Personalized cancer medicine and the future of pathology.

In February 2011, a group of pathologists from different departments in Europe met in Zurich, Switzerland, to discuss opportunities and challenges for pathology in the era of personalized medicine. The major topics of the meeting were assessment of the role of pathology in personalized medicine, its future profile among other biomedical disciplines with an interest in personalized medicine as well as the evolution of companion diagnostics. The relevance of novel technologies for genome analysis in clinical practice was discussed. The participants recognize that there should be more initiatives taken by the pathology community in companion diagnostics and in the emerging field of next-generation sequencing and whole genome analysis. The common view of the participants was that the pathology community has to be mobilized for stronger engagement in the future of personalized medicine. Pathologists should be aware of the challenges and the analytical opportunities of the new technologies. Challenges of clinical trial design as well as insurance and reimbursement questions were addressed. The pathology community has the responsibility to lead medical colleagues into embracing this new area of genomic medicine. Without this effort, the discipline of pathology risks losing its key position in molecular tissue diagnostics.

Lung-specific inactivation of CCAAT/enhancer binding protein alpha causes a pathological pattern characteristic of COPD.

The link between respiratory complications in prematurely born infants and susceptibility for developing chronic obstructive pulmonary disease (COPD) is receiving increasing attention. We have previously found that CCAAT/enhancer binding protein (C/EBP) activity in airway epithelial cells of COPD patients is decreased compared to healthy smokers, suggesting a previously unknown role for C/EBPs in COPD pathogenesis. To investigate the role of the transcription factor C/EBPalpha in lung development and its potential role in COPD, mice with a lung epithelial-specific disruption of the C/EBPalpha gene (Cebpa(DeltaLE)) were generated using Cre-mediated excision, and the resulting pathology was studied during development and into adulthood. Cebpa(DeltaLE) mice exhibit impaired lung development and epithelial differentiation, as well as affected vascularity. Furthermore, Cebpa(DeltaLE) mice that survive until adulthood develop a severe pathological picture with irregular emphysema; bronchiolitis, including goblet cell hyperplasia, bronchiolar metaplasia, fibrosis and mucus plugging; and an inflammatory cell and gene expression profile similar to COPD. Cebpa(DeltaLE) mice display lung immaturity during development, and adult Cebpa(DeltaLE) mice develop a majority of the histopathological and inflammatory characteristics of COPD. Cebpa(DeltaLE) mice could thus provide new valuable insights into understanding the long-term consequences of lung immaturity and the link to susceptibility of developing COPD.

Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) lacking expression of steroid receptors and human epidermal growth factor receptor 2, having chemotherapy as the only therapeutic option, is characterised by early relapses and poor outcome. We investigated intratumoural (i.t.) levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and survival in patients with TNBC compared with non-TNBC. PATIENTS AND METHODS: VEGF levels were determined by an enzyme immunosorbent assay in a retrospective series consisting of 679 consecutive primary breast cancer patients. RESULTS: Eighty-seven patients (13%) were classified as TNBC and had significantly higher VEGF levels; median value in TNBC was 8.2 pg/microg DNA compared with 2.7 pg/microg DNA in non-TNBC (P < 0.001). Patients with TNBC had statistically significant shorter recurrence-free survival [hazard ratio (HR) = 1.8; P = 0.0023], breast cancer-corrected survival (HR = 2.2; P = 0.004) and overall survival (HR = 1.8; P = 0.005) compared with non-TNBC. Patients with TNBC relapsed earlier than non-TNBC; mean time from diagnosis to first relapse was 18.8 and 30.7 months, respectively. The time between first relapse and death was also shorter in TNBC: 7.5 months versus 17.5 months in non-TNBC (P = 0.087). CONCLUSIONS: Our results show that TNBC have higher i.t. VEGF levels compared with non-TNBC. Ongoing clinical trials will answer if therapy directed towards angiogenesis may be an alternative way to improve outcome in this poor prognosis group.

Shorter survival-times following adjuvant endocrine therapy in oestrogen- and progesterone-receptor positive breast cancer overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor.

