Genetic Heterogeneity in Uveal Melanoma Assessed by Multiplex Ligation-Dependent Probe Amplification
|
|
- Ambrose Conley
- 5 years ago
- Views:
Transcription
1 Anatomy and Pathology Genetic Heterogeneity in Uveal Melanoma Assessed by Multiplex Ligation-Dependent Probe Amplification Justyna Dopierala, 1 Bertil E. Damato, 2 Sarah L. Lake, 1 Azzam F. G. Taktak, 3 and Sarah E. Coupland 1 From the 1 Department of Pathology, School of Cancer Studies, and the 3 Department of Medical Physics and Clinical Engineering, University of Liverpool, Liverpool, United Kingdom; and 2 St. Paul s Eye Hospital, Royal University Liverpool Hospital, Liverpool, United Kingdom. Supported by the Eye Tumor Research Fund (Royal Liverpool University Hospital), which provides a PhD stipend to JD; Eye Tumor Research Fund (RLBUHT) Grant CRR10416; and Fight-for-Sight UK Grant CRR Submitted for publication December 1, 2009; revised March 28 and April 18, 2010; accepted April 25, Disclosure: J. Dopierala, None; B.E. Damato, None; S.L. Lake, None; A.F.G. Taktak, None; S. E Coupland, None Corresponding author: Sarah E. Coupland, Department of Pathology, University of Liverpool, Liverpool, UK, s.e.coupland@liverpool.ac.uk. PURPOSE. To determine intratumor genetic heterogeneity in uveal melanoma (UM) by multiplex ligation dependent probe amplification (MLPA) in formalin-fixed, paraffin-embedded (FFPE) tumor tissues. METHODS. DNA was extracted from whole tumor sections and from two to nine different areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1, 3, 6, and 8 were tested with MLPA for copy number changes. The tumor was considered heterogeneous at a locus if (1) the difference in dosage quotients (DQs) of any two areas was 0.2 or more, and (2) the DQs of the areas belonged to different ranges. RESULTS. Comparison of MLPA data obtained from microdissected areas of the UMs showed heterogeneity in 1 to 26 examined loci in 24 (75%) tumors, with only 25% of the tumors being homogeneous. Intratumor heterogeneity of 3p12.2, 6p21.2, and 8q11.23 was most common, occurring in 30% of the UMs. Gains of chromosome 3 were observed in four UMs, with three of these tumors showing the highest degree of heterogeneity. Copy number variation was associated with differences in tumor cell type, but not with differences in tumor pigmentation or reactive inflammation. UMs with genetic heterogeneity across multiple sample sites showed equivocal MLPA results when the whole tumor section was examined. These results suggest that different clones dilute MLPA results. CONCLUSIONS. Heterogeneity of chromosomal abnormalities of chromosomes 1, 3, 6, and 8 is present in most UMs. This heterogeneity causes equivocal MLPA results. One random tumor sample may not be representative of the whole tumor and, therefore, may be insufficient for prognostic testing. (Invest Ophthalmol Vis Sci. 2010;51: ) DOI: / iovs In almost 50% of all patients with uveal melanoma (UM), metastatic disease develops that usually involves the liver and is almost inevitably fatal. 1 Such metastatic disease occurs almost exclusively in patients with tumors that show partial or complete deletion of chromosome Tumor dimensions at the time of initial ocular treatment and mitotic count give an indication of the likely survival time in the presence of monosomy 3. 5 Epithelioid melanoma cells, closed connective tissue loops, and a high mitotic count also suggest that a biopsy result indicating disomy 3 may be erroneous. 2,6 12 We have developed online tools for performing multivariate analyses of UM, to estimate, with a reasonable degree of reliability, the survival probability of individual patients. 13 Such personalized prognostication enables reassurance of UM patients with good prognoses while indicating more intensive care for those having a high risk of metastasis. Genetic typing of UMs should also facilitate studies of systemic adjuvant therapy by excluding patients with a low risk of metastasis. We and others have been typing UMs by using a variety of methods, such as cytogenetics and gene expression profiling. 14 For several years, we relied on fluorescence in situ hybridization (FISH), but this method required larger, fresh tumor samples and tested only one centromeric locus on chromosome 3, so that partial deletions were missed. 15 In late 2006, we replaced FISH with multiplex ligation-dependent probe amplification (MLPA), 16 which simultaneously tests 31 genomic sequences on chromosomes 1, 3, 6, and 8, requiring smaller tumor samples that can be either fresh or formalin-fixed and paraffin-embedded (FFPE). 17 In 2009, we validated this method in 73 UMs from patients treated between 1998 and This evaluation showed that equivocal (borderline) MLPA results for chromosome 3 loci indicate a high risk of metastasis, suggesting that this phenomenon could occur as a result of melanoma cell heterogeneity, with disomy 3 cells diluting monosomy 3 cell clones. Several research groups have reported on histologic and genetic intratumoral heterogeneity of UM Such heterogeneity gives rise to a risk of sampling error when performing microbiopsy. In these studies of genetic heterogeneity of UM, only chromosome 3 was tested, and usually only one locus on this chromosome was assessed. We thought it would be useful to study multiple loci, not only on chromosome 3 but also on chromosomes 1, 6, and 8, which are known to develop abnormalities of prognostic significance. The goals of this study, therefore, were to gain more knowledge on intratumoral heterogeneity in UM by assessing copy number variations within different areas of UM using MLPA on FFPE tumor tissue. If we could confirm such heterogeneity, we then proceeded to attempt to determine whether there was an association between the heterogeneous loci and equivocal results of the same loci detected in the whole tumor sections (i.e., a dilution effect). We hope that our findings will facilitate the interpretation of UM microbiopsy results of these tumors. Investigative Ophthalmology & Visual Science, October 2010, Vol. 51, No Copyright Association for Research in Vision and Ophthalmology
2 IOVS, October 2010, Vol. 51, No. 10 Tumor Heterogeneity in Uveal Melanoma 4899 METHODS Patients The present study was performed on archival UM tissue originating from 38 patients who underwent enucleation at the Liverpool Ocular Oncology Centre (LOOC) in 2007 and None of these patients underwent eye-saving treatment (e.g., radiotherapy) before enucleation. Selected UMs were large enough to allow sampling of several distinct areas, each measuring 0.6 mm in diameter for genetic analysis (i.e., a basal diameter exceeding 11.9 mm and a thickness exceeding 5 mm). Informed consent was obtained, and the study was conducted in accordance with the Declaration of Helsinki and with institutional review board approval. Histomorphologic Assessment Four-micrometer sections were cut from each FFPE UM block and stained with hematoxylin and eosin (H&E) and periodic acid-schiff (PAS), with and without hematoxylin counterstain. Histopathologic examination was performed as described elsewhere 15 for assessment of cell type 2 ; presence of PAS closed connective tissue loops; degree of pigmentation; mitotic count (number of mitoses per 40 high-power fields; one HPF 40 objective); and the presence of admixed reactive cells (macrophages and/or lymphocytes). Degrees of reactive inflammation and pigmentation were graded as follows: none, 0; mild, 1; moderate, 2; and strong/numerous, 3 (Fig. 1). This assessment was performed for the whole tumor as well as for each of the microdissected areas (described later). The H&E section was also used to guide microdissection for DNA extraction. Sampling of FFPE Tissue and DNA Extraction For DNA extraction, 20- m-thick whole tumor sections were cut, and two to nine tumor areas were either microdissected using a scalpel