Mammography screen-detected breast cancers have a better prognosis than predicted from established prognostic markers. A search for additional features that are characteristic for these tumours and their prognosis is needed to reduce overtreatment, a recognized challenge in breast cancer patient management today. Here, we have investigated the occurrence and importance of tumour elastosis.We performed a population based retrospective study of breast cancers detected in the Norwegian Breast Cancer Screening Programme in Vestfold County during 2004–2009. In total, 197 invasive screen-detected cancers and 75 interval cancers in patients aged 50–69 years were compared with regard to standard clinico-pathological parameters and tumour shape, as well as ER, PR, HER2 and Ki67 expression. In particular, the presence of elastotic material in tumours was graded on a 4-tiered scale (score 0–3).Screen-detected cancers had a significantly higher content of stromal elastosis than interval cancers (p < 0.001). High content of elastosis (score 3) correlated strongly with stellate tumour shape, low histological grade, and ER+/HER2- status. Further, high elastosis score was significantly associated with lower Ki67 expression. In survival analyses, cases with high elastosis demonstrated increased recurrence free (p = 0.03) and disease-specific survival (p = 0.11) compared to cases with low elastosis.There is a strong correlation between the presence of tumour elastosis, stellate tumour shape and mammography detection of breast cancers. To our knowledge, this is the first time elastosis has been studied in relation to breast cancer detection method. Presence of elastosis is associated with low tumour cell proliferation (Ki67) and a good prognosis.http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_230The virtual slide(s) for this article can be found here:  The value of population-wide mammography screening is controversial ,2. Thus,Breast cancers are morphologically diverse and show a wide spectrum of growth patterns and features such as expression of ER, PR, HER2 and tumour cell proliferation. Recently, studies have also focused on the stroma (microenvironment) surrounding tumour cells and providing growth conditions and support for malignant cells. The tumour stroma consists of several components, including extracellular matrix (composed of various proteoglycans and fibrous proteins) , certainElastin is normally expressed in significant quantities in skin, lung, cartilage, and large arteries, and it is produced by fibroblasts, smooth muscle cells and chondrocytes as a 72 kDa precursor tropoelastin, which is secreted into the extracellular microenvironment. This protein is cross-linked by lysyl oxidase (LOX) and complexes with several smaller proteins, including fibrillin, to form large structures with unique elastic properties . In breaElastosis was first studied in breast cancer decades ago by Shivas & Douglas and others, showing that tumours with large amounts of elastotic material had a better prognosis ,26. FollPatients were included from Vestfold County in Eastern Norway. Vestfold comprises 5% of the Norwegian population with around 230,000 inhabitants. The Norwegian Breast Cancer Screening Programme involves biannual mammography in the age-group 50–69 years, and was implemented in this county in 2004. The mean age of the actual patients at the time of diagnosis was 60 years (range 49–70 years). A total of 37,977 women participated during the study period March 2004 – June 2009 with attendance rates of 71% and 76%, respectively, during the first two screening rounds. During this period, a total of 285 patients with 202 invasive screen-detected cancers and 83 invasive interval tumours were diagnosed during the prevalent and subsequent rounds. The term interval cancer refers to a breast cancer diagnosed between two screening sessions. Ten patients  were excluded because only core biopsies were available from these locally advanced tumours; the evaluation of elastosis content was limited to cases where standard sections were available (cases treated by primary surgery). In addition, 3 other patients were excluded: 1 screen-detected cancer had no residual tumour tissue for further investigation; 1 screen-detected case was diagnosed as a malignant phyllodes tumour, and 1 patient with interval cancer suffered from multiple metastases at the time of diagnosis, and no biopsy or surgery of the breast was performed. Four patients  had simultaneous tumours in both breasts. To represent the case for this particular study, the Nottingham Prognostic Index (NPI) was used for three of them, to select the tumour with charateristics of the worst prognosis. The fourth patient had an identical NPI in both tumours; the tumour with highest Ki67 score was subsequently selected for our study. Thus, a total of 272 patients with 197 from the screening group and 75 from the interval group were included for this population-based study. Clinical data  were recorded from patient journals . The study was approved by the Regional Ethics Committee of Eastern Norway (registration number S-08685d).All cases were re-examined by microscopy by one of the authors (T.A.K.) by using standard HES stained sections. Tumours were classified as either ductal, lobular or other types of carcinomas. Histological grading was done according to the Nottingham criteria . Tumour Tissue microarray slides from the tumour set were used for Ki67 evaluation. The slides were dewaxed with xylene/ethanol before microwave antigen retrieval for 20 min in TRS buffer (pH9). The slides were incubated for 30 min with a monoclonal Ki67 antibody  (Dako), diluted 1:100. The staining was performed using the EnVision-labelled polymer method. 