested PCR for the diagnosis of M. tuberculosis infection on archived material in HIV-positive and HIV-negative patients.

[Detection of mycobacterium tuberculosis DNA using nested polymerase chain reaction in lymph nodes with sarcoidosis, fixed in formalin and embedded in paraffin]. / Ricerca del DNA del Mycobacterium tuberculosis mediante nested Polymerase Chain Reaction in linfonodi con sarcoidosi fissati in formalina e inclusi in paraffina.

An involvement of mycobacteria in the pathogenesis of sarcoidosis has often been hypothesized, but not confirmed reproducibly. In this study we applied a nested Polymerase Chain Reaction (PCR) to the insertion sequence IS6110 for detection of Mycobacterium tuberculosis (MT) DNA in formalin-fixed and paraffin-embedded tissues from patients with sarcoidosis, with tuberculosis and atypical mycobacteriosis confirmed by culture, and from negative control samples. MT-DNA could be detected in 2/30 samples of sarcoidosis, in 10/10 tuberculoses, in 0/5 atypical mycobacterioses and in 0/10 negative controls. Nested PCR confirmed its high sensitivity and specificity in detecting MT-DNA on archival histopathological specimens. From our results we conclude that in the granulomatous lesions of sarcoidosis MT-DNA is only sporadically demonstrable and probably it doesn't play a role in the pathogenesis of this disease.