or a 0.6-mm-diameter donor punch (manual arrayer; Beecher Instruments, Sun Prairie, WI). When possible, samples were obtained from the tumor apex and base as well as from anterior and posterior portions of the UM. Areas within or at the edge of a UM consisting purely of blood vessels, necrotic tumor, or dense macrophage/lymphocyte infiltrates were intentionally avoided in obtaining the samples. Extraocular melanoma was not sampled, as only two UMs demonstrated extraocular extension (Table 1). Our procedure resulted in a total of 187 UM samples. Nontumor controls comprised 18 FFPE normal choroid and 6 FFPE reactive tonsils. Tissue lysis and protein digestion were performed in 125 L of lysis buffer (50 mm Tris-HCl [ph 8.2], 1 mm EDTA, 100 mm NaCl, 0.5% Tween-20, 0.5% NP40, and 20 mm DTT). Proteinase K (Qiagen, Crawley, UK) was added to the final concentration of 0.8 mg/ml, and after 36 hours of incubation (24 hours at 56 C and 12 hours at 37 C), RNA was cleaved by the addition of RNase A (Sigma-Aldrich Co., Ltd., Gilingham, UK) to a final concentration of 20 g/ L. DNA was extracted (DNeasy Blood and Tissue protocol; Qiagen) and was then eluted in 40 to 50 L of AE buffer. DNA Quantification and Quality Assessment The DNA concentration and absorbance were measured with a spectrophotometer (NanoDrop; Thermo Scientific, Wilmington, DE) at 280 and 260 nm. Multiplex PCR was adapted from van Dongen et al., 23 using the RAG1, PLZF, AF4 exon 3, and AF4 exon 11 primers (Eurofins MWG Operon, London, UK), and was performed on samples with a concentration exceeding 40 ng/ L, to assess DNA quality with a thermal cycler (model TC-412; Techne, Staffordshire, UK). The reaction volume was 25 L and contained 100 ng of DNA and 1 highperformance buffer; 2 mm MgCl 2 ; 0.8 mm dntp mix; units of polymerase (ThermoStart; ABgene-Thermo Fisher Scientific); 0.5% BSA (Sigma-Aldrich Co., Ltd.); 0.1 M forward and reverse primers for RAG1, PLZF, and AF4 exon 11; and 0.2 M forward and reverse primers for AF4 exon 3. PCR products were visualized on 2% agarose gels (150 ma for 30 minutes) stained with 1 SYBR DNA gel stain (SYBR Safe; Invitrogen, Paisley, UK), with a gel-imaging system (Bio Doc-It; Ultra-Violet Products, Ltd., Cambridge, UK). MLPA Procedure and Sequencing The MLPA procedure and sequencing were performed in 32 tumors, as previously reported 14 (Salsa P027.B1 Uveal Melanoma kit; MRC-Holland, Amsterdam, The Netherlands). In brief, six nontumor control samples were used in each MLPA assay, with 200 ng of DNA being analyzed for both tumor and nontumor samples. MLPA reactions were performed on a thermal cycler (G-Storm GS1; Gene Technologies Ltd, Essex, UK) with fragment detection being performed on a genetic analyzer (ABI-3130XL and GeneMapper software; Applied Biosystems [ABI], Foster City, CA). Raw data were received as peak heights, as a measure of peak intensity for each of the 43 probes (31 test probes and 12 control probes). MLPA was performed in triplicate for each UM sample. FIGURE 1. The grading system used for degree of pigmentation and density of inflammatory cells within the UM: (a) UM with no pigmentation and no inflammatory infiltrate (grade 0 for both categories); UM with (b) mild inflammatory infiltrate and (c) mild pigmentation (grade 1); UM with (d) moderate reactive cell infiltrate and (e) moderate pigmentation (grade 2); and; UM with (f) numerous reactive lymphocytes and (g) heavy pigmentation (grade 3). MLPA Data Analysis Analysis was performed using an adapted version of a spreadsheet (Excel; Microsoft, Redmond, WA) designed by the National Genetics Reference Laboratory (NGRL), Manchester, UK ( ngrl.org.uk/manchester/), as described previously. 18 We modified this method to exclude UM control loci that seemed abnormal. Such abnormality was confirmed by manual analysis: abnormal probes (a dosage quotient [DQ] outside the range) that were not detected by the program were excluded, and those falsely excluded were retrieved. The MLPA data were considered reliable if the number of control probes within the normal range was 7. The DQ was categorized as suggested by the NGRL as a deletion (D), 0.65; equivocal deletion (E), ; normal diploid (N), ; equivocal amplification (Q), ; and amplification (A) 1.35.
3 4900 Dopierala et al. IOVS, October 2010, Vol. 51, No. 10 TABLE 1. Clinical and Histological Characteristics of 32 Uveal Melanomas Tested for Genetic Heterogeneity UM Age Sex FU (y) LBD EOM Closed Loops MC EC Cell Het Chr 3 Status* (Overall) Chr 3 Status (Microdissection) H02 79 F L 2 L H04 81 F N 3 G,1 N H05 68 M L 2 L H06 56 F L 4 L H07 49 F L 2 L H08 85 F G/N 2 G/N,2 L H09 69 M L 2 L H10 60 F L 3 G/N,2 L H12 63 F L 7 L,1 N H14 56 M N 4 N H15 71 M L 4 L H16 77 F L 3 L H17 60 F N 5 N H18 67 M N 3 N H20 59 M U 9 N H21 68 M N 4 N H23 67 M L 4 L H24 63 F L 5 L H25 81 F U 5 U H26 63 F L 3 L H27 74 F EL 4 EL H28 54 M L 8 L H29 94 F G 4 N/G H30 61 M L 4 L H41 72 F N 4 N M08 88 M L 3 L M12 72 M L 2 L M13 41 F N 2 N M16 65 F L 2 L M17 63 M L 2 L M18 58 M N 2 N M27 58 M L 2 L FU, follow-up; LBD, largest basal tumor diameter (measured in mm); EOM, extraocular melanoma growth; MC, mitotic count (number per 40/HPF); EC, epithelioid cells; Cell het, cellular heterogeneity. * Chromosome 3 status as determined with routine diagnostic MLPA analysis on a single tumor sample. Chromosome 3 status as determined with MLPA on multiple microdissected sites within the UM. The numbers indicate how many samples were microdissected in each tumor. The data highlighted in bold indicate a change in the interpretation of chromosome 3 status. L, loss; N, normal (diploid); G, gain; EL, equivocal loss; U, uninterpretable. Establishment by MLPA of the Criteria for Heterogeneity Samples taken from each UM were assessed for heterogeneity at each locus by calculating a difference between the maximum and minimum values of DQs for a locus. This method identified the tumor samples that showed the greatest intratumoral difference at each locus. Therefore, calculation of all the differences of all sample combinations was unnecessary. DQ heterogeneity was significant if the difference between two loci was 0.2. This value was the difference between the upper and lower limits of the ranges for deletion, equivocal deletion, equivocal amplification, and amplification. The difference between the upper and lower limit of the diploid range was 0.3. Consequently, a second condition for the definition of a heterogeneous locus was that the DQ difference of 0.2 or more had to belong to different DQ ranges. Correlation between Genetic and Histologic Heterogeneity Chromosomal and histologic heterogeneity findings were coded 0 for no heterogeneity and 1 for heterogeneity. Fisher s exact test was used to correlate chromosomal heterogeneity with histologic heterogeneity with respect to cell type, closed connective tissue loops, degree of pigmentation, and reactive inflammation (SPSS ver. 16.0; SPSS, Chicago, IL). Correlation of DQ Heterogeneity with MLPA Result from Whole Tumor Sections To determine whether equivocal MLPA results obtained from whole UM sections could be explained by intratumoral variation in copy number, we scored the DQs obtained from the whole tumor sections as follows: 1, for equivocal loss or gain; and 0, for unequivocal outcome (i.e., normal diploid and amplification or deletion). For the microdissected tumor area, we used the binary coding specified earlier. The 2 test was used to test for any association after dichotomization of the cohort for heterogeneity and equivocality. RESULTS Patients and Samples A total of 160 samples from 32 UMs were studied after 27 samples from 6 tumors were excluded because of inadequate DNA quality or poor MLPA reproducibility. The 32 UMs included in the study were from 15 men and 17 women who had a mean age of 67 years (range, 41 94). The largest basal tumor diameter (LBD) averaged 16.0 mm (range, ). Two UMs showed extraocular growth. Histologic examination showed epithelioid cells in 19 tumors, PAS closed connective tissue loops in 18 tumors, and a high mitotic count (i.e., exceeding 5/40 HPF) in 16 UMs (Table 1). Two tumors showed partial loss of chromosome 3, and
