Strong Notch activation hinders bevacizumab efficacy in advanced colorectal cancer.

AIM: To assess the role of Notch activation in predicting bevacizumab efficacy in colorectal cancer (CRC). MATERIALS & METHODS: Notch activation was evaluated by immunohistochemistry (IHC) on 65 CRC enrolled within randomized clinical trials assessing first-line bevacizumab-based chemotherapy and on 21 CRC treated with chemotherapy alone. RESULTS: Strong Notch (IHC 3+) activation was negatively associated with response (18 vs 62% in low Notch cases [IHC 0, 1, 2+]; p = 0.016), progression-free survival (4.9 vs 12.1 months; p = 0.002) and overall survival (19.3 vs 30.4 months; p = 0.039). No correlation was found between Notch activation and clinical outcome in CRC treated with chemotherapy alone. CONCLUSION: A potential role of Notch activation in the antitumor activity of bevacizumab could be hypothesized.

Notch-Jagged1 signaling and response to bevacizumab therapy in advanced colorectal cancer: A glance to radiomics or back to physiopathology?

Introduction: The Notch intracellular domain (NICD) and its ligands Jagged-1(Jag1), Delta-like ligand (DLL-3) and DLL4 play an important role in neoangiogenesis. Previous studies suggest a correlation between the tissue levels of NICD and response to therapy with bevacizumab in colorectal cancer (CRC). Another marker that may predict outcome in CRC is radiomics of liver metastases. The aim of this study was to investigate the expression of NICD and its ligands and the role of radiomics in the selection of treatment-naive metastatic CRC patients receiving bevacizumab. Methods: Immunohistochemistry (IHC) for NICD, Jag1 and E-cadherin was performed on the tissue microarrays (TMAs) of 111 patients with metastatic CRC treated with bevacizumab and chemotherapy. Both the intensity and the percentage of stained cells were evaluated. The absolute number of CD4+ and CD8+ lymphocytes was counted in three different high-power fields and the mean values obtained were used to determine the CD4/CD8 ratio. The positivity of tumor cells to DLL3 and DLL4 was studied. The microvascular density (MVD) was assessed in fifteen cases by counting the microvessels at 20x magnification and expressed as MVD score. Abdominal CT scans were retrieved and imported into a dedicated workstation for radiomic analysis. Manually drawn regions of interest (ROI) allowed the extraction of radiomic features (RFs) from the tumor. Results: A positive association was found between NICD and Jag1 expression (p < 0.001). Median PFS was significantly shorter in patients whose tumors expressed high NICD and Jag1 (6.43 months vs 11.53 months for negative cases; p = 0.001). Those with an MVD score ≥5 (CD31-high, NICD/Jag1 positive) experienced significantly poorer survival. The radiomic model developed to predict short and long-term survival and PFS yielded a ROC-AUC of 0.709; when integrated with clinical and histopathological data, the integrated model improved the predictive score (ROC-AUC of 0.823). Discussion: These results show that high NICD and Jag1 expression are associated with progressive disease and early disease progression to anti VEGF-based therapy; the preliminary radiomic analyses show that the integration of quantitative information with clinical and histological data display the highest performance in predicting the outcome of CRC patients.

Optimizing PD-L1 evaluation on cytological samples from advanced non-small-cell lung cancer.

Aim: Programmed cell death-ligand 1 (PD-L1) predicts response to immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) patients. Most NSCLCs are diagnosed at an advanced stage and using minimally invasive diagnostic procedures that yield small biopsies or cytological samples. Methods: Cytological smears and paired histological samples from 52 advanced NSCLC patients were tested for PD-L1 expression by immunocyto/histochemistry (ICC/IHC) and for PD-L1 gene status by FISH. Results:PD-L1 was overexpressed in 9/52 (17%) cytological samples and in seven (13.5%) matched biopsies. The concordance between immunocytochemistry and IHC was 92.3% (48/52; p < 0.001). The concordance between PD-L1 gene status on cytology and histology was 69.2% (18/26; p < 0.001). No correlation between IHC and fluorescence in situ hybridization results was found. Conclusion: Our data support the feasibility and reliability of PD-L1 protein and PD-L1 gene assessment on direct cytological smears from NSCLC patients whenever histological sample are inadequate.