500 tumour cell nuclei were counted, and the Ki67 positive fraction was calculated.Further histological evaluation was done in conjunction by two authors (Y.C. and J.L.) in a blinded manner for the following parameters:The amount of elastotic stroma in the tumour, which was evaluated in cases with standard sections available, was graded by microscopy in a semiquantitive manner from 0 – 3, according to Shivas & Douglas . This meThe contour shape of the tumour was evaluated in whole tumour slides. If tumour processes (containing tumour cells) were radiating into surrounding tissue, the tumour shape was classified as stellate ,35. TumoPresence of necrotic areas in the infiltrating tumour tissue (not necrosis in DCIS areas) was noted. A necrotic focus was defined as a confluent area of necrosis detectable at intermediate magnification (x40), containing at least 10–15 necrotic cells .The presence of a large collagenous area devoid of carcinoma cells, usually in the center of the tumour, was noted. This has previously been linked to worse prognosis in breast cancer ,38. A fi2 test or Kendall´s test. Mann–Whitney U test was used for analysis of age, tumour diameter and Ki67 between categories. Univariate survival analyses of time to death due to breast cancer  and time to recurrence for patients without metastases at the time of diagnosis , were performed using the Kaplan–Meier method (log-rank test). Entry date was the time of diagnosis. Patients who died from other causes were censored at the date of death in the analyses of disease specific survival. For statistical analyses, tumour elastosis was dichotomized; high elastosis (grade 3) and low elastosis (grade 0–2). A total of 272 patients were accessible for survival analysis in the current study.All statistical analyses were performed with IBM SPSS Statistics, version 21.0 . Two-sided p-values of <0.05 were considered statistically significant. Associations between different categorical variables were assessed by Pearsons χFigure Table Further, screen-detected cancers were significantly associated with star-shape Figure  and abseHigh stromal elastosis was associated with ER and PR expression and HER2 negativity Table . Also, tThe median follow-up period was 71 months (range 2–117 months). Among 272 patients finally included, distant metastases or local tumour recurrence were observed at follow-up in 31 (12 %), and 22 patients (8 %) died of breast cancer.In univariate survival analyses, high elastosis content was significantly associated with longer recurrence-free survival as compared to cases with low elastosis  is a frequent finding in breast cancer. By comparing screen-detected and interval cancers in a population-based material from the Norwegian Breast Cancer Screening Programme, we here found a strong correlation between screening detection and tumour elastosis. High content of elastosis in turn showed strong covariation with other predictors of good prognosis such as low histological grade, hormone receptor expression, HER2 negative tumours, and low tumour cell proliferation by the Ki67 index. Also, significantly improved recurrence free survival was observed in tumours with high elastin content. To our knowledge, this is the first time elastosis content is studied in relation to breast cancer detection method.Tumour elastosis was associated with stellate shape, which is frequent in lesions detected by mammography. Mammographic spiculation has previously been found to correlate with good prognostic factors ,40. MoreIn contrast to screen-detected breast cancer in our study, interval cancers typically had low or absent elastosis content. They tended to have a rounded shape, and displayed necrosis more frequently, the latter reflecting their more aggressive phenotype. These tumours also had higher histological grade and Ki67 index and were more often hormone receptor negative and HER2 positive, which is in accordance with previous studies .What is the biological significance of aggregation of large quantities of elastin fibres in breast cancer stroma? Is there a mechanistic link between the presence of elastin and a better prognosis? The angiostatic molecule endostatin has been reported to accumulate on elastin fibres , and thiMammography screening has been criticized for only a small reduction in breast cancer mortality, although studies differ in their estimates -47. At tOur results indicate a strong correlation between the presence of tumour elastosis and mammography detection of breast cancers, and there was also an association with stellate tumour shape, a frequent detection criterion by mammography. The presence of elastosis was associated with low-grade tumours and good prognosis. Whether and how elastosis is mechanistically involved in tumour development and progress requires further study.