4 IOVS, October 2010, Vol. 51, No. 10 Tumor Heterogeneity in Uveal Melanoma 4901 LOCI TESTED BY MLPA 60% 50% UVEAL MELANOMAS EXAMINED FIGURE 2. Distribution of heterogeneity of 31 loci across chromosomes 1, 3, 6, and 8, tested by MLPA (P027.B1). Purple: heterogeneous loci; blue: loss; light blue: equivocal loss; green: normal; orange bars: equivocal gain; red bars: gain; x: unreliable results; FR, frequency of the heterogeneous loci; INF, number of heterogeneous loci per tumor/across tumors; HET LOCI, number of heterogeneous loci per tumor. 21 showed complete monosomy 3 in at least one microdissected area. The median postoperative follow-up time was 1.2 years (range, ). Five patients (M8, H12, H23, H24, and H28) developed metastatic disease by the time of study closure, and one of these patients (M8) died during the study period. Intratumoral Chromosomal Heterogeneity According to the criteria for the study, genetic heterogeneity was detected in 24 (75%) of the 32 UMs in between 1 and 26 of the tested 31 loci across chromosomes 1, 3, 6, and 8 (Fig. 2). The percentage of loci that were heterogeneous varied among the UMs (Fig. 3). The most heterogeneous loci, in decreasing frequency, were CDKN1A (6p21.2) in 11 (35%) of 31 (95% confidence interval [CI], 19% 52%); RP1 (8q11.23) in 11 (34%) of 32 (95% CI, 18% 51%); and ROBO1 (3p12.2) in 10 (31%) of 32 (95% CI, 15% 47%) of tumors. (Fig. 4). Percentage of tumors 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% -5% -10% 0% 1-10% 11-20% 21-30% 31-40% 41-60% 61-70% 71-80% 81-90% Percentage of heterogenous loci per tumor FIGURE 3. The percentage of tumors according to the percentage of heterogeneous loci per tumor. INCREASING FREQUENCY OF HETEROGENOUS LOCI Percent 40% 30% 20% 10% 0% -10% Gene Locus FIGURE 4. Frequency of the heterogeneous loci on chromosomes 1, 3, 6, and 8. Eight (25%) of the evaluated 32 melanomas were homogeneous for all 31 loci tested by MLPA (Fig. 2). Three of these were of disomy 3 type. They had a mean LBD of 16.3 mm, with a median mitotic count of 3 of 40 HPFs, and epithelioid cells in one tumor only. Five UMs showed monosomy 3, and these had a median LBD of 15.2 mm, a median mitotic count of 9 of 40 HPFs, and epithelioid cells in two tumors. There were no tumors with partial deletion of chromosome 3 in this group. There was no significant difference in the mean LBD (t-test P 0.533), mean mitotic count (t-test P 0.54), prevalence of epithelioid cellularity (Fisher s exact test: P 0.219), and the prevalence of monosomy 3 (Fisher s exact test: P 1.00), when homogeneous and heterogeneous UMs were compared. Heterogeneity of Chromosome 3 Fifteen (47%) of the examined 32 UMs showed intratumoral variation of at least one locus on chromosome 3 (Fig. 2). Of these, 13 also demonstrated heterogeneity for at least one locus on chromosomes 1, 6, and/or 8. The most common heterogeneous loci on chromosome 3 were ROBO1 (3p12.2), VHL (3p25.3), and FANCD2 (3p25.3), which occurred in 31% (95%, CI 15% 47%), 28% (95% CI, 0.16% 44%), and 22% (95% CI, 14% 36%) of all tumors, respectively (Fig. 2). In 11 (73%) of the 15 UMs with heterogeneity of chromosome 3, the variation involved one to six scattered loci (i.e., they were not clustered in similar chromosomal regions). In the remaining four UMs, 11 of the 13 tested loci on chromosome 3 showed heterogeneity (Fig. 2). When UMs heterogeneous for chromosome 3 loci were compared with those tumors with homogeneous chromosome 3 loci, there was no significant difference between the mean LBD, the mean mitotic rate, the presence of epithelioid cells, and the presence of partial or total chromosome 3 loss for any of the chromosome 3 loci (Table 2). Of note, three of the four UMs with chromosome 3 heterogeneity of 11 loci showed gain of one or both arms of chromosome 3 in at least one part of the tumor (Fig. 5). Of the remaining 17 UMs that were homogenous for all 13 loci on chromosome 3, 5 were of the disomy 3 type, whereas 12 showed monosomy 3 and none showed partial chromosome 3 loss. Correlation between Genetic and Histomorphologic Heterogeneity Intratumoral histomorphologic heterogeneity for melanoma cell type was detected in 10 of 32 UMs. Such cellular heterogeneity correlated with intratumoral heterogeneity of VHL (3p25.3), CDKN1A, and RP1 (Table 3), as well as heterogeneity of connective tissue loops (P 0.001). Heterogeneity of the
5 4902 Dopierala et al. IOVS, October 2010, Vol. 51, No. 10 TABLE 2. A Comparison of UMs with Heterogeneous Chromosome 3 Loci with Those Tumors with Homogeneous Chromosome 3 Loci FANCD2 (i) (3p25.3) FANCD2 (ii) (3p25.3) VHL (3p25.3) MLH1 (3p22.1) CTNNB1 (3p22) SEMA3B (3p21.3) FHIT(i) (3p14.2) FHIT(ii) (3p14.2) ROBO1 (3p12.2) CPO (3q12) RHO (3q21.3) MME (3q25.1) OPA1 (3q29) LBD Mitotic rate Epithelioid cells Monosomy 3 (MLPA) P-values were obtained from the Fisher s exact test and the t-test. There was no significant difference between the mean LBD, the mean mitotic rate, the presence of epithelioid cells, and the presence of partial or total chromosome 3 loss for any of the chromosome 3 loci. latter also correlated with an intratumoral copy number difference in CDKN1A. There was no correlation between genetic heterogeneity and variation in reactive inflammation or tumor pigmentation (Table 3). Distribution of Chromosomal Changes across Uveal Melanomas Not Showing Heterogeneity at a Particular Locus The most common abnormalities in our cohort of UMs were deletions of chromosome 3 and gains of the long arm of chromosome 8. Loss of loci located on chromosome 3 was present in between 25% (for VHL, 3p25.3) and 58% (for FHIT(ii), 3p14.2) of the UMs. Gains of loci on 8q were present in between 41% (for RP1, 8q11.23) and 66% (DDEF1, 8q24.2) of the tumors. Depending on which locus was considered, amplifications on 6p occurred in 19% to 26% of the UMs. Deletions of loci were seen in 13% to 28% and 19% of the tumors on 1p and 6q, respectively. Duplications on 1p, 3, and 6q and deletions on 6p were rare (Fig. 6). Correlation between Genetic Heterogeneity and Equivocal MLPA Results Equivocal results obtained from the whole tumor sections correlated with the underlying intratumoral copy number differences ( 2 test, P 0.001). DISCUSSION The main finding of our investigation using MLPA on FFPE material is that 75% of the analyzed UMs showed intratumoral heterogeneity of chromosomes 1, 3, 6, or 8. Almost 50% of tumors showed intratumoral heterogeneity of at least one locus of chromosome 3. The loci showing heterogeneity most commonly were ROBO1, CDKN1A, and RP1. Most of the UMs showing heterogeneity of chromosome 3 also showed heterogeneity of loci on the other examined chromosomes. Intratumoral heterogeneity of some loci correlated with variation in cell type but not with reactive inflammation or degree of pigmentation. Genetic heterogeneity correlated with equivocal MLPA results obtained from whole tumor sections. To our knowledge, no studies have been undertaken to investigate the intratumoral heterogeneity of multiple gene loci in UM. In this cohort of UMs, 24 (75%) of 32 tumors showed intratumoral heterogeneity of 1 to 26 loci across chromosomes 1, 3, 6, and 8. Only one quarter of the studied UMs were homogeneous for all 31 loci tested by MLPA. Small tumors (LBD 11.9 mm; thickness 5 mm) were excluded from the study because it was not possible to sample multiple sites from such cases. We plan, however, to examine increasingly smaller UMs and to compare the MLPA results with those in the present study. The degree of genetic heterogeneity varied