High levels of Notch intracellular cleaved domain are associated with stemness and reduced bevacizumab efficacy in patients with advanced colon cancer.

δ­like ligand 4 (DLL4)­Notch signaling is associated with tumor resistance to anti­vascular endothelial growth factor (VEGF) therapy. Furthermore, Notch signaling is critical for the maintenance of colon cancer stem cells (CSCs), which are relevant in drug resistance and tumor angiogenesis. CD44 is a transmembrane glycoprotein and is considered a putative marker of CSCs. To assess the association of Notch intracellular cleaved domain (NICD), DLL4 and CD44 expression with the efficacy of anti­angiogenic drugs, a series of samples derived from patients with advanced colon cancer enrolled in prospective clinical trials were analyzed. Histological samples from 51 primary tumors that originated from patients treated with bevacizumab­based first­line chemotherapy were analyzed by immunohistochemistry for NICD, DLL4 and CD44 expression, and CD31 for microvessel count. The expression levels of genes relevant for angiogenesis [angiopoietin (ANGPT)1, ANGPT2, fibroblast growth factor (FGF)1, FGF2, epidermal growth factor, placental growth factor, VEGFA and DLL4] were detected by reverse transcription­quantitative PCR using RNA extracted from the frozen tissues of four tumors with low and four tumors with high NICD expression. Strong NICD levels were observed in 12/51 (24%) of the patients, whereas 16/51 (31%) of the colon cancer subjects exhibited high CD44 expression. Strong CD44 staining was associated with high NICD levels compared with the CD44 expression levels noted in samples with low NICD levels (67 vs. 20%, P=0.005). No association was observed with regards to the expression levels of NICD, CD44 and the other aforementioned biomarkers. High expression levels of NICD and CD44 predicted reduced progression­free survival (P<0.001) and overall survival (P=0.002). No significant differences in the expression of angiogenesis­related genes were detected between low and high NICD­expressing tumors. In conclusion, NICD and CD44 tissue levels exhibited an association and may be related to a reduced survival rate in patients with advanced colon cancer treated with bevacizumab.

Buccal mucosa cells as in vivo model to evaluate gefitinib activity in patients with advanced non small cell lung cancer.

PURPOSE: To evaluate the role of pretreatment and posttreatment expression in buccal mucosa cells of signal transduction proteins activated by epidermal growth factor receptor, including phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated mitogen-activated protein kinase (p-MAPK), and phosphorylated AKT (p-AKT), in predicting gefitinib activity in advanced non-small cell lung cancer patients. Expression of the same proteins was also assessed on corresponding tissue samples for comparison. Moreover, EGFR gene mutations and copy number were analyzed. EXPERIMENTAL DESIGN: Protein expression was evaluated by standard immunocytochemistry in buccal smears, obtained by scraping immediately before and after 2 weeks of gefitinib treatment, and in the available archival tumor specimens. EGFR gene mutations were evaluated by direct sequencing and gene copy number was determined by fluorescence in situ hybridization. Data were correlated with gefitinib toxicity and objective response. RESULTS: Fifty-eight patients with pretreated advanced non-small cell lung cancer were enrolled and nine of these patients (15%) showed an objective response to gefitinib (including two complete responses). Toxicity (P = 0.025) and baseline p-AKT expression in buccal mucosa cells (P = 0.061) showed a potential predictive role. On the contrary, the probability of achieving an objective response was not affected by pretreatment expression of EGFR, p-EGFR, and p-MAPK, either in buccal mucosa or in tumor tissue. Responders showed a nonstatistically significant trend toward a more pronounced reduction in the expression of p-EGFR, p-MAPK, and p-AKT after gefitinib treatment. Among responders, five of six (83%) tumors showed EGFR gene mutation, whereas none of the tumors from patients with stable or progressive disease did (P < 0.001). CONCLUSIONS: Epithelial cells obtained from buccal mucosa may be used to assess the pharmacodynamic effect of EGFR-targeted agents, and pretreatment p-AKT expression may be a possible predictive biomarker of in vivo gefitinib activity.

Fingerprint Change: Not Visible, But Tangible.