Tumour targeting of the CCK2 receptor-binding minigastrin analogue PP-F11 was determined in a tumour-bearing mouse model at increasing peptide amounts. The optimal therapy was analysed for PP-F11 labelled with 111In-PP-F11 was determined in nude mice bearing CCK2 receptor-transfected A431 xenografts at 1 and 4 h post-injection for escalating peptide masses of 0.03 to 15 nmol/mouse. The absorbed tumour dose was estimated, assuming comparable biodistributions of the 90Y, 177Lu or 213Bi radiolabelled peptides. The linear-quadratic (LQ) model was used to calculate the tumour control probabilities (TCP) as a function of tumour mass and growth.Tumour uptake of 90Y and 177Lu were 400 MBq 90Y/nmol and 120 MBq177Lu/nmol. Both the large elution volume from the 220 MBq 225Ac generator used and reaction kinetics diminished the maximum achieved 213Bi SA in practice: 40 MBq 213Bi/nmol. Tumour uptakes decreased rapidly with increasing peptide amounts, following a logarithmic curve with ED50 = 0.5 nmol. At 0.03 nmol peptide, the (300 mg) tumour dose was 9 Gy after 12 MBq 90Y-PP-F11, and for 111In and 177Lu, this was 1 Gy. A curative dose of 60 Gy could be achieved with a single administration of 111 MBq 90Y labelled to 0.28 nmol PP-F11 or with 4 × 17 MBq 213Bi (0.41 nmol) when its α-radiation relative biological effectiveness (RBE) was assumed to be 3.4. Repeated dosing is preferable to avoid complete tumour receptor saturation. Tumours larger than 200 mg are curable with 90Y-PP-F11; the other radionuclides perform better in smaller tumours. Furthermore, 177Lu is not optimal for curing fast-growing tumours.Practically achievable maximum SAs for PP-F11 labelled with 90Y, despite its longer β-particle range in tissue, certainly for tumours larger than 300 mg. The predicted TCPs are of theoretical nature and need to be compared with the outcome of targeted radionuclide experiments.Receptor saturation, specific radiopharmaceutical activities and absorbed doses in the tumour together favour therapy with the CCK2 receptor-binding peptide PP-F11 labelled with  Indeed in nude mice, 111In-PP-F11 showed low retention in the kidneys in combination with a high uptake in A431 xenografts transfected with the CCK2 receptor in comparison with other minigastrin analogues. The tumour and physiological organ uptake was measured at 1 and 4 h after injection of 370 kBq/0.03 nmol 111In-labelled PP-F11. Moreover, in vitro cell uptake experiments showed a high affinity of PP-F11 for the CCK2 receptor and a high internalisation rate into CCK2 receptor-transfected A431 cells [PP-F11, a minigastrin analogue, was identified to be one of the most promising cholecystokinin 2 (CCK2) receptor-binding peptides to target medullary thyroid cancer (MTC) and small cell lung cancer lesions in a comparative search by the EU COST Action BM0607 [3, but due to different growth patterns, tumours in different animals may show large variation in size.Preclinical PRRT studies are typically being performed in mice with tumours bearing an initial size of 8 to 9 mm in diameter, which corresponds to a spherical volume of 270 to 380 mm177Lu and 131I, are considered favourable to maximise the absorbed dose in tumours of this small size. For pragmatic reasons, the choice for a therapeutic radionuclide is often between the beta-emitters 90Y and 177Lu, as both are readily available. The latter choice (177Lu) seems to be optimal, as the mean beta-energy of 0.133 MeV yields a high probability of cure in the 1- to 3-mm (0.5 to 14 mm3) size range [90Y (0.933 MeV) shows optimal tumour control in the 28- to 42-mm range. Neither 213Bi nor any other α-emitter was evaluated in the paper by O'Donoghue et al., but with the availability of 225Ac generators [213Bi has become a very interesting radionuclide for PRRT as well.Low-energy beta-emitting radionuclides, such as  0.5 to 1 mm3 size177Lu-DOTA,Tyr3-octreotate (DOTAtate) and 90Y-DOTA,Tyr3-octreotide (DOTAtoc) in large and small tumours implanted in rats [90Y, the ideal tumour size for cure was found to be 3 to 9 cm2 , which corresponds to spherical tumours with diameters between 20 and 34 mm and masses between 4 and 20 g. The absorbed dose to these tumours was between 48 and 60 Gy. With 177Lu, a more favourable response rate was found in rats bearing smaller tumours (size <1 cm or mass <0.5 g) compared with those bearing larger tumour masses. The absorbed dose to these small tumours was about 58 Gy.The theoretical prediction of  was conf in rats ,6. With We reasoned for the PRRT study that the ideal β-emitting radionuclide for labelling PP-F11 should be selected taking into account not only the absorbed radiation dose, but also the relation between the uptake kinetics in various sizes of tumours and the peptide amount to be injected. The specific activity achievable for each radionuclide dictates the peptide amount to be injected for a tumour radiation dose of 60 Gy; tumour cure is not always possible with the same peptide amount of 0.03 nmol (corresponding to 0.1 μg) as was used in the biodistribution study. Especially, neutron capture reactor-produced radionuclides show a limitation in the maximum achievable specific activity . The spe90Y, 177Lu or 213Bi. The absorbed dose in small tumours was determined, assuming the same biodistribution profile for all radiolabelled analogues and taking the influence of peptide amount on its pharmacokinetics into account as well as the radiation transport of the emitted spectra. The aim of this study was to