between the examined UMs (Fig. 3). Slight variation involving up to six scattered loci was seen in seven UMs, whereas five tumors showed heterogeneity of 40% of loci on at least three of the four chromosomes examined. The heterogeneity of single isolated loci should be interpreted with caution, as these differences could be an artifact, as a result of DNA degradation after formalin fixation. However, copy number differences involving several loci across a large chromosomal area are associated with true heterogeneity between these regions. The most heterogeneous loci detected using MLPA in decreasing frequency were CDKN1A (35% of UMs), RP1 (34%), and ROBO1 (31%). The least heterogeneous locus was MYCBP, showing a copy number variation in only three tumors. Because of the short follow-up period and the small number of patients with metastatic disease, the clinical relevance of these findings has yet to be determined. With respect to chromosome 3 only, approximately half of the examined UMs showed heterogeneity of at least one locus, with 11 of the tumors demonstrating copy number variation in up to 6 loci. When compared with baseline MLPA data used for classifying the UM for chromosome 3 status, this heterogeneity in the loci between areas did not result in a change in interpretation (Table 1). Four of these UMs, however, showed marked heterogeneity involving 11 of the 13 loci examined on chromosome 3. The intratumoral heterogeneity in these four UMs was enough to result in contradictory interpretation of the chromosomal status in different parts of the same tumor (Table 1). For example, one microdissected area of the UMs indicated monosomy 3, and another area showed amplification of several loci on chromosome 3 (Figs. 5F J). Other groups have reported chromosome 3 heterogeneity in 6.6% 20 to 14% 21 of tumors; however, they applied only one chromosome 3 probe, using FISH or chromogenic (C)ISH, and so the prevalence of this phenomenon is probably underreported. The advantage of MLPA over these cytogenetic methods is that it allows for the simultaneous examination of 13 chromosome 3 loci. Some researchers have suggested that chromosome 3 abnormalities are more common at the tumor base 22 and are associated with melanoma cell type 19 and presence of PAS connective tissue loops. 21 In the present study, we demonstrated a strong correlation between genetic heterogeneity of some chromosomal loci and differences in cell type, as well as the presence or absence of PAS connective tissue loops. Because the copy number variation affected numerous different loci on chromosome 3 and the number of UMs examined in this cohort was relatively small, the statistical analysis for a potential association between the geographic location of a microdissected sample (i.e., apex versus base or anterior versus posterior) and copy number variation was not possible. An interesting finding was that genetic heterogeneity of chromosomes 1, 3, 6, and 8 did not correlate with intratumoral variation in reactive inflammation. This result suggests that the copy number variation observed using MLPA was most likely to be caused by the presence of melanoma cell clones differing in chromosome 3 copy number. This impression is supported by our finding that equivocal MLPA data from whole tumor sec-
6 Tumor Heterogeneity in Uveal Melanoma IOVS, October 2010, Vol. 51, No. 10 A B C 4903 D ii I iii area (i) E area (ii) area (iii) 1.8 GAIN CHROMOSOME 1p CHROMOSOME 3 CHROMOSOME 6 CHROMOSOME LOSS NORMAL area (i) F area (ii) G area (iii) H I ii I area (ii) 2.5 GAIN area (i) J 2.0 CHROMOSOME 1p CHROMOSOME 3 CHROMOSOME 6 CHROMOSOME 8 NORMAL 1.5 LOSS FIGURE 5. MLPA results for three different areas of UM H04, and for two different areas of UM H08. Images and MLPA data for similar areas within these two tumors are not depicted. (A E) UM H04. Area (ii) showed two copies of 3; normal 1, and 6; and loss of 8p and gains on 8q. Areas (i) and (iii) showed gains of multiple loci on both arms of 3 and differences in the status of 1p, 6p, and 8. Area (iv) (not shown) was similar to (i). There were no significant differences in histomorphology in the areas examined. (F J) UM H08. Area (i) showed monosomy 3 and loss of chromosome 1. Area (iii) was similar (not shown). Area (ii) showed a partial gain of 3p25.3-3p22 and two copies of 3q, a partial loss of 1p, and gains of 6p and 8q. Area (iv) (not shown) was similar to (ii). Histologically, there are differences only in the degree of pigmentation of the spindle cells tions were strongly associated with copy number variation in the microdissected areas (P 0.001). However, to support the hypothesis that admixed reactive cells in UMs play a minor role in producing heterogeneous MLPA data, we are currently investigating macrophage- and lymphocyte-rich UMs by using fluorescence-activated cell sorting methods to separate neoplastic from nonneoplastic cell populations. Also of particular interest was that four of the UMs analyzed demonstrated gains on chromosome 3, which consisted of partial or total gain of one or both arms of chromosome 3. To our knowledge, this finding has not been reported. Although MLPA cannot detect polyploidy, we believe that the gains observed on chromosome 3 were aneuploid aberrations, since area (i) area (ii) loss of 1p was also present in three of these UMs, and loss of 6q and 8p was found in one other UMs. Three of these four UMs showed marked intratumor heterogeneity of chromosome 3. Monosomy 3 is considered an early event in UM24; therefore, UMs showing monosomy 3 in at least one area of the tumor and two or more copies of chromosome 3 in other regions of the same tumor are particularly interesting, suggesting that additional copies of chromosome 3 were gained during tumor progression. In summary, we have demonstrated that (1) genetic heterogeneity of chromosomes 1, 3, 6, and 8 is present in most FFPE UMs, so that a single random sample may not be representative of the whole tumor, with the result that false
7 4904 Dopierala et al. IOVS, October 2010, Vol. 51, No. 10 TABLE 3. Probabilities Obtained by Fisher s Exact Test Cell Type Closed Connective Tissue Loops Pigmentation Admixed Reactive Cells Loops Pigmentation Reactive cells MFN2 (1p36.22) NBL1 (1p36.13) PTAFR (1p34) MYCBP (1p34) MUTYH (1p33) RPE65 (1p31) NOTCH2 (1p11.2) FANCD2 (i) (3p25.3) FANCD2 (ii) (3p25.3) VHL (3p25.3) MLH1 (3p22.1) CTNNB1 (3p22) SEMA3B (3p21.3) FHIT (i) (3p14.2) FHIT (ii) (3p14.2) ROBO1 (3p12.2) CPO (3q12) RHO (3q21.3) MME (3q25.1) OPA1 (3q29) PEC1 (6p25.2) DCDC2 (6p22.2) CDKN1A (6p21.2) RUNX2 (6p21) CTGF (6q23.1) IGF2R (6q26) NRG1 (8p12) RP1 (8q11.23) MYC (i) (8q24.12) MYC (ii) (8q24.12) DDEF1 (8q24.2) The null hypothesis was that there is no association between intratumoral heterogeneity of histomorphologic variables (cell type, closed connective tissue loops, pigmentation of tumor cells, and admixed reactive cells) and heterogeneity of 31 loci tested by MLPA. Bold values indicate strong evidence against the null hypothesis (i.e., an association between a variable in a column with a variable in a row). reassurance regarding the survival prognosis is provided; (2) although genetic heterogeneity for chromosome 3 occurred in 47% of the UMs, it involved only a few scattered loci in most tumors and did not change the interpretation of chromosome 3 status between the microdissected areas; (3) UMs containing clones with gains in chromosome 3 occur and tend to show a high degree of heterogeneity; and (4) equivocal MLPA results are likely to be caused by genetically Percent 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% HET GAIN EQ GAIN NORMAL EQ LOSS LOSS Gene locus FIGURE 6. Distribution of chromosomal abnormalities. Het, heterogeneous loci; gain, gain in copy number; eq gain, equivocal gain; normal, normal copy number; eq loss, equivocal loss; loss, loss in copy number.
