Morten T. Andersen

Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.

OBJECTIVE To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment. DESIGN Retrospective analysis of data in a clinical project. SETTING University hospital laboratories. PATIENT(S) In total, 26 patients diagnosed with leukemia, who had ovarian tissue cryopreserved before potentially sterilizing chemotherapy and conditioning. INTERVENTION(S) Ovarian cortex from each patient was examined with histology and immunohistochemistry. In addition, in eight cases a specific chromosomal abnormality could be used as a genetic marker for detection of malignant cells by polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S) Evidence of malignant cells by immunohistochemistry or PCR. RESULT(S) Histology and immunohistochemistry did not reveal malignant cell infiltration in the ovarian cortex of any of the patients. In six of the eight patients (75%) with chromosomal abnormalities in the malignant cells, PCR showed evidence of leukemic cells in the ovarian tissue. CONCLUSION(S) Immunohistochemistry was unable to locate leukemic cells in the ovarian cortex; however, PCR detected potentially malignant cells in the majority of cases. The viability and malignancy of these cells remains to be determined. At present, reimplantation of ovarian cortex to leukemia patients cannot be recommended.

Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

Diagnostic potential of miR-126, miR-143, miR-145, and miR-652 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is difficult to distinguish from reactive mesothelial proliferations (RMPs). It is uncertain whether miRNAs are useful biomarkers for differentiating MPM from RMPs. Thus, we screened with a quantitative RT-PCR (RT-qPCR)-based platform the expression of 742 miRNAs in formalin-fixed, paraffin-embedded, preoperative diagnostic biopsy samples, surgically resected MPM specimens previously treated with chemotherapy, and corresponding non-neoplastic pleura (NNP), from five patients. miR-126, miR-143, miR-145, and miR-652 were significantly down-regulated (≥twofold) in resected MPM and/or chemotherapy-naïve diagnostic tumor biopsy samples. The miRNA expression pattern was validated by RT-qPCR in a cohort of 40 independent MPMs. By performing binary logistic regression on the RT-qPCR data for the four miRNAs, the established four-miRNA classifier differentiated MPM from NNP with high sensitivity and specificity (area under the curve, 0.96; 95% CI, 0.92-1.00). The classifiers optimal logit(P) value of 0.62 separated NNP and MPM samples with a sensitivity of 0.95 (95% CI, 0.89-1.00), a specificity of 0.93 (95% CI, 0.87-0.99), and an overall accuracy of 0.94 (95% CI, 0.88-1.00). The level of miR-126 in MPM was inversely correlated with that of the known target, the large neutral amino acid transporter, small subunit 1 (r = -0.38; 95% CI, -0.63 to -0.06). Overall, these results indicate that these four miRNAs may be suitable biomarkers for distinguishing MPM from RMPs.

The prognostic effect of American Joint Committee on Cancer staging and genetic status in patients with choroidal and ciliary body melanoma.

PURPOSE To evaluate the prognostic effect of a combination of American Joint Committee on Cancer (AJCC) staging (7th edition) and genetic status in patients with posterior uveal melanoma. METHODS A consecutive cohort of 153 patients with posterior uveal melanoma treated at Copenhagen University Hospital from January 1, 2009 through December 31, 2012 was followed until October 2014. Survival, AJCC stage, and cytogenetic data were registered. The AJCC stage was available for all patients, and cytogenetic information for chromosomes 3 and 8 was available for 139 patients. The individual and joint prognostic effects of AJCC staging and cytogenetic changes were evaluated by cumulative incidence curves and Cox proportional hazard models. RESULTS An overall 5-year survival rate of 62% (95% confidence interval [CI]: 0.50-0.73) was observed. A normal genetic status of chromosomes 3 and 8, as found in 42 patients (30%), minimized the additional prognostic effect of AJCC staging. The frequency of tumors with normal genetic status decreased with increasing AJCC stage. Both AJCC stage III (hazard ratio [HR]: 11.0, 95% CI: 1.4-85.6) and abnormal copy number of chromosomes 3 (HR: 6.3, 95% CI: 1.4-28.3) and 8 (HR: 2.8, 95% CI: 1.03-7.8) were identified as significant predictors of a poor prognosis in the multivariate Cox regression analysis. CONCLUSIONS Identification of a normal genetic status of chromosomes 3 and 8 minimized the prognostic effect of AJCC staging, while a combination of genetic status and AJCC staging provided the most accurate prediction of survival in patients with an abnormal chromosomal status.