determine which radionuclide is capable of reaching tumour-sterilizing absorbed radiation doses to the tumour of at least 60 Gy, as such a dose is expected to lead to a high probability of tumour cure over a wide range of tumour sizes according to the linear-quadratic (LQ) model (from 1 to 500 mm3) [Estimation of the optimal therapy setting has been performed in this study for preclinical testing of PP-F11, assumed to be labelled with either The synthesis of the peptide PP-F11 has been described before by Kolenc-Peitl et al. . In summ111In by incubation with 111InCl3  in 0.25 M ammonium acetate buffer, pH 5.0 under strict metal-free conditions. To reduce oxidation of the peptides during radiolabelling, 100 μg (10 μl) selenomethionine was added. The labelling mixture was incubated for 30 min at 95°C. After incubation, 50 mM EDTA was added to a final concentration of 5 mM. Radiochemical purity and the presence of oxidized peptide were checked on an HPLC system  equipped with an Alltima RP-C18 column  and an in-line NaI radiodetector . A gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in acetonitrile was used. The maximum specific activity of the peptide was 11 GBq/μmol.DOTA-conjugated peptides were radiolabelled with 111In-labelled PP-F11 were studied in female athymic BALB/c mice with subcutaneous tumours induced by inoculation of A431 cells. Mice were inoculated with 2 × 106 A431-CCK2R cells (0.2 ml) in the left flank and with A431 mock-transfected cells in the right flank. After approximately 10 days, when the tumours had reached a weight of approximately 100 to 200 mg, the mice were randomly divided into groups of five mice. The mice were injected intravenously with 370 kBq (0.03 nmol) of 111In-labelled peptide via the lateral tail vein. In an additional experiment, six groups of mice were injected with 370 kBq/0.05 nmol 111In-PP-F11 supplemented with an increasing amount  of molar excess of the unlabelled PP-F11 to determine the receptor saturation effect as well as the non-receptor specific uptake in CCK2 receptor-expressing tissues. Mice were killed by CO2 asphyxiation at 1 and 4 h after administration in the biodistribution study and after 2 h in the receptor saturation study. A blood sample was drawn, and organs of interest and the tumour were dissected, weighed and counted in a gamma counter. The animal experiments were approved by the local animal welfare committee and performed according to national regulations.Biodistribution and tumour targeting of the 90Y, 111In, 177Lu, 213Bi and its daughters were determined in a spherical geometry using the Monte Carlo code MCNP5  of the radioactivity in the tumour [The absorbed energy distribution was determined in each spherical tumour by using 25 equal-volume concentric spherical shells within the source volume and 25 equal-volume spherical shells around the tumour extending to a radial distance of 1 cm as a consistency check of the electron transport model used. Absorbed dose rate per unit activity e tumour .Ã is based on the 111In-PP-F11 biodistribution data at 1 and 4 h for a peptide mass of 0.03 nmol (0.1 μg)  as a function of peptide mass (Mp) was fitted to the sigmoid-shaped one-site competition curve:The tumour uptake concentration data  needed to obtain an absorbed dose D in a tumour with mass m and using the biodistribution for a peptide mass of Mp = 0.03 nmol is obtained by:The amount of activity needed with each radionuclide to obtain a radiation dose of 60 Gy in a 300-mg tumour is derived for the dosimetry according to the 0.03 nmol peptide mass biodistribution. The dosimetry is adjusted according to the peptide mass needed to label the peptide at a practically achievable specific activity using the logarithmic receptor saturation function. Traditionally, the activity A0(Mp) as a function of the peptide mass Mp changes for the maximum (achievable) specific activity Asp at both sides of the equation:The relation for Mp needed with specific activity Asp that will deliver a dose D in a tumour with mass m. Not only the absorbed dose but also the amount of peptide, the specific activity used and the total number of receptor binding sites available are of importance. It is assumed the shape of the kinetics does not change with peptide mass, just the uptake value itself. The maximum and practically achievable specific activities for labelling peptides with 111In, 90Y and 177Lu are indicated in Table 111In, and 90Y shows an almost four times higher specific activity (SA) than 177Lu. The administered amount of peptide per injection was maximised at 0.5 nmol (1.02 μg) to stay within practically feasible limits in both volume and radioactivity. The practical limit in specific activity of 213Bi-labelled peptides was obtained at our institute with a 220 MBq 225Ac generator.This equation can be solved to obtain the amount of peptide mass 213Bi with peptide is much lower than after labelling with 90Y, 111In or 177Lu, due to the at least twice higher reaction volume with 213Bi. The elution volume of the generator is fixed at approximately 600 μL, dependent on the generator bed volume in the preclinical setting using a 220 MBq 225Ac generator. Generators containing higher amount of 225Ac activity have larger bed volumes and higher elution volumes [213Bi, a longer reaction time will result in decay of activity. By reducing the elution volume and reaction time of 213Bi labelling, a higher SA can be achieved. Presently, the time between elution and injection in the animal