First case report of M1 macrophage polarization in an untreated symptomatic patient with toxoplasmosis.

BACKGROUND: In immunocompetent patients, acute toxoplasmosis is usually asymptomatic. We identified M1 macrophages in a case of symptomatic acute Toxoplasma gondii infection that resolved without treatment. M1 macrophages have been demonstrated in animal models of toxoplasmosis, but not in humans. CASE PRESENTATION: A 63-year-old woman presented with laterocervical and axillary bilateral lymphadenopathy. Her anamnesis defined an episode of high fever and prolonged asthenia 4 months previously, which suggested an infectious disease. Following laboratory, radiological, and pathological analyses, she was diagnosed with toxoplasmosis. Immunohistochemical analyses were performed on lymph node sections. More than 50% of the macrophages in the lymph node microgranulomas were M1 macrophages, defined by CD68+/p-Stat1+ staining, and the presence of T helper 1 lymphocytes indicated an immune response known to induce M1 macrophage polarization. Activated endothelial cells were found only in inflamed areas. No therapy was administered before or after diagnosis, and the lymphadenopathy resolved after a follow-up of 5 months. CONCLUSIONS: This is the first report to demonstrate the presence of M1 macrophages in human toxoplasmosis. Our findings contribute to the understanding of the pathogenesis of toxoplasmosis, and encourage further studies on the role of macrophage polarization in human toxoplasmosis.

The lack of epithelial interleukin-7 and BAFF/BLyS gene expression in prostate cancer as a possible mechanism of tumor escape from immunosurveillance.

PURPOSE: The human prostate is endowed with intraepithelial and stromal lymphocytes, which may develop lymphoid follicles (LF) and allow a local immune response. We sought to investigate whether interleukin (IL)-7 and BAFF/BLyS, two fundamental survival factors for T and B cells, are expressed in the normal and neoplastic prostate and affect intraprostatic lymphocyte homeostasis. EXPERIMENTAL DESIGN: We have used real-time reverse transcription-PCR of microdissected prostatic glands and confocal microscopy to detect cytokine production, combined with immunohistochemistry to characterize intraprostatic lymphocytes. RESULTS: Prostatic epithelia constitutively produce IL-7 and, to a lesser extent, BAFF/BLyS. Indeed, we show that IL-7 receptor alpha is expressed by intraepithelial T lymphocytes and parafollicular T cells, whereas BAFF-R is found on periglandular B lymphocytes and mantle zone B cells of LFs. Prostate-homing B and T lymphocytes are scarcely proliferating, whereas most of them express the antiapoptotic protein bcl-2 and reveal a low apoptotic index in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The transition from normal to neoplastic glands in prostate cancer (PCa) is marked by a dramatic decline of IL-7 and BAFF/BLyS production. Accordingly, PCa is characterized by a significant reduction of intraepithelial lymphocytes and loss of LFs. B-cell and T-cell expression of bcl-2 decrease, whereas the apoptotic events increase. The remaining PCa-infiltrating lymphocytes are mostly CD8(+) T cells that lack terminal differentiation and barely penetrate neoplastic glands. CONCLUSIONS: These results suggest that epithelial IL-7 and BAFF/BLyS production support intraprostatic lymphocyte survival. Its loss in PCa is associated with a severe depletion of prostate-associated lymphocytes and points to a novel tumor escape mechanism.

Frequent mutations in the neurotrophic tyrosine receptor kinase gene family in large cell neuroendocrine carcinoma of the lung.

The neurotrophic tyrosine receptor kinase (NTRK) family is potentially implicated in tumorigenesis and progression of several neoplastic diseases, including lung cancer. We investigated a large number of pulmonary neuroendocrine tumors (PNETs) and non-small cell lung carcinomas (NSCLCs) without morphological evidence of neuroendocrine differentiation for mutations in the NTRK gene family. A total of 538 primary lung carcinomas, including 17 typical carcinoids (TCs), 10 atypical carcinoids (ACs), 39 small cell lung carcinomas (SCLCs), 29 large cell neuroendocrine carcinomas (LCNECs), and 443 NSCLCs were evaluated by single-strand conformation polymorphism (SSCP) and sequencing of the tyrosine kinase domain (TKD) of NTRK1, NTRK2, and NTRK3. The NTRK1 gene was never found to be mutated. A total of 10 somatic mutations were detected in NTRK2 and NTRK3, mostly located in the activating and catalytic loops. NTRK mutations were seen in 9 (10%) out of 95 PNETs but in 0 out of 443 NSCLCs investigated. No mutations were observed in TCs, ACs, and SCLCs. Interestingly, all the mutations were restricted to the LCNEC histotype, in which they accounted for 31% of cases. A mutational analysis, performed after microdissection of LCNECs combined with adenocarcinoma (ADC), showed that only neuroendocrine areas were positive, suggesting that NTRK mutations are involved in the genesis of the neuroendocrine component of combined LCNECs. Our data indicate that somatic mutations in the TKD of NTRK genes are frequent in LCNECs. Such mutational events could represent an important step in the cancerogenesis of these tumors and may have potential implications for the selection of patients for targeted therapy.

Multicenter Comparison of 22C3 PharmDx (Agilent) and SP263 (Ventana) Assays to Test PD-L1 Expression for NSCLC Patients to Be Treated with Immune Checkpoint Inhibitors.