8 IOVS, October 2010, Vol. 51, No. 10 Tumor Heterogeneity in Uveal Melanoma 4905 different melanoma cell clones and not by reactive cells, although further investigation using fresh tumor material is needed to confirm this impression. Acknowledgments The authors thank Helen Kalirai for fruitful discussion of the results and Roger Mountford (Molecular Genetics Department, Liverpool Women s Hospital) for the use of the sequencing facilities. References 1. Kujala E, Makitie T, Kivela T. Very long-term prognosis of patients with malignant uveal melanoma. Invest Ophthalmol Vis Sci. 2003; 44: Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jockel KH, Becher R. Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996;347: Kilic E, van Gils W, Lodder E, et al. Clinical and cytogenetic analyses in uveal melanoma. Invest Ophthalmol Vis Sci. 2006;47: Scholes AG, Damato BE, Nunn J, Hiscott P, Grierson I, Field JK. Monosomy 3 in uveal melanoma: correlation with clinical and histologic predictors of survival. Invest Ophthalmol Vis Sci. 2003; 44: Damato B, Coupland SE. A reappraisal of the significance of largest basal diameter of posterior uveal melanoma. Eye. 2009;23: White VA, Chambers JD, Courtright PD, Chang WY, Horsman DE. Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer. 1998;83: McLean IW, Foster WD, Zimmerman LE, Gamel JW. Modifications of Callender s classification of uveal melanoma at the Armed Forces Institute of Pathology. Am J Ophthalmol. 1983;96: Coupland SE, Campbell I, Damato B. Routes of extraocular extension of uveal melanoma: risk factors and influence on survival probability. Ophthalmology. 2008;115: Folberg R, Pe er J, Gruman LM, et al. The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: a matched case-control study. Hum Pathol. 1992;23: Kivela T, Makitie T, Al-Jamal RT, Toivonen P. Microvascular loops and networks in uveal melanoma. Can J Ophthalmol. 2004;39: Aalto Y, Eriksson L, Seregard S, Larsson O, Knuutila S. Concomitant loss of chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8 in metastasizing primary uveal melanoma. Invest Ophthalmol Vis Sci. 2001;42: Parrella P, Sidransky D, Merbs SL. Allelotype of posterior uveal melanoma: implications for a bifurcated tumor progression pathway. Cancer Res. 1999;59: Damato B, Eleuteri A, Fisher AC, Coupland SE, Taktak AF. Artificial neural networks estimating survival probability after treatment of choroidal melanoma. Ophthalmology. 2008;115: Onken MD, Worley LA, Person E, Char DH, Bowcock AM, Harbour JW. Loss of heterozygosity of chromosome 3 detected with single nucleotide polymorphisms is superior to monosomy 3 for predicting metastasis in uveal melanoma. Clin Cancer Res. 2007;13: Damato B, Duke C, Coupland SE, et al. Cytogenetics of uveal melanoma: a 7-year clinical experience. Ophthalmology. 2007; 114: Damato B, Coupland SE. Translating uveal melanoma cytogenetics into clinical care. Arch Ophthalmol. 2009;127: Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30:e Damato B, Dopierala J, Klaasen A, van Dijk M, Sibbring J, Coupland S. Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death. Invest Ophthalmol Vis Sci. 2009;50: Sandinha T, Farquharson M, McKay I, Roberts F. Correlation of heterogeneity for chromosome 3 copy number with cell type in choroidal melanoma of mixed-cell type. Invest Ophthalmol Vis Sci. 2006;47: Mensink HW, Vaarwater J, Kilic E, et al. Chromosome 3 intratumor heterogeneity in uveal melanoma. Invest Ophthalmol Vis Sci. 2009;50: Maat W, Jordanova ES, van Zelderen-Bhola SL, et al. The heterogeneous distribution of monosomy 3 in uveal melanomas: implications for prognostication based on fine-needle aspiration biopsies. Arch Pathol Lab Med. 2007;131: Schoenfield L, Pettay J, Tubbs RR, Singh AD. Variation of monosomy 3 status within uveal melanoma. Arch Pathol Lab Med. 2009;133: van Dongen JJ, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT Leukemia. 2003;17: Prescher G, Bornfeld N, Friedrichs W, Seeber S, Becher R. Cytogenetics of twelve cases of uveal melanoma and patterns of nonrandom anomalies and isochromosome formation. Cancer Genet Cytogenet. 1995;80:40 46.
MRC-Holland MLPA. Description version 13;
SALSA MLPA probemix P027-C1 Uveal Melanoma Lot C1-0211: A large number of probes have been replaced by other probes in the same chromosomal regions as compared to previous lots, and several reference probes
More informationUniversity of Groningen
University of Groningen Multiplex Ligation-Dependent Probe Amplification of Uveal Melanoma Damato, Bertil; Dopierala, Justyna; Klaasen, Annelies; van Dijk, Marcory; Sibbring, Julie; Coupland, Sarah E.
More informationTransvitreal Retinochoroidal Biopsy Provides a Representative Sample From Choroidal Melanoma for Detection of Chromosome 3 Aberrations
Anatomy and Pathology/Oncology Transvitreal Retinochoroidal Biopsy Provides a Representative Sample From Choroidal Melanoma for Detection of Chromosome 3 Aberrations Mette Bagger, 1,2 Morten T. Andersen,
More informationUveal melanoma is the most common primary intraocular
Genetics Higher Percentage of FISH-Determined Monosomy 3 and 8q Amplification in Uveal Melanoma Cells relate to Poor Patient Prognosis Thomas van den Bosch, 1,2 Jackelien G. M. van Beek, 2,3 Jolanda Vaarwater,
More informationProduct Description SALSA MLPA Probemix P027-C2 Uveal melanoma To be used with the MLPA General Protocol.
Product Description SALSA Probemix P027-C2 Uveal melanoma To be used with the MLPA General Protocol. Version C2. As compared to version C1, three reference probes have been replaced and the lengths of
More informationGenotypic Profiling of 452 Choroidal Melanomas with Multiplex Ligation-Dependent Probe Amplification
Imaging, Diagnosis, Prognosis Clinical Cancer Research Genotypic Profiling of 452 Choroidal Melanomas with Multiplex Ligation-Dependent Probe Amplification Bertil Damato 1, Justyna A. Dopierala 2, and
More informationUveal melanoma (UM) is a deadly tumor associated with loss. Genomic Profile of 320 Uveal Melanoma Cases: Chromosome 8p-Loss and Metastatic Outcome
Genetics Genomic Profile of 320 Uveal Melanoma Cases: Chromosome 8p-Loss and Metastatic Outcome Kathryn G. Ewens, 1 Peter A. Kanetsky, 2 Jennifer Richards-Yutz, 1 Saad Al-Dahmash, 3 Maria Carla De Luca,
More informationUveal melanoma (UM) is the most common primary intraocular
A R T I C L E S Clinical and Cytogenetic Analyses in Uveal Melanoma Emine Kilic, 1 Walter van Gils, 1,2 Elisabeth Lodder, 2 H. Berna Beverloo, 2 Marjan E. van Til, 2 Cornelia M. Mooy, 3 Dion Paridaens,
More informationABOUT 50% OF PATIENTS
CLINICAL SCIENCES Translating Uveal Melanoma Cytogenetics Into Clinical Care Bertil Damato, MD, PhD, FRCOphth; Sarah E. Coupland, MBBS, PhD, FRCPath Objective: To report our experience in translating uveal
More informationAlmost 50% of all patients with uveal melanoma (UM) die of
Anatomy and Pathology Comparison of Formalin-Fixed and Snap-Frozen Samples Analyzed by Multiplex Ligation-Dependent Probe Amplification for Prognostic Testing in Uveal Melanoma Sarah L. Lake, 1,2 Helen
More informationUveal melanoma remains the most common primary intraocular. Monosomy 3 Predicts Death but Not Time until Death in Choroidal Melanoma
Monosomy 3 Predicts Death but Not Time until Death in Choroidal Melanoma Maria T. Sandinha, 1 Maura A. Farquharson, 2 Ian C. McKay, 3 and Fiona Roberts 4 PURPOSE. To study whether monosomy 3 can predict
More informationLiverpool Ocular Oncology Biobank
Liverpool Ocular Oncology Biobank Ocular Oncology NHS Specialised Services - national organisation responsible for the commissioning of specialised services that help improve the lives of children and
More informationFrequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma
954 & 2011 USCAP, Inc. All rights reserved 0893-3952/11 $32.00 Frequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma Mohamed H Abdel-Rahman
More informationUveal melanoma is the most frequently occurring
Effect of Heterogeneous Distribution of Monosomy 3 on Prognosis in Uveal Melanoma Inge H. G. Bronkhorst, MD; Willem Maat, MD; Ekaterina S. Jordanova, PhD; Wilma G. M. Kroes, MD; Nicoline E. Schalij-Delfos,
More informationEnhancing survival prognostication in patients with choroidal melanoma by integrating pathologic, clinical and genetic predictors of metastasis
18 Int. J. Biomedical Engineering and Technology, Vol. 8, No. 1, 2012 Enhancing survival prognostication in patients with choroidal melanoma by integrating pathologic, clinical and genetic predictors of
More informationIn the early 1990s, nonrandom genetic abnormalities
Genetics Chromosome 3 Status in Uveal Melanoma: A Comparison of Fluorescence In Situ Hybridization and Single- Nucleotide Polymorphism Array Arun D. Singh, 1 Mary E. Aronow, 1 Yang Sun, 2 Gurkan Bebek,
More informationEnhancing survival prognostication in. integrating pathologic, clinical and genetic predictors of metastasis
The Royal Liverpool and Broadgreen University Hospitals NHS Trust NHS Enhancing survival prognostication in patients with choroidal melanoma by integrating pathologic, clinical and genetic predictors of
More informationUveal melanoma is the most common form of primary
Concurrent Loss of Chromosome Arm 1p and Chromosome 3 Predicts a Decreased Disease-Free Survival in Uveal Melanoma Patients Emine Kilic, 1,2,3 Nicole C. Naus, 1,3 Walter van Gils, 2 Caroline C. Klaver,
More informationPrognostication in UM
Prognostication in UM Prognostication in UM 1. Is there a preferred prognostic tool? 2. What is the role of prognostic biopsy? Prognostic parameters in uveal melanoma Clinical Histomorphological Immunohistochemical
More informationGene Expression Profiling has been proposed as a method of risk stratification for uveal melanoma.