is in the order of 30 min, by which already 36% of the initial 213Bi radioactivity has decayed.The practical achievable SA of  volumes ,19. HighThe suitability of the radionuclide for treating a tumour xenograft is investigated by constraining the tumour control probability (TCP) to at least 90% according to ,20,21:4\S of clonogenic cells as a function of dose D in the tumour is described by the linear-quadratic model. The dose per fraction D/N is used in the quadratic term to allow correction for fractionation of the therapy. The dose prolongation or Lea-Catcheside factor G is used to adjust the quadratic part of the LQ model for radionuclide-induced dose rates. During the exposure with effective half-life Teff, sub-lethal damage is being repaired according to an exponential function with repair half-life Tμ. The single-exponential clearance with effective half-life Teff is indicated in Equation G. Equation T1 and T2 and amplitudes A1 and A2. The second expression for G (4b) can be modified to take the uptake kinetics into account, with A2 = −A1 and The survival 9/g. The following LQ model parameters were considered: α = 0.27 Gy−1, α/β = 6 Gy and Tμ = 0.5 h . The rells, see .TD during the dose accumulation was made by including the tumour growth in the LQ model equation for cell survival:Correction of TCP for tumour growth with doubling time γ = ln(2)/TD and T is the irradiation time. Overall, the TCP is adjusted by taking the power with eγTirr the growth of the tumour during the effective irradiation time Tirr when the slope, or derivative, of the cell survival curve remains positive, as the radiation effect outweighs the tumour growth [Tirr has passed can be considered effective in cell kill. Therefore, both the absorbed dose and the TCP were corrected for the efficacious dose Deff and the tumour growth during the irradiation time, with:where r growth . The tumr growth . Only thIn the case that it was impossible to give curative doses with the radionuclide in one administration, a treatment interval of 24 h between subsequent injections was assumed with unaltered growth in the treatment gap in between.90Y and 213Bi in comparison to 177Lu. An additional clearance compartment was assumed with a clearance half-life of 150 h, being a median curve between the single exponential and the situation with just physical decay after 4 h, indicated in Figure 213Bi  [The effect of increasing PP-F11 peptide mass on the uptake of the radiolabelled peptide in the tumour and the normal tissues is shown in Figure S-values) were calculated for 90Y, 111In, 177Lu, 213Bi, 213Po, 209Pb and 209Tl in small spherical tumours and are indicated in Table 111In and 177Lu, almost the complete emitted internal conversion electron and beta-energy are absorbed in the largest sphere, whereas for 90Y, only 61% of the emitted energy is absorbed. In the 1-mg sphere the energy absorption ϕ = 10% for 90Y and ϕ = 80% and 69% for 111In and 177Lu, respectively. The absorbed energy fractions by the α-particles from 213Bi and 213Po are more than 95% in the smallest sphere and reach 100% in the 100-mg spheres.Absorbed dose rates per MBq activity  injections with 90Y, 111In, 177Lu and 213Bi labelled to PP-F11, together with the absorbed doses in other tumour sizes, varying from 1 to 500 mg.Taking the single-exponential clearance rate into account, an absorbed dose of 60 Gy in a 300-mg tumour can only be achieved by administering 5 nmol of PP-F11 peptide labelled with 2 GBq of 90Y, 74% for 111In, and 48% for 177Lu when given in one fraction, according to Equation 90Y), 16% (111In) and 12% (177Lu), despite the same dose of 60 Gy. Both reductions in TCP are caused by the lower dose rates, either by the radionuclides' half-lives or by the fractionation scheme. The TCP for 213Bi PP-F11 depends on the relative biological effectiveness (RBE) of its α-radiation (93% of the absorbed dose), which is not clear yet. A RBE higher than 1.2 will already result in a TCP of more than 90% in all tumours below 300 mg. Previously, the RBE for cell killing by 213Bi-DOTA-octreotate has been shown to be 3.4 [213Bi-PP-F11, 90% TCP will already be reached with 4 × 17 MBq (0.41 nmol).The TCPs for absorbed doses of 60 Gy in a 300-mg tumour are 77% for o be 3.4 . If thisG-factor shows a rise: 60 Gy yields higher TCPs of 99% (90Y), 32% (111In) and 20% (177Lu) vs. double-exponential clearance kinetics  are applied, the absorbed dose will be reduced by 18%, whereas for the fast uptake (T1/2 = 7 min), only a 4.2% reduction in dose is observed in comparison to the absorbed dose for instantaneous uptake. Compensation for this possible dose reduction can be obtained by administering either one additional therapy cycle or 9% more radioactivity per cycle.The dose effect by Tirr indicates the time until the radiation cell kill outnumbers the tumour growth. With 111 MBq 90Y (sufficient to cure a static 300-mg tumour), Tirr can vary between 184 h (Tdoubling of 2 days) and 354 h (Tdoubling of 28 days). When 90Y is given in 4 × 20 MBq with 24-h interval, Tirr ranges between 218 and 389 h. The irradiation times for 5 × 359 MBq 111In are comparable to those of 90Y: 237 and 414 h. With 6 × 47 MBq 177Lu, the tumour growth takes over at later times: its irradiation times vary between 317 h (Tdoubling of 2 days) and 611 h (Tdoubling of 28 days). For therapy with 213Bi, the influence of the tumour doubling time is much less (16.8 MBq 213Bi: 6.7 h (for Tdoubling of 2 days) and 11 h (for Tdoubling of 28 days)). The