Hand-foot syndrome, a chemotherapy-induced cutaneous toxicity, can cause an alteration in fingerprints causing a setback for cancer patients due to the occurrence of false rejections. A colon cancer patient was fingerprinted after not having been able to use fingerprint recognition devices after 6 months of adjuvant chemotherapy. The fingerprint images were digitally processed to improve fingerprint definition without altering the papillary design. No evidence of skin toxicity was present. Two months later, the situation returned to normal. The fingerprint evaluation conducted on 15 identification points highlighted the quantitative and qualitative fingerprint alteration details detected after the end of chemotherapy and 2 months later. Fingerprint alteration during chemotherapy has been reported, but to our knowledge, this particular case is the first ever reported without evident clinical signs. Alternative fingerprint identification methods as well as improved biometric identification systems are needed in case of unexpected situations.

Age dependent differences in the kinetics of γδ T cells after influenza vaccination.

Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age-related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19-30 years) and 23 elderly (53-67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67+ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy.

A Rare Case of Acute Myeloid Leukemia with a t(2;3) Chromosomal Translocation Characterized by Thrombophilia and Chemoresistance.

We hereby report a case of acute myeloid leukemia with translocation t(2;3) and involvement of the ectopic virus integration site-1 (EVI1) gene. Like most other 3q26-related disorders reported thus far, we describe a phenotype with elevated platelet counts and dysmegakaryopoesis. The clinical course of our patient was complicated by symptomatic thrombophilia and chemoresistance. In addition, our case exhibited FLT3 (Fms-related tyrosine kinase 3) internal tandem duplication. Although anagrelide was successful in controlling elevated platelet counts, allogeneic stem cell transplantation failed to overcome chemoresistance due to simultaneous graft-versus-host-disease and relapse of acute myeloid leukemia. Given the dismal outcome of our case and previously reported cases, we propagate the implementation of targeted therapies to newly diagnosed patients with acute myeloid leukemia t(2;3). Preclinical models indicate drugs that plausibly target the EVI1-related molecular vulnerability as candidates for basket trials. Anagrelide exhibited a hopeful signal of activity in 3q26-related thrombocytosis and should be evaluated for implementation as supportive care.

Highly Predictive Model for a Protective Immune Response to the A(H1N1)pdm2009 Influenza Strain after Seasonal Vaccination.

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young (<31 years, N = 17) versus elderly (≥50 years, N = 20) donors sero-negative to the newly emerged A(H1N1)pdm09 influenza virus strain and correlated it with the serological response to that specific strain after seasonal influenza vaccination. Extensive multi-chromatic FACS analysis (36 lymphocyte sub-populations measured) was used to quantitatively assess the cellular immune status before vaccination. We identified CD4+ T cells, and amongst them particularly naive CD4+ T cells, as the best correlates for a successful A(H1N1)pdm09 immune response. Moreover, the number of influenza strains a donor was sero-negative to at baseline (NSSN) in addition to age, as expected, were important predictive factors. Age, NSSN and CD4+ T cell count at baseline together predicted sero-protection (HAI≥40) to A(H1N1)pdm09 with a high accuracy of 89% (p-value = 0.00002). An additional validation study (N = 43 vaccinees sero-negative to A(H1N1)pdm09) has confirmed the predictive value of age, NSSN and baseline CD4+ counts (accuracy = 85%, p-value = 0.0000004). Furthermore, the inclusion of donors at ages 31-50 had shown that the age predictive function is not linear with age but rather a sigmoid with a midpoint at about 50 years. Using these results we suggest a clinically relevant prediction model that gives the probability for non-protection to A(H1N1)pdm09 influenza strain after seasonal multi-valent vaccination as a continuous function of age, NSSN and baseline CD4 count.

HER-2/neu evaluation by fluorescence in situ hybridization on destained cytologic smears from primary and metastatic breast cancer.

OBJECTIVE: To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain. STUDY DESIGN: Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears. RESULTS: HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears. CONCLUSION: HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.

FGFR as potential target in the treatment of squamous non small cell lung cancer.