INTRODUCTION: Among the several agents targeting the programmed cell death 1 (PD-1) pathway, pembrolizumab is currently the only one approved for the treatment of patients with NSCLC in association with a companion diagnostic assay, the anti-PD-L1 immunohistochemical (IHC) 22C3 PharmDx (Agilent Technologies, Santa Clara, CA) using the Dako Autostainer (Dako, Carpinteria, CA). However, the Dako platform is not present in each pathology department, and this technical limitation is a major problem for the diffusion of the PD-L1 IHC predictive test for pembrolizumab. METHODS: The Italian Society of Anatomic Pathology and Cytopathology and the Italian Association of Medical Oncology in an independent, multicenter study compared the in vitro diagnostics PD-L1 IHC 22C3 pharmDx test (Agilent) on the Dako Autostainer and the in vitro diagnostics Ventana PD-L1 (SP263) test on the Ventana BenchMark platform (Ventana Medical Systems, Tucson, AZ). Using serial sections from tissue microarrays, 100 lung adenocarcinomas were locally stained and scored in four centers with the same antibody batches. RESULTS: A high analytical correlation (more than 90% at the lower 95% confidence interval [CI] value) between PD-L1 expression levels obtained with the 22C3 and SP263 assays was observed. At the proposed clinically relevant cutoffs (≥50% and ≥1%), the overall concordances between 22C3 and SP263 data were 0.99 (95% CI: 0.96-1) and 0.80 (95% CI: 0.68-0.91), respectively. The lower agreement between data obtained with the 22C3 and SP263 clones at the cutoff of 1% or higher was mainly related to the lower (about 80%) interrater agreement at this cutoff with each clone. CONCLUSIONS: These results indicate a high correlation between PD-L1 IHC expression data obtained with the Agilent PD-L1 IHC 22C3 pharmDx and the Ventana PD-L1 (SP263) tests in NSCLC and suggest that the two assays could be utilized interchangeably as an aid to select patients for first-line and second-line treatment with pembrolizumab and potentially with other anti-PD-1/PD-L1 checkpoint inhibitors.

ROS1 Gene Fusion in Advanced Lung Cancer in Women: A Systematic Analysis, Review of the Literature, and Diagnostic Algorithm.

PURPOSE: Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non-small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. MATERIAL AND METHODS: A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. RESULTS: ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement (P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). CONCLUSION: Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

Int6 expression can predict survival in early-stage non-small cell lung cancer patients.

PURPOSE: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up. EXPERIMENTAL DESIGN: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues. RESULTS: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P=0.0006) and disease-free (P=0.024) survival. CONCLUSIONS: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.

ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm.

INTRODUCTION: Recent regulatory changes have allowed the diagnostic use of immunohistochemical (IHC) analysis for the identification of patients with non-small cell lung cancer who are eligible for treatment with anaplastic lymphoma receptor tyrosine kinase (ALK) inhibitors. The U.S. Food and Drug Administration has approved the VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) as companion diagnostics, and the Italian Medicines Agency has recognized IHC analysis as a diagnostic test indicating an algorithm for patient selection. METHODS: On the basis of the new regulations, we compared two commonly used IHC assays on 1031 lung adenocarcinomas: the VENTANA ALK (D5F3) CDx Assay with the OptiView Amplification Kit (Ventana Medical Systems) and a standard IHC test with the clone 5A4 (Novocastra, Leica Biosystems, Newcastle Upon Tyne, United Kingdom) along with their interpretative algorithms. Fluorescence in situ hybridization (FISH) was performed in all cases. Next-generation sequencing was performed in FISH/IHC analysis-discordant samples. RESULTS: FISH gave positive results in 33 (3.2%) cases. When FISH was used as a reference, the VENTANA ALK (D5F3) CDx assay had a sensitivity of 90.9% ± 2.6%, a specificity of 99.8% ± 0.6%, and positive and negative predictive values of 93.8% ± 2.1% and 99.7% ± 0.6%, respectively. The clone 5A4-based IHC test showed a sensitivity of 90.9% ± 2.6%, a specificity of 98.3% ± 1.3%, and positive and negative predictive values of 63.8% ± 4.2% and 99.7% ± 0.6%, respectively. Five cases with IHC analysis/FISH-discordant results in our series were analyzed together with those previously reported in the literature. Overall, data from 35 patients indicate a response rate to ALK inhibitors in 100% of FISH-negative/IHC analysis-positive cases (seven of seven) and 46% of FISH-positive/IHC analysis-negative cases (13 of 28), respectively. CONCLUSIONS: Our results confirm the difficulty in managing an IHC test without amplification in the absence of confirmatory FISH analysis, as well as the possibility of performing a direct diagnosis in approximately 90% of patients by the VENTANA ALK (D5F3) CDx Assay. On the basis of the recent regulatory changes, the data that have emerged from the literature, and the results of the present study, a new algorithm for ALK assessment in non-small cell lung cancer has been devised.

Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.

OBJECTIVES: Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3-5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests. MATERIAL AND METHODS: A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing. RESULTS: Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively. CONCLUSIONS: Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.

Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications.

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16(INK4a)/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16(INK4a)/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16(INK4a)/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing.

PURPOSE: The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells. EXPERIMENTAL DESIGN: We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS. RESULTS: In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS. CONCLUSIONS: The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.