build-up of the dose over this irradiation time is reduced in comparison to the build-up to infinity; for 213Bi, the reduction is below 0.5% and negligible like the maximal 5% reduction with 90Y, but with 177Lu, it is 18% at Tdoubling of 2 days and 5% at Tdoubling of 28 days. Both the effects by reduction in dose and tumour growth during the irradiation have influence on the tumour cure probabilities, as shown in Figure The tumour doubling time influences the tumour cure possibilities in therapies which need the double-exponential clearance kinetics to reach therapeutic doses. The irradiation time The reduction in tumour size as a function of time is shown in Figure 90Y and 213Bi make both radionuclides very suitable for therapy of 200- to 300-mg tumours, using lower peptide mass and thus higher tumour uptake. Yttrium (90Y) is capable of controlling larger-sized (>200 mg) tumours, whereas 213Bi is capable of curing over the whole range of masses considered, but most optimal in the smallest sizes.Based on studies with 12 CCK2 receptor-binding analogues, the biodistribution of the CCK2 analogue PP-F11 shows a very favourable uptake in CCK2 receptor-expressing tumours and relatively low renal retention, which makes it potentially suitable for therapeutic application. This study aimed to investigate its possibilities and hurdles for successful radionuclide therapy with this peptide analogue in mice with s.c. CCK2 receptor-expressing tumours. The particle range of the selected radionuclides has an important impact on the cure capability of the radioloabelled peptide for various sizes of tumours . Since t177Lu-DOTA-octreotate has not been observed, whereas the receptor-mediated uptake in the liver and spleen did show a decrease by the therapeutic amount of peptide [This receptor saturation effect has been observed previously with somatostatin receptor-targeted peptides, both in preclinical biodistribution studies , as in c peptide .213Bi shows ideal properties to cure small lesions, with most of its absorbed dose coming from its short half-life daughter 213Po. The RBE of 213Bi-PP-F11 is not known, but when the effects are comparable to the RBE found for 213Bi-DOTΑ-octreotate of 3.4 [90Y-DOTA-octreotide [111In and 177Lu in the case of single-exponential clearance , 213Bi shows high TCPs, taking an enhancement by increased RBE from its α-radiation into account.For tumour xenografts in almost the whole size range (<500 mg), 177Lu, whereas 213Bi is most strongly influenced by the uptake kinetics.The influence of the clearance kinetics is the largest with 213Bi.Ongoing growth of the tumour during the absorbed dose build-up reduces the TCP for longer half-life radionuclides and has hardly any influence on the cure options for


Neuroblastoma (NBL) is the most common extra-cranial tumour in childhood. It can present as an abdominal mass, but is usually metastatic at diagnosis so the symptomatology can be varied. Nephroblastoma, also more commonly known as a Wilms tumour, is the commonest renal tumour in childhood and more typically presents as abdominal pathology with few constitutional symptoms, although rarely haematuria can be a presenting feature. The pathophysiology and clinical aspects of both tumours including associated risk factors and pathologies are discussed. Oncogenetics and chromosomal abnormalities are increasingly recognised as important prognostic indicators and their impact on initial management is considered. Imaging plays a pivotal role in terms of diagnosis and recent imaging advances mean that radiology has an increasingly crucial role in the management pathway. The use of image defined risk factors in neuroblastoma has begun to dramatically change how this tumour is characterised pre-operatively. The National Wilms Tumour Study Group have comprehensively staged Wilms tumours and this is reviewed as it impacts significantly on management. The use of contrast-enhanced MRI and diffusion-weighted sequences have further served to augment the information available to the clinical team during initial assessment of both neuroblastomas and Wilms tumours. The differences in management strategies are outlined. This paper therefore aims to provide a comprehensive update on these two common paediatric tumours with a particular emphasis on the current crucial role played by imaging. Neuroblastoma (NBL) is the most common extra-cranial tumour in childhood. It can present as an abdominal mass, but it is often metastatic at diagnosis, so can manifest in a variety of other ways often in an unwell child. Nephroblastoma, also more commonly known as a Wilms tumour, is the commonest renal tumour in childhood and more typically presents as abdominal pathology in an otherwise asymptomatic patient. The natural histories and typical clinical courses of these common tumours are very different. This paper reviews the clinical aspects and imaging characteristics of both tumours, summarising the key differences to aid the paediatrician and radiologist in their diagnosis and management.Neuroblastoma (NBL) is the most common solid extra-cranial tumour in childhood . NBL ariTypically, NBLs occur in early childhood with up to 90% diagnosed by 6 years of age . IncidenThe vast majority of cases are sporadic. Approximately 1% are familial, displaying an autosomal dominant pattern of inheritance with incomplete penetrance . There aNBLs have a variable prognosis; tumour stage, patient age, tumour oncogenes and DNA content are all known to be