Different pulmonary adenocarcinoma growth patterns significantly affect survival.

OBJECTIVE: Adenocarcinoma (AC) is the number one pathological entity of lung cancer with approximately 30-40% of cases. It is known to be heterogeneous and has 5 histopathological growth patterns. We evaluated the long-term survival rates of patients with predominant subtypes. METHODS: 290 patients with AC underwent pulmonary resection between 2012 and 2017 at our institution. We excluded all patients with lymph node involvement and distant metastases. Hence, 163 patients were included for further analysis. Predominant growth pattern was defined if more than 10% of cells showed a growth pattern. 1, 3, and 5-year survival rates were evaluated. Survival was assessed by Kaplan-Meier curves and the Cox proportional hazards model was used to identify prognostic factors for overall survival. RESULTS: Predominant growth patterns >10% were compared to <10% growth patterns of the same subtype. 1-year, 3-year, and 5-year overall survival rates of patients with predominant solid tumor growth >10% differed significantly from patients with <10% (88.4% vs. 97.6%, p = 0.04; 65.8% vs. 87.4% p = 0.001, 36.4% vs. 65.9% p = 0.01). Survival rates did not differ between >10% papillary and acinar growth compared to <10%. Kaplan-Meier curves showed reduced overall survival for patients with solid tumor growth >10% (log-rank 0.002). Solid tumor growth >10% was an independent prognostic factor for worse long-term survival (Hazard ratio: 3.05, p = 0.01). CONCLUSION: Our study demonstrates that the presence of a predominant solid pattern in pulmonary adenocarcinoma is a factor for an unfavorable prognosis. This should be kept in mind in daily clinical practice.

Postoperative long-term survival of non-small cell lung cancer patients with skip-N2 metastases.

OBJECTIVE: Radical lymphadenectomy is crucial in operations for non-small cell lung cancer (NSCLC). Usually pN1 and pN2 lymph nodes are affected consecutively (N1N2). Nevertheless, pN2 metastases may also occur in the absence of pN1 as skip-N2 metastases (N0N2). Here we compare the long-term survival of N1N2- and N0N2 patients. MATERIALS AND METHODS: 464 patients underwent surgery for NSCLC at our institution between 2012 and 2017. We retrospectively reviewed data of pN2 stage patients (n = 68). Patients with N1N2 (n = 39) were compared to N0N2 (n = 29) patients. 1-, 3-and 5-year survival rates were measured. Survival was assessed by Kaplan-Meier curves and the cox proportional hazards model was used to identify prognostic factors for overall survival. All patients received adjuvant chemoradiation therapy according to European guidelines. RESULTS: The baseline characteristics did not differ between groups. We observed no differences in the histology, localization, or gender in our cohort. N0N2 patients showed significantly better 1- (N1N2: 82.4% vs. N0N2 100%; p = 0.001), 3- (14.7% vs. 63.6%; p=<0.001) and 5-year (9.4% vs. 43.8%; p = 0.001) survival rates. Tumor size (Hazard ratio (HR) 1.46, Confidence interval (CI 95%) 1.03-2.04; p = 0.03) and the occurrence of N1N2 (HR 4.26, CI 2.04-8.91; p < 0.0001) were independent prognostic factors for worse long-term survival. The Kaplan-Meier curves showed a reduced overall survival for N1N2 patients (log-rank N1N2, N0N2 p < 0.0001). CONCLUSION: N1N2 patients have a significantly worse prognosis compared to N0N2 patients. This will aid to classify the heterogeneous pN2-NSCLC patient population more precisely. Further, multimodal therapy should be considered for N1N2 patients.

LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway.

Lysine-specific demethylase 1 (LSD1) is a histone modifier that is highly overexpressed in lung adenocarcinoma, which results in aggressive tumor biology. Tumor cell proliferation and migration analysis after LSD1 inhibition in the lung adenocarcinoma cell line PC9, using the LSD1 inhibitor HCI-2509 and siRNA, demonstrated that LSD1 activity was essential for proliferation and migration capacities of tumor cells. Moreover, reduced proliferation rates after LSD1 inhibition were shown to be associated with a cell-cycle arrest of the tumor cells in the G2-M-phase. Expression profiling followed by functional classification and pathway analysis indicated prominent repression of the polo-like kinase 1 (PLK1) pathway upon LSD1 inhibition. In contrast, transient overexpression of exogenous PLK1 plasmid rescued the LSD1 inhibition-mediated downregulation of PLK1 pathway genes. Mechanistically, LSD1 directly regulates expression of PLK1 by binding to its promoter region that subsequently affects expression of its downstream target genes. Notably, using lung adenocarcinoma TCGA datasets a significant correlation between LSD1 and PLK1 along with its downstream targets was observed. Furthermore, the LSD1/PLK1 linkage was confirmed by IHC analysis in a clinical lung adenocarcinoma cohort (n = 43). Conclusively, this is the first study showing a direct transcriptional link between LSD1 and PLK1. IMPLICATIONS: These findings point to a role of LSD1 in regulating PLK1 and thus efficient G2-M-transition-mediating proliferation of tumor cells and suggest targeting the LSD1/PLK1 axis as a novel therapeutic approach for lung adenocarcinoma treatment.