Last Review Status/Date: September 2014 Description Page: 1 of 5 Gene Expression Profiling has been proposed as a method of risk stratification for uveal melanoma. Background Uveal melanoma Uveal melanoma,
More informationRoutes of Extraocular Extension of Uveal Melanoma
Routes of Extraocular Extension of Uveal Melanoma Risk Factors and Influence on Survival Probability Sarah E. Coupland, MBBS, PhD, 1 Ian Campbell, MD, FRCS, 2 Bertil Damato, MD, PhD 3 Purpose: To correlate
More informationFEP Medical Policy Manual
FEP Medical Policy Manual Last Review: December 2016 Effective Date: January 15, 2017 Related Policies None Gene Expression Profiling for Uveal Melanoma Summary Uveal melanoma is associated with a high
More informationTransvitreal Fine Needle Aspiration Biopsy of Choroidal Melanoma via Pars Plana Vitrectomy
Surgical Technique Is pars plana vitrectomy a safe method for performing fine needle aspiration biopsy of choroidal melanoma? What are the rates of complications? Clinical Characteristics Do tumor thickness
More information(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish
More informationMetastatic Disease in Polyploid Uveal Melanoma Patients Is Associated With BAP1 Mutations
Anatomy and Pathology/Oncology Metastatic Disease in Polyploid Uveal Melanoma Patients Is Associated With BAP1 Mutations Serdar Yavuzyigitoglu, 1,2 Hanneke W. Mensink, 3 Kyra N. Smit, 1,2 Jolanda Vaarwater,
More informationMRC-Holland MLPA. Description version 06; 23 December 2016
SALSA MLPA probemix P417-B2 BAP1 Lot B2-1216. As compared to version B1 (lot B1-0215), two reference probes have been added and two target probes have a minor change in length. The BAP1 (BRCA1 associated
More informationHigh-Resolution Array CGH Analysis Identifies Regional Deletions and Amplifications of Chromosome 8 in Uveal Melanoma
Anatomy and Pathology/Oncology High-Resolution Array CGH Analysis Identifies Regional Deletions and Amplifications of Chromosome 8 in Uveal Melanoma David W. Hammond, 1 Nawal S. D. Al-Shammari, 1,2 Sarah
More informationUveal melanoma (UM) is the most common primary. Radiation Treatment Affects Chromosome Testing in Uveal Melanoma. Anatomy and Pathology/Oncology
Anatomy and Pathology/Oncology Radiation Treatment Affects Chromosome Testing in Uveal Melanoma Mehmet Dogrusöz, 1 Wilma G. M. Kroes, 2 Sjoerd G. van Duinen, 3 Carien L. Creutzberg, 4 Mieke Versluis, 1
More informationFEP Medical Policy Manual
FEP Medical Policy Manual Effective Date: July 15, 2018 Related Policies: 8.01.10 Charged-Particle (Proton or Helium Ion) Radiotherapy for Neoplastic Conditions Gene Expression Profiling for Uveal Melanoma
More informationWhole-Genome Microarray Detects Deletions and Loss of Heterozygosity of Chromosome 3 Occurring Exclusively in Metastasizing Uveal Melanoma
Anatomy and Pathology Whole-Genome Microarray Detects Deletions and Loss of Heterozygosity of Chromosome 3 Occurring Exclusively in Metastasizing Uveal Melanoma Sarah L. Lake, 1 Sarah E. Coupland, 1 Azzam
More informationIdentification of monosomy 3 in choroidal melanoma by chromosome in situ hybridisation
1527 SCIENTIFIC REPORT Identification of monosomy 3 in choroidal melanoma by chromosome in situ hybridisation M T Sandinha, M A Farquharson, F Roberts... Background/aims: In uveal melanoma monosomy 3 is
More informationDoes ocular treatment of uveal melanoma influence survival?
British Journal of Cancer (2) 3, 285 29 All rights reserved 7 92/ www.bjcancer.com Minireview Does ocular treatment of uveal melanoma influence survival? *,1 1 Ocular Oncology Service, Royal Liverpool
More informationGender Differences in Clinical Presentation and Prognosis of Uveal Melanoma METHODS. Patients
Anatomy and Pathology Gender Differences in Clinical Presentation and Prognosis of Uveal Melanoma Ofira Zloto, Jacob Pe er, and Shahar Frenkel PURPOSE. We examined the clinical differences in manifestation
More informationGastric Carcinoma with Lymphoid Stroma: Association with Epstein Virus Genome demonstrated by PCR
Gastric Carcinoma with Lymphoid Stroma: Association with Epstein Virus Genome demonstrated by PCR Pages with reference to book, From 305 To 307 Irshad N. Soomro,Samina Noorali,Syed Abdul Aziz,Suhail Muzaffar,Shahid
More informationConjunctival melanoma (CoMs) is a rare malignancy, representing
Anatomy and Pathology Multiplex Ligation-Dependent Probe of Conjunctival Melanoma Reveals Common BRAF V600E Gene Mutation and Gene Copy Number Changes Sarah L. Lake, 1,2 Fidan Jmor, 1,2 Justyna Dopierala,
More informationSALSA MS-MLPA KIT ME011-A1 Mismatch Repair genes (MMR) Lot 0609, 0408, 0807, 0407
SALSA MS-MLPA KIT ME011-A1 Mismatch Repair genes (MMR) Lot 0609, 0408, 0807, 0407 The Mismatch Repair (MMR) system is critical for the maintenance of genomic stability. MMR increases the fidelity of DNA
More informationSupplementary Online Content
Supplementary Online Content Fumagalli D, Venet D, Ignatiadis M, et al. RNA Sequencing to predict response to neoadjuvant anti-her2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
More informationAbstract. Optimization strategy of Copy Number Variant calling using Multiplicom solutions APPLICATION NOTE. Introduction
Optimization strategy of Copy Number Variant calling using Multiplicom solutions Michael Vyverman, PhD; Laura Standaert, PhD and Wouter Bossuyt, PhD Abstract Copy number variations (CNVs) represent a significant
More informationSupplementary Figure 1
Supplementary Figure 1 Supplementary Fig. 1: Quality assessment of formalin-fixed paraffin-embedded (FFPE)-derived DNA and nuclei. (a) Multiplex PCR analysis of unrepaired and repaired bulk FFPE gdna from
More informationGene Expression Profiling for Melanoma
Medical Policy Manual Genetic Testing, Policy No. 29 Gene Expression Profiling for Melanoma Next Review: April 2019 Last Review: April 2018 Effective: June 1, 2018 IMPORTANT REMINDER Medical Policies are
More informationUveal melanoma (UM) is the most common intraocular malignant
Genomic Profiling and Identification of High-Risk Uveal Melanoma by Array CGH Analysis of Primary Tumors and Liver Metastases Julien Trolet, 1,2,3 Philippe Hupé, 1,2,3,4 Isabelle Huon, 5 Ingrid Lebigot,
More informationPatricia Chevez-Barrrios AAOOP-USCAP /12/2016
Biomarkers in Ocular Melanoma Patricia Chévez-Barrios, MD Pathology and Genomic Medicine, Houston Methodist Hospital Professor of Pathology and Laboratory Medicine and Ophthalmology, Weill Cornell Medical
More informationSALSA MLPA KIT P060-B2 SMA
SALSA MLPA KIT P6-B2 SMA Lot 111, 511: As compared to the previous version B1 (lot 11), the 88 and 96 nt DNA Denaturation control fragments have been replaced (QDX2). Please note that, in contrast to the
More informationDecreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival
British Journal of Cancer (2002) 87, 1308 1313 ª 2002 Cancer Research UK All rights reserved 0007 0920/02 $25.00 www.bjcancer.com Decreased endothelin receptor B expression in large primary uveal melanomas
More informationUveal Melanoma. Protocol applies to malignant melanoma of the uvea.