PURPOSE: To investigate the possible correlation between expression of HER2 and vascular endothelial growth factor (VEGF), and to determine the predictive value of these factors in patients receiving adjuvant endocrine therapy including the group with a breast cancer (BC) positive for both oestrogen receptor (ER) and progesterone receptor (PgR). MATERIAL AND METHODS: By enzyme immuno-sorbent assays (ELISA) tumour levels of HER2 and VEGF proteins were determined in 679 consecutive primary BC patients, median age 63 years, median follow-up time 92 months. A total of 404 patients received adjuvant endocrine therapy, mainly tamoxifen, out of them 295 had an ER and PgR positive BC. In 160 patients, HER2 status was also determined by immunohistochemistry (IHC) using the monoclonal antibody CB11. RESULTS: Overexpression of HER2 by IHC was found in 15% of the patients. Overexpression of HER2 by ELISA correlated with HER2 by IHC (P < 0.001) and a higher VEGF expression (P = 0.004). Patients receiving adjuvant endocrine therapy with high VEGF (RFS P = 0.0087, BCCS P = 0.0012) or over-expressing HER2 (RFS P = 0.0116, BCCS P = 0.0036) had significantly shorter survival. Factors retaining statistical significance in multivariate analyses for recurrence-free survival (RFS) were nodal status (P < 0.001), tumour size (P = 0.005) and VEGF (P = 0.032) and for breast cancer corrected survival (BCCS) nodal status (P < 0.001), tumour size (P = 0.001), ER status (P = 0.022), and VEGF (P = 0.016). Both factors were significantly correlated with survival in the group with a BC positive for both ER and PgR; VEGF (RFS P = 0.0177, BCCS P = 0.0321) and HER2 (RFS P = 0.0143, BCCS P = 0.0311). In multivariate analyses, nodal status (P < 0.001) and VEGF (P = 0.021) were independent factors for RFS. Nodal status (P < 0.001) and tumour size (P = 0.016) retained independent factors for BCCS. Combined analysis identified a high-risk group (HER2 positive and high VEGF) with significantly reduced survival. CONCLUSION: The results from this retrospective analysis suggest that overexpression of HER2 and higher VEGF expression may add information on patient's outcome after adjuvant endocrine therapy in ER and PgR positive BC.

Ki-67 expression predicts locoregional recurrence in stage I oral tongue carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) is an aggressive cancer associated with poor prognosis. Methods for determining the aggressiveness of OTSCC from analysis of the primary tumour specimen are thus highly desirable. We investigated whether genomic instability and proliferative activity (by means of Ki-67 activity) could be of clinical use for prediction of locoregional recurrence in 76 pretreatment OTSCC paraffin samples (stage I, n=22; stage II, n=33; stage III, n=8; stage IV, n=13). Eleven surgical tumour specimens were also analysed for remnants of proliferative activity after preoperative radiotherapy. Ninety-seven percent of cases (n=72) were characterised as being aneuploid as measured by means of image cytometry. Preoperative radiotherapy (50-68 Gy) resulted in significant reduction of proliferative activity in all patients for which post-treatment biopsies were available (P-value=0.001). Proliferative activity was not associated with response to radiation in stage II patients. However, we report a significant correlation between high proliferation rates and locoregional recurrences in stage I OTSCC patients (P-value=0.028). High-proliferative activity is thus related to an elevated risk of recurrence after surgery alone. We therefore conclude that Ki-67 expression level is a potentially useful clinical marker for predicting recurrence in surgically treated stage I OTSCC.

Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome.