Evolution analysis of heterogeneous non-small cell lung carcinoma by ultra-deep sequencing of the mitochondrial genome.

Accurate assessment of tumour heterogeneity is an important issue that influences prognosis and therapeutic decision in molecular pathology. Due to the shortage of protective histones and a limited DNA repair capacity, the mitochondrial (mt)-genome undergoes high variability during tumour development. Therefore, screening of mt-genome represents a useful molecular tool for assessing precise cell lineages and tracking tumour history. Here, we describe a highly specific and robust multiplex PCR-based ultra-deep sequencing technology for analysis of the whole mt-genome (wmt-seq) on low quality-DNA from formalin-fixed paraffin-embedded tissues. As a proof of concept, we applied the wmt-seq technology to characterize the clonal relationship of non-small cell lung cancer (NSCLC) specimens with multiple lesions (N = 43) that show either different histological subtypes (group I) or pulmonary adenosquamous carcinoma as striking examples of a mixed-histology tumour (group II). The application of wmt-seq demonstrated that most samples bear common mt-mutations in each lesion of an individual patient, indicating a single cell progeny and clonal relationship. Hereby we show the monoclonal origin of histologically heterogeneous NSCLC and demonstrate the evolutionary relation of NSCLC cases carrying heteroplasmic mt-variants.

Outcome in neonates with necrotizing enterocolitis and patent ductus arteriosus.

BACKGROUND: There is no agreement of the influence of patent ductus arteriosus (PDA) on outcomes in patients with necrotizing enterocolitis (NEC). In this study, we assessed the influence of PDA on NEC outcomes. METHODS: A retrospective study of 131 infants with established NEC was performed. Outcomes (death, disease severity, need for surgery, hospitalization duration), as well as multiple clinical parameters were compared between NEC patients with no congenital heart disease (n=102) and those with isolated PDA (n=29). Univariate, multivariate and stepwise logistic regression analyses were performed. RESULTS: Birth weight and gestational age were significantly lower in patients with PDA [median (95% CI): 1120 g (1009-1562 g), 28.4 wk (27.8-30.5 wk)] than in those without PDA [median (95% CI): 1580 g (1593-1905 g), 32.4 wk (31.8-33.5 wk); P<0.05]. The risk of NEC-attributable fatality was higher in NEC patients with PDA (35%) than in NEC patients without PDA (14%)[univariate odds ratio (OR)=3.3, 95% CI: 1.8-8.6, P<0.05; multivariate OR=2.4, 95% CI: 0.82-2.39, P=0.111]. Significant independent predictors for non-survival within the entire cohort were advanced disease severity stage III (OR=27.9, 95% CI: 7.4-105, P<0.001) and birth weight below 1100 g (OR=5.7, 95% CI: 1.7-19.4, P<0.01). CONCLUSIONS: In patients with NEC, the presence of PDA is associated with an increased risk of death. However, when important differences between the two study groups are controlled, only birth weight and disease severity may independently predict mortality.

Localized amyloid light-chain amyloidosis and extramedullary plasmacytoma of the mitral valve.

An unusual case of localized amyloid light-chain (AL) amyloidosis and extramedullary plasmacytoma of the mitral valve is described. The worsening of a mitral regurgitation led to investigations and surgery. The valve presented marked distortion and thickening by type AL amyloid associated with a monotypic CD138+ immunoglobulin lambda plasma cell proliferation. Systemic staging showed a normal bone marrow and no evidence of amyloid deposition in other localizations. The patient's outcome after mitral valve replacement was excellent. To our knowledge, this is the first description of a localized AL amyloidosis as well as of a primary extramedullary plasmacytoma of the mitral valve.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Tumor architecture exerts no bias on nuclear grading in breast cancer diagnosis.