Uveal Melanoma Protocol applies to malignant melanoma of the uvea. Protocol revision date: January 2005 Based on AJCC/UICC TNM, 6 th edition Procedures Cytology (No Accompanying Checklist) Biopsy (No Accompanying
More informationI have no relevant conflicts of interest to disclose. John T. Seykora MD PhD Departments of Dermatology & Pathology and Laboratory Medicine
Molecular Characterization of Stage 1-3 Melanoma: Are we close to accurate prognostication and prediction? I have no relevant conflicts of interest to disclose. John T. Seykora MD PhD Departments of Dermatology
More informationMRC-Holland MLPA. Description version 19;
SALSA MLPA probemix P6-B2 SMA Lot B2-712, B2-312, B2-111, B2-511: As compared to the previous version B1 (lot B1-11), the 88 and 96 nt DNA Denaturation control fragments have been replaced (QDX2). SPINAL
More informationPrediction of prognosis in patients with uveal melanoma using fluorescence in situ hybridisation
1440 Department of Surgical and Anaesthetic Sciences, Royal Hallamshire Hospital, SheYeld KAPatel Institute for Cancer Studies, University of SheYeld, SheYeld N D Edmondson Academic Unit of Ophthalmology
More informationAssessment of Breast Cancer with Borderline HER2 Status Using MIP Microarray
Assessment of Breast Cancer with Borderline HER2 Status Using MIP Microarray Hui Chen, Aysegul A Sahin, Xinyan Lu, Lei Huo, Rajesh R Singh, Ronald Abraham, Shumaila Virani, Bal Mukund Mishra, Russell Broaddus,
More informationRetina Center of Oklahoma Sam S. Dahr, M.D. Adult Intraocular Tumors
Adult Intraocular Tumors Sam S. Dahr, M.D. Retina Center of Oklahoma www.retinacenteroklahoma.com www.rcoklahoma.com Table of Contents Posterior uveal malignant melanoma Uveal metastasis Uveal melanoma
More informationMRC-Holland MLPA. Description version 08; 30 March 2015
SALSA MLPA probemix P351-C1 / P352-D1 PKD1-PKD2 P351-C1 lot C1-0914: as compared to the previous version B2 lot B2-0511 one target probe has been removed and three reference probes have been replaced.
More informationRNA preparation from extracted paraffin cores:
Supplementary methods, Nielsen et al., A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor positive breast cancer.
More informationSurveillance following treatment of primary ocular melanoma
Surveillance following treatment of primary ocular melanoma Introduction 50% of UM patients relapse with predominantly liver metastases Risk of metastatic disease can be predicted relatively accurately
More informationIn Situ Hybridization: Market Strategies and Forecasts, US,
In Situ Hybridization: Market Strategies and Forecasts, US, 2018-2024 Table of Contents In Situ Hybridization: Executive Summary The study is designed to give a comprehensive overview of the In Situ Hybridization
More informationSupplemental Data: Detailed Characteristics of Patients with MKRN3. Patient 1 was born after an uneventful pregnancy. She presented in our
1 2 Supplemental Data: Detailed Characteristics of Patients with MKRN3 Mutations 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Patient 1 was born after an uneventful pregnancy. She presented
More informationSingle Cell Quantitative Polymer Chain Reaction (sc-qpcr)
Single Cell Quantitative Polymer Chain Reaction (sc-qpcr) Analyzing gene expression profiles from a bulk population of cells provides an average profile which may obscure important biological differences
More informationApplications of IHC. Determination of the primary site in metastatic tumors of unknown origin
Applications of IHC Determination of the primary site in metastatic tumors of unknown origin Classification of tumors that appear 'undifferentiated' by standard light microscopy Precise classification
More informationMRC-Holland MLPA. Description version 12; 13 January 2017
SALSA MLPA probemix P219-B3 PAX6 Lot B3-0915: Compared to version B2 (lot B2-1111) two reference probes have been replaced and one additional reference probe has been added. In addition, one flanking probe
More informationVitreoretinal surgical management In ocular oncology
www.ophtalmique.ch Vitreoretinal surgical management In ocular oncology Pournaras Jean-Antoine C Vitreoretinal Surgery Unit 1. Surgical resection after proton beam therapy 2. Ocular Biopsy 3. RD in advanced
More informationHER2 CISH pharmdx TM Kit Interpretation Guide Breast Cancer
P A T H O L O G Y HER2 CISH pharmdx TM Kit Interpretation Guide Breast Cancer FROM CERTAINTY COMES TRUST For in vitro diagnostic use HER2 CISH pharmdx Kit HER2 CISH pharmdx Kit is intended for dual-color
More informationDECISIONDx BIOMARKER TESTS
DECISIONDx BIOMARKER TESTS Non-Discrimination Statement and Multi-Language Interpreter Services information are located at the end of this document. Coverage for services, procedures, medical devices and
More informationMRC-Holland MLPA. Description version 29;
SALSA MLPA KIT P003-B1 MLH1/MSH2 Lot 1209, 0109. As compared to the previous lots 0307 and 1006, one MLH1 probe (exon 19) and four MSH2 probes have been replaced. In addition, one extra MSH2 exon 1 probe,
More informationReview Article Genetics of Uveal Melanoma and Cutaneous Melanoma: Two of a Kind?
Hindawi Publishing Corporation Dermatology Research and Practice Volume 2010, Article ID 360136, 13 pages doi:10.1155/2010/360136 Review Article Genetics of Uveal Melanoma and Cutaneous Melanoma: Two of
More informationRNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using
Supplementary Information Materials and Methods RNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using Trizol reagent (Invitrogen,Carlsbad, CA) according to the manufacturer's instructions.
More informationNew: P077 BRCA2. This new probemix can be used to confirm results obtained with P045 BRCA2 probemix.
SALSA MLPA KIT P045-B2 BRCA2/CHEK2 Lot 0410, 0609. As compared to version B1, four reference probes have been replaced and extra control fragments at 100 and 105 nt (X/Y specific) have been included. New:
More informationMATERIALS AND METHODS
A R T I C L E S Detection of c-myc Amplification in Uveal Melanoma by Fluorescent In Situ Hybridization Paola Parrella, 1,2 Otavia L. Caballero, 2 David Sidransky, 2 and Shannath L. Merbs 3 PURPOSE. Genetic
More informationSALSA MLPA KIT P050-B2 CAH
SALSA MLPA KIT P050-B2 CAH Lot 0510, 0909, 0408: Compared to lot 0107, extra control fragments have been added at 88, 96, 100 and 105 nt. The 274 nt probe gives a higher signal in lot 0510 compared to
More informationRole of FISH in Hematological Cancers
Role of FISH in Hematological Cancers Thomas S.K. Wan PhD,FRCPath,FFSc(RCPA) Honorary Professor, Department of Pathology & Clinical Biochemistry, Queen Mary Hospital, University of Hong Kong. e-mail: wantsk@hku.hk
More informationLong-Term Survivors with Metastatic Uveal Melanoma
The Open Ophthalmology Journal, 2012, 6, 49-53 49 Long-Term Survivors with Metastatic Uveal Melanoma Open Access Dominic M. Buzzacco,1, Mohamed H. Abdel-Rahman,1,2, Stanley Park 1, Frederick Davidorf 1,
More information6/22/2015. Original Paradigm. Correlating Histology and Molecular Findings in Melanocytic Neoplasms
6 Correlating Histology and Molecular Findings in Melanocytic Neoplasms Pedram Gerami MD, Associate Professor of Dermatology and Pediatrics at Northwestern University Disclosures: I have been a consultant
More informationGene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics
Plasseraud et al. Diagnostic Pathology (2017) 12:59 DOI 10.1186/s13000-017-0650-3 RESEARCH Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic
More informationCLINICAL SCIENCES. Metastatic Melanoma Death Rates by Anatomic Site After Proton Beam Irradiation for Uveal Melanoma
CLINICAL SCIENCES Metastatic Melanoma Death Rates by Anatomic Site After Proton Beam Irradiation for Uveal Melanoma Wenjun Li, MS; Evangelos S. Gragoudas, MD; Kathleen M. Egan, ScD Background: Ciliary
More informationMost severely affected will be the probe for exon 15. Please keep an eye on the D-fragments (especially the 96 nt fragment).