BACKGROUND: Our aim was to use quantitative real-time PCR (Q-PCR) and RNA expression profiles (RNA-EPs) to investigate HER2 status in relation to outcome. PATIENTS AND METHODS: Cut-off levels for Q-PCR and RNA-EP were established in relation to immunohistochemistry (IHC) validated by FISH in a test set of frozen tissue samples from 40 primary breast cancers. The HER2 status was subsequently studied in another validation set of 306 tumors, where Q-PCR and RNA-EP results were compared with previously carried out IHC that we had validated by chromogenic in situ hybridization (CISH). RESULTS: Q-PCR and RNA-EP offered similar sensitivity (90% versus 77%), specificity (93% versus 95%), and negative (99% versus 98%) and positive (63% versus 61%) predictive values for HER2 determinations. Analyses of relapse-free survival (RFS) and overall survival on the basis of 5 and 10 years of follow-up indicated equivalent hazard ratios for all three techniques. In contrast to IHC/CISH, both Q-PCR and RNA-EP analyses of HER2 also gave statistically significant results regarding RFS and breast cancer-corrected survival after 10 years of follow-up. CONCLUSION: The use of RNA-EP and Q-PCR to analyze HER2 in frozen and formalin-fixed breast cancer samples may be an alternate approach to IHC in combination with FISH/CISH.

Local production of nitric oxide in patients with tuberculosis.

Nitric oxide (NO), produced by the inducible nitric oxide synthase (iNOS), is important in host defence against Mycobacterium tuberculosis in rodents, but the presence of high-output NO production in human tuberculosis has been controversial. We investigated iNOS and nitrotyrosine (Ntyr) expression in pleural (n = 7), pulmonary (n = 5) and lymph node biopsies (n = 5) from untreated, newly diagnosed tuberculosis patients. Many iNOS and Ntyr reactive macrophages were observed in granulomas, including Langhans giant cells, indicating high-output NO production at the primary site of disease in tuberculosis.

Aspartic proteinase napsin is a useful marker for diagnosis of primary lung adenocarcinoma.

Napsin A is an aspartic proteinase expressed in lung and kidney. We have reported that napsin A is expressed in type II pneumocytes and in adenocarcinomas of the lung. The expression of napsin was examined in 118 lung tissues including 16 metastases by in situ hybridisation. Napsin was expressed in the tumour cell compartment in 33 of 39 adenocarcinomas (84.6%), in two of 11 large cell carcinomas and in one lung metastasis of a renal cell carcinoma. Expression of napsin was found to be associated with a high degree of differentiation in adenocarcinoma. Immunohistochemistry was performed for three proteins currently used as markers for lung adenocarcinoma : surfactant protein-A, surfactant protein-B and thyroid transcription factor-1. Thyroid transcription factor-1 showed the same sensitivity (84.6%) as napsin for adenocarcinoma, whereas surfactant protein-A and surfactant protein-B showed lower sensitivities. Among these markers, napsin showed the highest specificity (94.3%) for adenocarcinoma in nonsmall cell lung carcinoma. We conclude that napsin is a promising marker for the diagnosis of primary lung adenocarcinoma.

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure.

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.

Chromosomal numerical aberrations detected by fluorescence in situ hybridization on bladder washings from patients with bladder cancer.

OBJECTIVE: Previous studies on touch biopsy specimens have determined numerical or structural changes involving many different chromosomes in bladder cancer. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) assay in bladder washings as an objective technique to detect chromosomal numerical aberrations in bladder cancer. The main advantages of bladder washings are that they can be easily collected during the clinical follow-up of patients with superficial bladder cancer and they do not contain so many degenerate cells as urine samples. METHODS: We collected specimens from 25 patients who underwent transurethral resection of bladder tumors. Double target FISH assays with centromeric labeled probes for chromosomes 7, 8, 9 and 11 were used on the bladder washings and on the touch biopsy slides. The results were compared to flow cytometry and tumor grade and stage. RESULTS: We found monosomy 9 and trisomy 7, 8, 9 and 11 in 28, 32, 36, 28 and 25% respectively of the patients. FISH analysis of bladder washing versus touch biopsy specimens were concordant in approximately 90% of the slides. Total DNA aneuploidy correlated well with numerical aberrations of chromosomes 7, 8 and 11, but not with chromosome 9. CONCLUSION: Although better hybridization efficiency was obtained on touch biopsy slides, the results in bladder washings were in high concordance. FISH analysis on bladder washing samples may become a simple tool to improve the accuracy of cytology.