To date therapeutic options for squamous cell lung cancer patients remain scarce because no druggable targets have been identified so far. Aberrant signaling by FGFs (fibroblast growth factors) and FGFRs (fibroblast growth factors receptors) has been implicated in several human cancers and, particularly, in squamous non-small cell lung cancer (NSCLC). FGFR gene amplifications, somatic missense mutations, chromosomal translocations are the most frequent mechanisms able to induce aberrant activation of this pathway. Data from literature have established that the presence of an aberrant FGFR signaling has to be considered a possible negative prognostic factor but predictive of potential sensitivity to FGFR inhibitors. In the last years, clinical research efforts allowed to identify and evaluate promising FGFR inhibitors, such as monoclonal antibodies, ligand traps, non-selective or selective tyrosine kinase inhibitors. This review summarizes the current knowledge about FGFR alterations in NSCLC and the relative inhibitors in development, in particular in squamous NSCLC.

Effects of aging on human leukocytes (part I): immunophenotyping of innate immune cells.

Immunosenescence is a hallmark of the aging immune system, leading to increased susceptibility to infections in the aged population and decreased ability to eradicate infectious pathogens. These effects, in turn, result in an increased burden on the healthcare system due to elevated frequency and duration of hospital visits. Growing evidence suggests that cells of the innate immune system are central modulators for the initiation and maintenance of an adequate pathogen-specific response through the adaptive immune system. While there are many reports on age-dependent alterations and dysfunctions of the adaptive immune system, the underlying mechanisms and effects of natural aging on the composition of the innate immune system remain unknown. Here, we present the results obtained from the comprehensive immunophenotyping of innate leukocyte populations, examined for age-related alterations within different sub-populations assessed using multi-parametric flow cytometry. We compared peripheral blood mononuclear cells from 24 young (aged 19-30 years) and 26 elderly (aged 53-67 years) donors. For classical CD16(+)CD56(dim) NK cells, the fraction of CD62L(+)CD57(+) was diminished in the elderly donors compared with young individuals, while the other investigated NK subsets remained unaffected by age. Both transitional monocytes and non-classical CD14(+-)CD16(++) monocytes were increased in the elderly compared with the young. The populations of pDCs and mDC2 were decreased among the elderly. These data demonstrate that the dynamics of the mDC subsets might counteract decreased virus surveillance. Furthermore, these data show that the maturation of NK cells might gradually slow down.

Effects of aging on human leukocytes (part II): immunophenotyping of adaptive immune B and T cell subsets.

Immunosenescence results from a continuous deterioration of immune responses resulting in a decreased response to vaccines. A well-described age-related alteration of the immune system is the decrease of de novo generation of T and B cells. In addition, the accumulation of memory cells and loss of diversity in antigen specificities resulting from a lifetime of exposure to pathogens has also been described. However, the effect of aging on subsets of γδTCR(+) T cells and Tregs has been poorly described, and the efficacy of the recall response to common persistent infections in the elderly remains obscure. Here, we investigated alterations in the subpopulations of the B and T cells among 24 healthy young (aged 19-30) and 26 healthy elderly (aged 53-67) individuals. The analysis was performed by flow cytometry using freshly collected peripheral blood. γδTCR(+) T cells were overall decreased, while CD4(+)CD8(-) cells among γδTCR(+) T cells were increased in the elderly. Helios(+)Foxp3(+) and Helios(-)Foxp3(+) Treg cells were unaffected with age. Recent thymic emigrants, based on CD31 expression, were decreased among the Helios(+)Foxp3(+), but not the Helios(-)Foxp3(+) cell populations. We observed a decrease in Adenovirus-specific CD4(+) and CD8(+) T cells and an increase in CMV-specific CD4(+) T cells in the elderly. Similarly, INFγ(+)TNFα(+) double-positive cells were decreased among activated T cells after Adenovirus stimulation but increased after CMV stimulation. The data presented here indicate that γδTCR(+) T cells might stabilize B cells, and functional senescence might dominate at higher ages than those studied here.

ALK and ROS1 rearrangements tested by fluorescence in situ hybridization in cytological smears from advanced non-small cell lung cancer patients.