SALSA MLPA probemix P343-C3 Autism-1 Lot C3-1016. As compared to version C2 (lot C2-0312) five reference probes have been replaced, one reference probe added and several lengths have been adjusted. Warning:
More informationSALSA MLPA probemix P315-B1 EGFR
SALSA MLPA probemix P315-B1 EGFR Lot B1-0215 and B1-0112. As compared to the previous A1 version (lot 0208), two mutation-specific probes for the EGFR mutations L858R and T709M as well as one additional
More informationBin Liu, Lei Yang, Binfang Huang, Mei Cheng, Hui Wang, Yinyan Li, Dongsheng Huang, Jian Zheng,
The American Journal of Human Genetics, Volume 91 Supplemental Data A Functional Copy-Number Variation in MAPKAPK2 Predicts Risk and Survival of Lung Cancer Bin Liu, Lei Yang, Binfang Huang, Mei Cheng,
More informationDr. dr. Primariadewi R, SpPA(K)
Curriculum Vitae Dr. dr. Primariadewi R, SpPA(K) Education : Medical Doctor from UKRIDA Doctoral Degree from Faculty of Medicine University of Indonesia Pathologist Specialist and Consultant from Faculty
More informationPRADER WILLI/ANGELMAN
SALSA MS-MLPA probemix ME028-B2 PRADER WILLI/ANGELMAN Lot B2-0811: As compared to version B1 (lot B1-0609, B1-1108), the 88 and 96 nt control fragments have been replaced (QDX2). PRADER-WILLI SYNDROME
More informationExpressArt FFPE Clear RNAready kit
Features and Example Results General problems with FFPE samples Formalin-fixation of tissues results in severe RNA fragmentation, as well as in RNA RNA, RNA-DNA and RNA protein cross-linking, which impairs
More informationIT S ABOUT TIME. IQFISH pharmdx Interpretation Guide THREEHOURSTHIRTYMINUTES. HER2 IQFISH pharmdxtm. TOP2A IQFISH pharmdxtm
I N T E R P R E TAT I O N IQFISH pharmdx Interpretation Guide TM HER2 IQFISH pharmdxtm TOP2A IQFISH pharmdxtm Breast carcinoma (FFPE) stained with HER2 IQFISH pharmdx Breast carcinoma (FFPE) stained with
More informationHER2 FISH pharmdx TM Interpretation Guide - Breast Cancer
P A T H O L O G Y HER2 FISH pharmdx TM Interpretation Guide - Breast Cancer For In Vitro Diagnostic Use FDA approved as an aid in the assessment of patients for whom Herceptin TM (trastuzumab) treatment
More informationMalignant melanoma of the uvea disseminates purely hematogenously, Very Long-Term Prognosis of Patients with Malignant Uveal Melanoma
Very Long-Term Prognosis of Patients with Malignant Uveal Melanoma Emma Kujala, 1 Teemu Mäkitie, 2 and Tero Kivelä 1,2 From the 1 Ocular Oncology Service and 2 Ophthalmic Pathology Laboratory, Department
More informationMultiple Fibroadenomas Harboring Carcinoma in Situ in a Woman with a Familty History of Breast/ Ovarian Cancer
Multiple Fibroadenomas Harboring Carcinoma in Situ in a Woman with a Familty History of Breast/ Ovarian Cancer A Kuijper SS Preisler-Adams FD Rahusen JJP Gille E van der Wall PJ van Diest J Clin Pathol
More informationncounter Data Analysis Guidelines for Copy Number Variation (CNV) Molecules That Count NanoString Technologies, Inc.
ncounter Data Analysis Guidelines for Copy Number Variation (CNV) NanoString Technologies, Inc. 530 Fairview Ave N Suite 2000 Seattle, Washington 98109 www.nanostring.com Tel: 206.378.6266 888.358.6266
More informationThe development of clonality testing for lymphomas in the Bristol Genetics Laboratory. Dr Paula Waits Bristol Genetics Laboratory
The development of clonality testing for lymphomas in the Bristol Genetics Laboratory Dr Paula Waits Bristol Genetics Laboratory Introduction The majority of lymphoid malignancies belong to the B cell
More informationReporting cytogenetics Can it make sense? Daniel Weisdorf MD University of Minnesota
Reporting cytogenetics Can it make sense? Daniel Weisdorf MD University of Minnesota Reporting cytogenetics What is it? Terminology Clinical value What details are important Diagnostic Tools for Leukemia
More information> 6000 Mutations in Melanoma. Tests That Cay Be Employed. FISH for Additions/Deletions. Comparative Genomic Hybridization
Winter Clinical 2017: The Assessment and Diagnosis of Melanoma Whitney A. High, MD, JD, MEng Associate Professor, Dermatology & Pathology Director of Dermatopathology (Dermatology) University of Colorado
More informationSALSA MLPA KIT P078-B1 Breast Tumour Lot 0210, 0109
SALSA MLPA KIT P078-B1 Breast Tumour Lot 0210, 0109 This P078-B1 Breast Tumour probemix contains probes for several genes (including ERBB2, BIRC5, MYC, TOP2A, ESR1, MTDH, CCND1, CCNE1, EGFR and C11orf30)
More informationTable S1. Primers and PCR protocols for mutation screening of MN1, NF2, KREMEN1 and ZNRF3.
Table S1. Primers and PCR protocols for mutation screening of MN1, NF2, KREMEN1 and ZNRF3. MN1 (Accession No. NM_002430) MN1-1514F 5 -GGCTGTCATGCCCTATTGAT Exon 1 MN1-1882R 5 -CTGGTGGGGATGATGACTTC Exon
More informationProtocol for the Examination of Specimens From Patients With Uveal Melanoma
Protocol for the Examination of Specimens From Patients With Uveal Melanoma Version: Protocol Posting Date: June 2017 Includes ptnm requirements from the 8 th Edition, AJCC Staging Manual For accreditation
More informationP323-B1 CDK4-HMGA2-MDM2
SALSA MLPA probemix P323-B1 CDK4-HMGA2-MDM2 Lot B1-0714, B1-0711. As compared to previous test version (lot A1-0508), this probemix has been completely redesigned. Probes for HMGA2 and several other genes
More informationA complete next-generation sequencing workfl ow for circulating cell-free DNA isolation and analysis
APPLICATION NOTE Cell-Free DNA Isolation Kit A complete next-generation sequencing workfl ow for circulating cell-free DNA isolation and analysis Abstract Circulating cell-free DNA (cfdna) has been shown
More informationAssessment Run B HER2 IHC
Assessment Run B24 2017 HER2 IHC Material The slide to be stained for HER2 comprised the following 5 materials: IHC: HER2 Score* (0, 1+, 2+, 3+) FISH: HER2 gene/chr 17 ratio** 1. Breast carcinoma, no.
More informationMolecular Diagnosis. Nucleic acid based testing in Oncology
Molecular Diagnosis Nucleic acid based testing in Oncology Objectives Describe uses of NAT in Oncology Diagnosis, Prediction, monitoring. Genetics Screening, presymptomatic testing, diagnostic testing,
More informationNucleic Acid Testing - Oncology. Molecular Diagnosis. Gain/Loss of Nucleic Acid. Objectives. MYCN and Neuroblastoma. Molecular Diagnosis
Nucleic Acid Testing - Oncology Molecular Diagnosis Nucleic acid based testing in Oncology Gross alterations in DNA content of tumors (ploidy) Gain/Loss of nucleic acids Markers of Clonality Oncogene/Tumor
More informationMedical Policy An independent licensee of the Blue Cross Blue Shield Association
Gene Expression Profiling for Uveal Melanoma Page 1 of 14 Medical Policy An independent licensee of the Blue Cross Blue Shield Association Title: Gene Expression Profiling for Uveal Melanoma Professional
More informationMAPK Pathway. CGH Next Generation Sequencing. Molecular Tools in Care of Patients with Pigmented Lesions 7/20/2017
Molecular Tools in Care of Patients with Pigmented Lesions Tammie Ferringer, MD Geisinger Medical Center, Danville, PA tferringer@geisinger.edu DISCLOSURE OF RELATIONSHIPS WITH INDUSTRY Tammie Ferringer,
More informationRare melanoma: Are the options improving? Dr Neil Steven Consultant in Medical Oncology University Hospital Birmingham University of Birmingham
Rare melanoma: Are the options improving? Dr Neil Steven Consultant in Medical Oncology University Hospital Birmingham University of Birmingham Classifying melanoma Melanoma (site of origin, thickness,
More informationDako IT S ABOUT TIME. Interpretation Guide. Agilent Pathology Solutions. ALK, ROS1 and RET IQFISH probes (Dako Omnis) MET IQFISH probe (Dako Omnis)
INTERPRETATION Dako Agilent Pathology Solutions IQFISH Interpretation Guide ALK, ROS1 and RET IQFISH probes (Dako Omnis) MET IQFISH probe (Dako Omnis) IT S ABOUT TIME For In Vitro Diagnostic Use ALK, ROS1,
More informationSupplementary Figure 1. mrna targets were found in exosomes and absent in free-floating supernatant. Serum exosomes and exosome-free supernatant were
Supplementary Figure 1. mrna targets were found in exosomes and absent in free-floating supernatant. Serum exosomes and exosome-free supernatant were separated via ultracentrifugation and lysed to analyze
More informationManagement and Outcome of Uveal Melanoma in a Single Tertiary Cancer Center in Jordan
Original Article doi:./tjpath.. Management and Outcome of Uveal Melanoma in a Single Tertiary Cancer Center in Jordan Ahmed Zewar, Ibrahim Nawaiseh, Imad Jaradat, Jakub Khzouz, Khaleel AlRawashdeh, Ghadeer
More information