implicated. The MYCN oncogene is responsible for providing the code used by proteins in tissue development. If this mutates, which can be signalled by abnormal amplification, cancerous cells can develop and the resulting mass is more resistant to treatment, thus it has a more unfavourable outcome . This neOther markers which can affect management include chromosomes and nerve receptors. Alterations in two chromosomes, namely a deletion on the short arm of chromosome 1 (1p) seen in around a quarter of NBLs, and deletion of chromosome 11q have a poorer prognosis [Presenting features are diverse and very much dependent on the anatomical location of the tumour. Most commonly, NBLs are located within the adrenal gland, but can be found in sympathetic ganglia of the retroperitoneum, posterior mediastinum, neck or pelvis . The orgAbdominal masses usually cause pain due to their mass effect, as well as abdominal distension . They ofParaneoplastic syndromes may be associated with non-metastatic disease. One of these syndromes is opsomyoclonus, complicating 2-4% of presentations ,6. The oPlain films are non-specific for NBL and are largely unhelpful in the diagnostic pathway. Tumours of the chest and neck can be incidentally picked up on radiographs performed for other reasons Figure . FeatureUltrasound (US) is often the first line investigation in paediatrics, particularly for those presenting with an abdominal mass. NBLs appear as solid, heterogeneous masses with calcification, but are rarely cystic on US . For surMRI should now be the cornerstone imaging modality for all primary NBL tumours Figure , whether99mTc-diphosphonate bone scintigraphy is currently recommended to look for bony disease [Nuclear medicine studies are utilised in the diagnostic pathway to detect occult disease and assess for distant bony spread. Metaiodobenzylguanidine (MIBG) scintigraphy Figure  is sensi disease . MIBG in disease ,11. Gall disease . The pep disease . DOTATATBone marrow biopsy is also necessary, alongside imaging, to confirm bony disease .The International Neuroblastoma Staging System (INSS) has been used since 1986 to stage NBL Table . Also, iThe entire number of IDRFs are summarised in Table Preliminary evidence suggests absence of IDRFs leads to more complete resection, with the presence of IDRFs resulting in more post-operative morbidity . It is uThe COG stratify risk as low, intermediate or high based on prognostic factors and the INSS staging system. Low-risk patients have a 5-year survival rate > 95% with intermediate and high risk group rates of 90-95% and 40-50% respectively . IntermeNephroblastoma is more commonly known as a Wilms tumour after Dr Max Wilms, the German surgeon who first described it in 1899. It is the most common renal malignancy in childhood and accounts overall for 6% of malignancies in children . It typiWilms tumour is an undifferentiated mesodermal tumour, containing a variable amount of embryonic renal elements  . There aNephroblastomatosis, which consists of immature metanephric tissue (nephrogenic rests), is considered a precursor to Wilms tumour Figure . The exaSyndromes associated with nephroblastomatosis include trisomies 13 and 18, Beckwith-Weidemann  and Drash syndromes .Other conditions associated with Wilms tumour include hemihypertrophy (WT2 gene), WAGR syndrome , sporadic non-familial aniridia, neurofibromatosis type 1 and cerebral gigantism (Sotos syndrome) ,22.Presentation is usually with a large, painless abdominal mass and very little in the way of constitutional symptoms . Up to 1Plain abdominal radiographs are non-specific for Wilms tumours. If performed, a feature that may be seen is a soft tissue mass displacing loops of bowel.Imaging and diagnosis of Wilms tumour generally begins with US Figure , which cAlthough yet to be conclusively shown to be superior to CT, the preferred imaging modality at diagnosis is now without doubt contrast-enhanced MRI, in these children with such a favourable long-term outcome. Like CT, MRI can also readily demonstrate the ‘claw sign’ of normal renal tissue around the tumour Figure . The tumMR findings in Wilms tumour are low signal intensity on T1W, with variable/high signal intensity on T2W . Foci ofSome surgeons may quite reasonably prefer later pre-operative CT prior to surgery, and this would generally be a local preference Figure . Post-trThere is continuing uncertainty about the role of pre-operative staging chest CT in the diagnosis of small pulmonary metastases in Wilms tumour. Certainly CT is superior to chest radiography for the detection of small lesions, but these may not always represent metastases. For patients with unilateral Wilms tumour with lung lesions only visible on chest CT, not seen on plain chest radiographs (but deemed metastases and treated as such), their overall survival and event-free survival was no different to those patients whose lung lesions were not deemed metastatic . HoweverPET-CT currently has no role in the initial diagnosis of Wilms tumours as the general prognosis is excellent and radiation exposure should be minimised. In patients who have relapsed, routine PET-CT may be of benefit as their prognosis is more guarded and the best chance of cure is at the first relapse. Accurate staging and discovery of the full extent of metastatic disease would therefore aid survival.Wilms tumour classically follows the “rule of 10’s”: up to 10% may have unfavourable histology, 10% are bilateral, 10% have vascular invasion, 10% have