BACKGROUND: The identification of ALK and ROS1 rearrangements and the availability of an effective target therapy, such as crizotinib, represent a new option in the treatment of advanced non-small cell lung cancer (NSCLC) patients. In light of recent advances in non-invasive diagnostic procedures, we aimed to demonstrate that direct cytological smears are suitable for assessing ALK and ROS1 rearrangements in patients with NSCLC. METHODS: Fifty-five patients with a cytological diagnosis of lung adenocarcinoma (ADC) were evaluated for ALK rearrangements by fluorescence in situ hybridization (FISH) and 12 patients for ROS1 FISH rearrangements. Seventeen of the 55 cytological samples tested for ALK were obtained from the primary tumor and 38 from metastatic lesions. Ten of 12 samples evaluated for ROS1 were obtained from metastatic sites and two from the primary tumor. RESULTS: ALK FISH was successful in 49/55 (89%) cytological ADC samples and ROS1 FISH in all 12 cytological samples. ALK rearrangements were found in 3/13 (23%) primary tumors and 7/36 (19%) metastatic sites. ROS1 rearrangements were found in one of the two primary tumors and in two of the 10 metastases. Two of the three rearranged cases were tested on cytology after knowing that they were rearranged on histology in order to increase representativeness of ROS1 rearranged cases in this study. CONCLUSION: Whenever cytology represents the only available material for diagnosis and biological characterization of NSCLC, minimally invasive procedures may provide an additional important source of cellular material for FISH assessment of ALK and ROS1 rearrangements.

Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method.

INTRODUCTION: The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. MATERIALS AND METHODS: Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. RESULTS: Based on morphology (nuclear dimension ≥10 µm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. CONCLUSIONS: Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

Biological parameters on computed tomography guided fine needle aspiration biopsy from peripheral primary non-small cell lung cancer.

Computed tomography (CT) guided fine needle aspiration biopsy (CT-guided FNAB) represents the procedure of choice for diagnosing peripheral primary lung cancer before surgery. The aim of the present study was to assess the reliability of the immunocytochemical evaluation of biological parameters and DNA flow cytometry on cellular material obtained from non-small cell lung cancer (NSCLC) patients by CT-guided FNAB. Thirty consecutive CT-guided FNABs obtained from NSCLC patients were submitted both to the immunocytochemical evaluation of p53, Ki67, bcl-2 and to flow cytometric DNA analysis. p53, Ki67 and bcl-2 were assessable in 60% (18/30), 53% (16/30) and 48% (10/21) of the cases, respectively. Flow cytometric DNA analysis was performed in 19 out of the 30 cases and 74% (14/19) of the histograms were evaluable. Cytofluorimetric S-phase fraction (SPF), was obtained in 57% (8/14) of the cases. The results of the current study suggest that CT-guided FNAB from primary NSCLC patients may represent an effective practice for the evaluation of biologic parameters and could be useful as a preoperative procedure. The role of neoadjuvant chemotherapy in operable NSCLC is still under debate. We suppose that in the future the presurgical characterization of NSCLC could suggest the opportunity of a neoadjuvant systemic treatment aimed to improve the clinical outcome. Moreover, in locally advanced or metastatic NSCLC immunocytochemistry could help to predict the response to chemotherapy and/or radiotherapy, avoiding ineffective treatments and supporting the development of more rational therapies.

Immunocytochemical evaluation of HER-2/neu on fine-needle aspirates from primary breast carcinomas.

Detection of HER-2/neu alterations is increasingly used in breast cancer patients for therapeutic purposes. This study examines the reliability of HER-2/neu immunocytochemical assessment on 66 cytospin smears obtained by fine-needle aspiration biopsy from breast cancer patients. Results were compared with those obtained by both fluorescence in situ hybridization (FISH) on fine-needle aspirate (FNA) and immunohistochemistry (IHC) on matched histologic section. Concordance between immunocytochemistry (ICC) and FISH was 78% and between ICC and IHC was 84%. Discordance mainly concerned seven unamplified cases that resulted positive by ICC and four cases scored negative by IHC but positive by ICC. Simultaneous assessment of HER-2/neu by ICC, IHC, and FISH was available in 24 cases; the concordance was 75%. In this study, the false positivity of immunocytochemical technique represents the major criticism. In our experience, FISH remains the most objective and powerful technique for HER-2/neu assessment on breast cancer FNAs.

CT-guided biopsy of pulmonary nodules: is pulmonary hemorrhage a complication or an advantage?