MicroRNA expression analysis and Multiplex ligation‐dependent probe amplification in metastatic and non‐metastatic uveal melanoma

To determine the association of microRNA expression and chromosomal changes with metastasis and survival in uveal melanoma (UM).

Are differentially expressed microRNAs useful in the diagnostics of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is radiologically and histologically difficult to distinguish from reactive mesothelial proliferations (RMPs), partly because proposed MPMmarkers have not shown enough specificity and reproducibility or need further validation (1–3). MicroRNAs (miRs) are small non-coding RNA-strands (~22 nt) post-transcriptionally regulating gene-expression and playing increasingly evident roles in several diseases (4). MiRs might potentially be attractive biomarkers for MPM, as MiR-expression in other cancers has shown prognostic and diagnostic significance (5). Furthermore, miRs are more stable and much smaller than mRNAs, thus they can be reliably detected in formalin-fixed paraffin-embedded (FFPE) tissues (6). However, it is unclear whether currently published miR-data may provide biomarker candidates for differentiating MPM from RMP, as it has been implied (7–11). The only study published to date comparing miR-expression in patientmatched MPM and adjacent non-neoplastic pleura (NP) samples, was based on few analyzed miRs (7). Recently, more extensive in vitro analysis compared miR-expression in normal mesothelial cell-cultures and commercially available MPM-cell-lines (8). Other comprehensive studies in vivo instead focused on miRsignatures differentiating MPM histological subtypes (epithelioid, sarcomatoid, and biphasic) or discriminating MPM from lung adenocarcinoma (9–11). For instance, Busacca et al. reported that the expression of miR-17-5p and miR-30c, already known to be related to other malignancies (5), was associated with mesothelioma subtypes (11). Others recently confirmed miR-17-5p up-regulation in MPM-cell-lines, in association with reduced expression of one of its targets, the cyclin-dependent kinase-inhibitor (CDKI) p21 (8, 12). In contrast miR-221 and -222, known negative regulators of the CDKI p27 and the tumor suppressor PTEN, were reported as down-regulated in human MPM cell lines compared to non-tumorous immortalized mesothelial cells (11). This is somehow surprising given that loss of p27 and PTEN expression is frequent and associated with poor prognosis in MPM patients (4,12,13) To test whether miR-17-5p, -30c, -221, and 222 may be useful in differentiating MPM from RMP in vivo, we quantified and compared their expression in FFPE epithelioid MPM (stages I-IV) and NP tissue-samples collected from 13 patients (11 male patients, 2 female patients, ages 49–69) (Table 1) during extra-pleural pneumonectomy preceded by 1–3 cycles of chemotherapy (vinorelbine/cisplatin or gemcitabine/carboplatin). We quantified these miRs and reference RNU6B by realtime-PCR-based TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions using 7500 Real-Time PCR System (Applied Biosystems). Threshold-cycles (Ct) were determined with related system-software (SDS v1.2.2, Applied Biosystems) and PCR-data analyzed comparatively with RNU6B as normalizer. Statistically significant (p < 0.05) differences between independent or paired groups were detected by nonparametric Mann–Whitney and Wilcoxon matched pairs tests, respectively.

Transvitreal Retinochoroidal Biopsy Provides a Representative Sample From Choroidal Melanoma for Detection of Chromosome 3 Aberrations