Leukocyte counts and macrophage phenotypes in induced sputum and bronchoalveolar lavage fluid from normal subjects.

It is unclear whether leukocytes in induced sputum (IS) and bronchoalveolar lavage (BAL) represent the same cell populations. To compare leukocyte counts and macrophage phenotypes and investigate any measurable dithiothreitol (DTT)-mediated effect on macrophage immunocytochemical staining results, IS and BAL samples from nine healthy smokers and seven nonsmokers were examined. BAL and IS samples were processed and cell viability and cell counts were assessed. The macrophages were characterized by seven monoclonal antibodies (RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR) using an indirect immunoalkaline phosphatase method. Intraindividual comparison of IS and BAL showed that IS samples from smokers and nonsmokers contained a lower total cell count (p<0.01 smokers, p<0.05 nonsmokers), a lower percentage of macrophages (both p<0.05) and a higher percentage of neutrophils (both p<0.05) than BAL samples. In addition, nonsmokers sputum samples contained a lower proportion of lymphocytes (p<0.05) than BAL. The macrophage expression of RFD7 and CD71 was higher in smokers sputum samples (both p<0.05) than in BAL, while nonsmokers sputum macrophages showed a higher expression of CD54 and CD71 (both p<0.05) than BAL macrophages. DTT-incubated BAL samples showed no difference in macrophage antigen expression from BAL samples not exposed to DTT. In conclusion, the relative proportions of leukocytes and the macrophage phenotypes differed between induced sputum and bronchoalveolar lavage suggesting that the methods provide samples from different lung compartments, inhabited by cells with different phenotypes.

Smoking alters the phenotype of macrophages in induced sputum.

To investigate the effect of chronic smoke exposure on pulmonary macrophages (PM), the expression of seven different surface and intracellular molecules of PM was studied in induced sputum (IS) samples from healthy volunteers--nine smokers and seven non-smokers. Sputum was induced by inhalation of nebulized saline (3.5% NaCl). Cell viability and total cell counts (TCC) were performed immediately. Cell differentials were determined on May-Grunwald Giemsa-stained cytospin preparations. The PM were immunologically characterized by use of the following monoclonal antibodies: RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR. The stainings were performed with a three-step, indirect immuno-alkaline phosphate method. Viability and TCC did not differ between the groups. Smokers had a higher percentage of macrophages (P < 0.05) and a lower proportion of neutrophils (P < 0.05). The percentage of macrophages expressing RFD1, HLA-DR, CD71 (P < 0.01 for all) and CD54 (P < 0.05) was significantly lower in smokers, whereas the remaining markers were expressed equally in the two groups. The results indicate that smoking induces a decrease in the expression by PM of surface molecules known to be associated with the antigen-presenting function.

Simultaneous interphase cytogenetic analysis and fluorescence immunophenotyping of dedifferentiated chondrosarcoma. Implications for histopathogenesis.

Cytogenetic analysis of four specimens (biopsy, definitive surgical, and two separately occurring lung metastases) of a dedifferentiated chondrosarcoma with a rhabdomyosarcomatous component revealed clonal karyotypic abnormalities in each. Anomalies seen in all specimens included a structurally aberrant chromosome 17 and extra copies of chromosomes 5, 7, 12, and 20. The derivation of the chromosomally abnormal cells was determined by a combined immunocytochemical/cytogenetic approach that allowed simultaneous assessment of cytogenetic aberrations and immunophenotypic features of individual cells. S-100 protein and desmin antibodies were used to evaluate the chondrosarcomatous and rhabdomyosarcomatous components, respectively. A chromosome 7-specific centromeric probe was used for determination of aneuploidy. In both specimens obtained from the primary lesion, S-100 protein and desmin-positive and -negative aneuploid cells were observed. These findings: 1) suggest that both the chondrocytic and rhabdomyoblastic cells arose from the same abnormal clone, 2) support the theory of a common primitive mesenchymal cell progenitor with the ability to differentiate or express features of more than one line of mesenchymal differentiation, and 3) indicate that the term dedifferentiated may be an inaccurate designation for this neoplasm.