PURPOSE: We aimed to assess the correlation between pulmonary hemorrhage and pneumothorax in computed tomography (CT)-guided transthoracic fine needle aspiration (TTFNA), particularly its possible value as protection against the development of pneumotorax. MATERIALS AND METHODS: We reviewed the CT images of 538 patients (364 males and 174 females, mean age 70 years, range 36-90 years) who underwent CT-guided TTFNA of pulmonary nodules between January 2008 and September 2013. The following CT findings were assessed: pulmonary hemorrhage (type 1, along the needle track; type 2, perilesional; low-grade, ≤6 mm; high-grade, >6 mm), pneumothorax, distance between the target nodule and the pleural surface, and emphysema. RESULTS: Pneumothorax occurred in 154 cases (28.6%) and pulmonary hemorrhage occurred in 144 cases (26.8%). The incidence of pneumothorax was lower in patients showing type 1 and high-grade pulmonary hemorrhage pattern. The incidence of pneumothorax in biopsies ≥30 mm from pleural surface was 26% (12/46) in cases showing this pattern, while it was 71.4% (30/42) when this pattern was not seen. Similarly, the incidence of pneumothorax in biopsies <30 mm from the pleural surface was 0% (0/28) in cases showing this hemorrhage pattern, while it was 19% (76/394) when this pattern was not seen. CONCLUSION: Pulmonary hemorrhage during TTFNA is a frequent event that protects against pneumothorax. A bleeding greater than 6 mm along the needle track is associated with lower incidence of pneumothorax, especially in biopsies deeper than 3 cm.

Epidermal growth factor receptor and Kras gene expression: reliability of mutational analysis on cytological samples.

Epidermal growth factor receptor (EGFR) and Kras gene mutations are crucial for discriminating patients responsive to anti-EGFR drugs in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), respectively. The majority of NSCLCs come to clinical attention at an advanced stage when surgery is no longer recommended and a considerable number of them are diagnosed by cytology only. A large number of metastatic CRCs are also diagnosed by imaging and minimally invasive techniques such as fine-needle aspiration biopsy. Here, we report our experience in the mutation analysis of EGFR and Kras on cytological material obtained from superficial and deep lesions of NSCLC and CRC. Our series included 63 cytological specimens from primary or metastatic lesions of 42 NSCLCs and 21 CRCs. The cytological material was adequate for the mutation analysis in 39/42 (93%) NSCLCs and in 20/21(95%) CRCs. EGFR and Kras mutations were found in 9 (23%) and 9 (23%) NSCLC cases, respectively. Kras mutations were found in 9/20 (45%) CRC specimens. Histological samples from the primary tumors were available in 9/42 NSCLCs and in 17/21 CRCs. The agreement of EGFR and Kras mutational status in cytological vs. histological samples was 100% for NSCLC and 88% for CRC. Our results suggest that standard cytology provides adequate material for the assessment of EGFR and Kras mutational status in NSCLC and CRC patients and could be specifically indicated in patients not eligible for surgery but candidate to anti-EGFR therapy.

Reliability of EGFR and KRAS mutation analysis on fine-needle aspiration washing in non-small cell lung cancer.

INTRODUCTION: Molecular profiling of advanced non-small cell lung cancer (NSCLC) has become essential for predicting customized medical treatment decision. In light of recent advances in non-invasive diagnostic procedures in NSCLC, we aimed to demonstrate the reliability of assessing molecular tests for epidermal growth factor receptor (EGFR) and KRAS genes on cytological samples by comparing the molecular profile obtained on cells from scraped smears with that on paired needle washing in a series of NSCLC cases. METHODS: Thirty-two cytological specimens obtained by fine-needle aspiration biopsy procedures from primary or metastatic lesions of NSCLCs were Giemsa stained for a rapid on-site evaluation and, in case of an adequate sampling, the cellular material obtained from needle washing was collected into a saline solution. Scraped smears and needle washings were tested for EGFR and KRAS by polymerase chain reaction followed by direct sequencing. RESULTS: The concordance between EGFR and KRAS mutational status in 29 paired scraped smears and needle washing was 100%, with 7 paired samples showing the same EGFR mutation (4 L858R mutation, 2 E746_A750 deletion and 1 A767_V769 duplication) and 8 paired samples showing the same KRAS mutations (4 G12D, 1 G12A, 1 G12V and 2 G12C). Three scraped smears, uninformative for poor DNA quality, resulted EGFR mutated on paired needle washings. CONCLUSIONS: Needle washing obtained in the course of NSCLC non-invasive fine needle diagnostic procedures allows reliable mutation testing and can be regarded as an additional important source of biological material for molecular profiling of advanced NSCLC.