PURPOSE To compare the status of chromosomes 3 and 8 in 25-gauge transvitreal retinochoroidal (TVRC) biopsy specimens and enucleated eyes in order to evaluate for genetic heterogeneity and the utility of TVRC biopsy to obtain an adequate sampling of the tumor. METHODS Genetic heterogeneity was evaluated in 27 patients treated at Rigshospitalet between 2009 and 2013. The TVRC biopsy was performed to confirm diagnosis prior to enucleation and was subsequently analyzed using two techniques for chromosomes 1p, 3, 6, and 8: Fluorescence in situ hybridization (FISH) in all patients, and multiplex ligation-dependent probe amplification (MLPA) in 16 patients. Biopsies were compared with histological sections from matched enucleated eyes, which were microdissected following a hexagonal grid and analyzed with MLPA. RESULTS Twenty-four tumors were available for analysis. The TVRC biopsy identified chromosome 3 aberrations with MLPA in all cases (sensitivity = 100%), while FISH missed two cases (sensitivity = 89%). Conversely, FISH analysis demonstrated polyploidy of chromosome 3 in three additional cases missed by MLPA. Chromosome 8 aberrations were detected in 75% of cases with MLPA and 68% of cases with FISH. Heterogeneity of chromosomes 3 and 8 was shown in 3 (13%) and 11 tumors (46%), respectively, with an increased frequency of genetic aberrations toward the base of the tumor (P = 0.049). The study showed no difference in tumor size between heterogeneous and homogenous melanomas (P = 0.82). CONCLUSIONS Regardless of genetic heterogeneity, the TVRC biopsy identified all patients with a high risk of developing metastatic disease when a combination of chromosome 3 and 8 status was assessed.

MicroRNAs as potential biomarkers in malignant pleural mesothelioma

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Current Biomarker Findings 2016:6 1–21 Current Biomarker Findings Dovepress

Abstract 3556: Diagnostic potential of microRNAs (miRs) in malignant pleural mesothelioma (MPM).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: MPM is histologically difficult to distinguish from reactive mesothelial proliferations (RMPs), partly because proposed MPM-markers are not specific, reproducible or validated enough (Zimling ZG, Jorgensen A & Santoni-Rugiu E, Histopathology 2012; 60(6B):E96-105). MiRs are small non-coding RNA-strands (∼22 nt) that post-transcriptionally regulate gene-expression, vital cellular processes and oncogenesis. However it is uncertain whether currently published miR-data may provide candidate diagnostic biomarkers for differentiating MPM from RMP. To pursue this goal, we performed a screening of miR-expression in FFPE diagnostic biopsies, surgically resected MPM-specimens and corresponding surrounding non-neoplastic pleura (NP). Materials and Methods: We performed a RT-qPCR-based (Exiqon®) screening of 742 human miRs’ expression in preoperative biopsies, epithelioid MPM-, and NP-specimens from 5 patients treated with extra-pleural pneumonectomy as part of trimodal protocol. The relevant identified differentially expressed miRs were validated on tissue samples from 41 independent MPM-patients by RT-qPCR TaqMan® MicroRNA Assays (Applied Biosystems). Quantification-cycles (Cq) were determined by the related 7500 RT-PCR system software, comparatively analyzing PCR-data with an internal reference-gene (RNU49). Significant (p < 0.05) differences between groups were detected by 1-way ANOVA with Bonferroni post-tests. Validated miR-targets were assessed by immunohistochemistry (IHC). Results: We identified significant difference in expression of 7 cancer-relevant miRs (down-regulation of miR-17-5p, -126, -143, -145 and -652 and up-regulation of miR-221 and -193a-3p) that correctly differentiated MPM from RMPs and was not influenced by chemotherapy (comparable miR-expression-levels in surgical samples and diagnostic biopsies). We furthermore tested if the 7 miRs could fulfill the recommendations of the International Mesothelioma Interest Group (IMIG) for a suitable MPM-marker (sensitivity/specificity > 80%) by generating ROC-curves using the RT-qPCR-data. The IMIG-criteria were not met for miR-17-5p, -221 and -143, while miR-126, -145, -193a-3p and -652 showed high sensitivity and specificity. Preliminary IHC-data of the validated miR-126 target L-type amino acid transporter 1 (LAT1) showed good correlation between miR-126 down-regulation and LAT1 up-regulation in MPM. Conclusion: The cancer-relevant miR-126, -145, -193a-3p and -652 have great potential as biomarkers for distinguishing MPM from RMPs, while miR-17-5p, -143 and -221, previously reported to be specific for MPMs histological subtypes and to play a pathogenic role in MPM, are not entirely suitable for this differential diagnostic purpose. Further studies by in situ-hybridization techniques will additionally corroborate the possible use of these miRs in MPM diagnostics. Citation Format: Morten Andersen, Morten Grauslund, Jesper Ravn, Jens B. Sorensen, Claus B. Andersen, Eric Santoni-Rugiu. Diagnostic potential of microRNAs (miRs) in malignant pleural mesothelioma (MPM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3556. doi:10.1158/1538-7445.AM2013-3556