We recently reported that nuclear grading in prostate cancer is subject to a strong confirmation bias induced by the tumor architecture. We now wondered whether a similar bias governs nuclear grading in breast carcinoma. An unannounced test was performed at a pathology conference. Pathologists were asked to grade nuclei in a PowerPoint presentation. Circular high power fields of 27 invasive ductal carcinomas were shown, superimposed over low power background images of either tubule-rich or tubule-poor carcinomas. We found (a) that diagnostic reproducibility of nuclear grades was poor to moderate (weighed kappa values between 0.07 and 0.54, 27 cases, 44 graders), but (b) that nuclear grades were not affected by the tumor architecture. We speculate that the categorized grading in breast cancer, separating tubule formation, nuclear pleomorphism, and mitotic figure counts in a combined three tier score, prevents the bias that architecture exerts on nuclear grades in less well-controlled situations.

What was I thinking? Eye-tracking experiments underscore the bias that architecture exerts on nuclear grading in prostate cancer.

We previously reported that nuclear grade assignment of prostate carcinomas is subject to a cognitive bias induced by the tumor architecture. Here, we asked whether this bias is mediated by the non-conscious selection of nuclei that "match the expectation" induced by the inadvertent glance at the tumor architecture. 20 pathologists were asked to grade nuclei in high power fields of 20 prostate carcinomas displayed on a computer screen. Unknown to the pathologists, each carcinoma was shown twice, once before a background of a low grade, tubule-rich carcinoma and once before the background of a high grade, solid carcinoma. Eye tracking allowed to identify which nuclei the pathologists fixated during the 8 second projection period. For all 20 pathologists, nuclear grade assignment was significantly biased by tumor architecture. Pathologists tended to fixate on bigger, darker, and more irregular nuclei when those were projected before kigh grade, solid carcinomas than before low grade, tubule-rich carcinomas (and vice versa). However, the morphometric differences of the selected nuclei accounted for only 11% of the architecture-induced bias, suggesting that it can only to a small part be explained by the unconscious fixation on nuclei that "match the expectation". In conclusion, selection of « matching nuclei ¼ represents an unconscious effort to vindicate the gravitation of nuclear grades towards the tumor architecture.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation.

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Absence of ectopic epithelial inclusions in 3,904 axillary lymph nodes examined in sentinel technique.

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 µm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.

Leukocyte-endothelial cell interaction is necessary for photodynamic therapy induced vascular permeabilization.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) affects vascular barrier function and thus increases vessel permeability. This phenomenon may be exploited to facilitate targeted drug delivery and may lead to a new clinical application of photodynamic therapy. Here, we investigate the role of leukocyte recruitment for PDT-induced vascular permeabilization. STUDY DESIGN/MATERIAL AND METHODS: Fluorescein isothiocyanate dextran (FITC-D, 2,000 kDa) was injected intravenously 120 minutes after focal PDT on striated muscle in nude mice bearing dorsal skinfold chambers (Visudyne® 800 µg/kg, fluence rate 300 mW/cm2 , light dose of 200 J/cm2). Leukocyte interaction with endothelial cells was inhibited by antibodies functionally blocking adhesion molecules ("MABS-PDT" group, n = 5); control animals had PDT but no antibody injection (group "PDT", n = 7). By intravital microscopy, we monitored leukocyte rolling and sticking in real-time before, 90 and 180 minutes after PDT. The extravasation of FITC-D from striated muscle vessels into the interstitial space was determined in vivo during 45 minutes to assess treatment-induced alterations of vascular permeability. RESULTS: PDT significantly increased the recruitment of leukocytes and enhanced the leakage of FITC-D. Neutralization of adhesion molecules before PDT suppressed the rolling of leukocytes along the venular endothelium and significantly reduced the extravasation of FITC-D as compared to control animals (156 ± 27 vs. 11 ± 2 (mean ± SEM, number of WBC/30 seconds mm vessel circumference; P < 0.05) at 90 minutes after PDT and 194 ± 21 vs. 14 ± 4 at 180 minutes after PDT). In contrast, leukocyte sticking was not downregulated by the antibody treatment. CONCLUSION: Leukocyte recruitment plays an essential role in the permeability-enhancing effect of PDT.

Asymptomatic intrapericardial mature teratoma.

Benign mature teratomas are the most common type of germ cell tumors that arise in the anterior mediastinum. Intrapericardial teratomas are a rare manifestation of this tumor type, which are generally diagnosed during infancy because of the heart compression symptoms they produce. Here we report a rare case of intrapericardial mature teratoma that was incidentally discovered in an asymptomatic 51-year-old woman.

[Predictive value of Her2/neu expression/amplification for the targeted treatment of breast cancer]. / Valeur prédictive de la surexpression/ amplification de Her2/neu pour un traitement ciblé du cancer du sein.

Her2/neu is a tyrosine kinase receptor which stimulates cell growth. The receptor is overexpressed in about 20% of breast cancers. Her2/neu expression is an indicator of poor prognosis but also the target of the treatment of breast cancer using humanised anti-Her2/ neu antibodies. Only cancers overexpressing the protein will respond to this therapy, but which has significant (cardiac) side effects and is expensive. It is therefore important to test for the overexpression of the protein on breast cancer cells. This paper discusses how this can be done and ongoing research into new therapeutic options targeting the involved signaling pathways.

Molecular imaging by micro-CT: specific E-selectin imaging.

The primary goal of this study was to design a fluorescent E-selectin-targeted iodine-containing liposome for specific E-selectin imaging with the use of micro-CT. The secondary goal was to correlate the results of micro-CT imaging with other imaging techniques with cellular resolution, i.e., confocal and intravital microscopy. E-selectin-targeted liposomes were tested on endothelial cells in culture and in vivo in HT-29 tumor-bearing mice (n = 12). The liposomes contained iodine (as micro-CT contrast medium) and fluorophore (as optical contrast medium) for confocal and intravital microscopy. Optical imaging methods were used to confirm at the cellular level, the observations made with micro-CT. An ischemia-reperfusion model was used to trigger neovessel formation for intravital imaging. The E-selectin-targeted liposomes were avidly taken up by activated endothelial cells, whereas nontargeted liposomes were not. Direct binding of the E-selectin-targeted liposomes was proved by intravital microscopy, where bright spots clearly appeared on the activated vessels. Micro-CT imaging also demonstrated accumulation of the targeted lipsomes into subcutaneous tumor by an increase of 32 + or - 8 HU. Hence, internalization by activated endothelial cells was rapid and mediated by E-selectin. We conclude that micro-CT associated with specific molecular contrast agent is able to detect specific molecular markers on activated vessel walls in vivo.

Transplantation tolerance induced by regulatory T cells: in vivo mechanisms and sites of action.

The mechanisms by which CD4(+)CD25(+)Foxp3(+) T (Treg) cells regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer model, we analyzed the in vivo expansion, trafficking, and effector function of alloreactive T cells and donor-specific Treg cells, in response to a full-thickness skin allograft. Fluorescent-labeled CD4(+)CD25(-) and antigen-specific Treg cells were transferred alone or co-injected into syngeneic BALB/c-Nude recipients transplanted with skins from (C57BL/6 x BALB/c) F1 donors. Treg cells divided in vivo, migrated and accumulated in the allograft draining lymph nodes as well as within the graft. The co-transfer of Treg cells did not modify the early activation and homing of CD4(+)CD25(-) T cells in secondary lymphoid organs. However, in the presence of Treg cells, alloreactive CD4(+)CD25(-) T cells produced significantly less IFN-gamma and were present in reduced numbers in the secondary lymphoid organs. Furthermore, time-course studies showed that Treg cells were recruited into the allograft at a very early stage after transplantation and effectively prevented the infiltration of effector T cells. In conclusion, suppression of rejection requires the early recruitment to the site of antigenic challenge of donor-specific Treg cells, which then mainly regulate the effector arm of T cell alloresponses.

Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?

OBJECTIVE: Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied. METHODS AND RESULTS: Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein-activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP expression, whereas rbCRP-positive males had significantly higher serum cholesterol levels than the rbCRP-negative counterparts. All mice exhibited extensive atherosclerotic lesions, as studied en face, and no differences were noted between rbCRP-negative and rbCRP-positive animals. Atherosclerotic luminal obstruction of aortic arch and first-order neck branches did not differ significantly between rbCRP-positive and rbCRP-negative mice. There was no correlation between rbCRP levels and atherosclerotic lesion formation. CONCLUSIONS: No marked effect of rbCRP on the formation of moderately advanced atherosclerotic lesions could be discerned in the apoE knockout mouse. Because of the oddities of the mouse complement system, however, this may not be a good model to investigate the role of CRP in human atherosclerosis.