calcifications on CT and 10% have pulmonary metastases at presentation .Staging of Wilms has been developed by the National Wilms Tumour Study (NWTS) and the current staging system is used by the COG Table . The samA unilateral Wilms tumour is treated with nephrectomy. Neoadjuvant chemotherapy is useful to shrink tumour size prior to surgery, but European (SIOP) and American application of this differs. SIOP prefer pre-operative chemotherapy as surgery is made easier as a consequence and there is less risk of tumour spillage. The result is less stage III disease, thus some patients are down-staged. Radiotherapy is indicated for local stage III, so down-staged patients avoid this and the long-term post-radiotherapy sequelae. There are slightly higher local relapse rates reported in SIOP studies, but these radiotherapy naïve patients do appear to have high salvage rates  The AmerIn children with bilateral disease, the therapeutic approach and philosophy are very different. Renal preservation surgery becomes paramount. Pre-operative chemotherapy is vital as each kidney is ultimately staged separately. Hemi-nephrectomy, wedge resections and nephron-sparing surgery require accurate pre-operative imaging. The surgical approach in bilateral disease aims to spare any normal renal parenchyma where possible.Treatment of Wilms tumour is hailed as one of the greatest success stories in modern oncology. Results from the NWTS Group has found overall 10-year survival rates for favourable histology of 96-89% for stages I-III disease (82-49% for unfavourable histology), 81% for stage IV disease (18% for unfavourable histology) and 78% for stage V disease .Neuroblastoma and Wilms tumour are both relatively common abdominal childhood cancers. Some notable key differences are summarised in Table


A pleomorphic invasive ductal carcinoma developed in a patient with Huntington's disease. The tumour showed marked nuclear pleomorphism and contained large number of bizarre tumour giant cells and abundant abnormal mitoses. Tumour cells showed nuclear vesicles and inclusions similar to those described in nuclei of neural cells in patients with Huntington's disease. The case suggests that, in some patients, tumour morphology may reflect specific individual features. Huntington's disease is an autosomal-dominant progressive neurodegenerative disorder characterised by chorea, dystonia, and cognitive decline . PatholoI here present a case of breast carcinoma developing in a patient with Huntington's disease. The tumour was of the uncommon pleomorphic invasive ductal type and showed unusual microscopic features which probably reflect the patient's polyglutamine disease.The patient presented with a 5 cm mass in the left breast. The patient had a long standing history of Huntington's disease. A breast core biopsy showed a poorly differentiated carcinoma which proved to be negative for oestrogen and progesterone receptors as well as for HER2 and cytokeratin 5 but positive for cytokeratin 7. No DCIS elements were present. The tumour was so undifferentiated that it warranted considering the possibility of a metastasis. Further immunohistochemistry showed that the tumour was negative for TTF1 and WT1, and clinical investigations failed to demonstrate any other tumours. The tumour was considered to be a primary grade 3 invasive ductal carcinoma of the breast, and mastectomy with axillary lymph node clearance was recommended.The mastectomy specimen showed a fairly well defined hard tumour mass, 48 mm in maximum dimension that appeared completely excised. Microscopically, the tumour showed marked nuclear pleomorphism involving more than 50% of tumour cells as well as scattered large numbers of multinucleated tumour cells, features which are consistent with pleomorphic invasive ductal carcinoma –11. The Because of the patient's general health, no further treatment was given. The patient died 15 months later from “complications of Huntington's disease.”Although many of the features seen in this case are similar to those described in pleomorphic invasive ductal carcinoma, there are additional features including peculiar cytoplasmic and nuclear changes that are more akin to those described in neural cells of patients with Huntington's disease. This raises the possibility that, in some patients, tumour morphology may reflect specific personal features, leading to a form of “individualised tumour morphology.” The significance of the positive cytoplasmic staining obtained with the huntingtin antibody used is not clear, as it is possible that the antibody may react with the normal as well as the abnormal protein which is known to be ubiquitous. Examination of more tissues is needed to reach a conclusion in that respect.


It is unclear whether the fluid status, as determined by bioimpedance vector analysis (BIVA) combined with serum N-terminal pro-B-type natriuretic peptides (NT-pro-BNP) measurement, is associated with treatment outcome among patients receiving continuous renal replacement therapy (CRRT). Our objective was to answer this question.Patients who were in the intensive care units of a university teaching hospital and who required CRRT were screened for enrollment. For the enrolled patients, BIVA and serum NT-pro BNP measurement were performed just before the start of CRRT and 3 days afterward. According to the BIVA and NT-pro BNP measurement results, the patients were divided into four groups according to fluid status type: type 1, both normal; type 2, normal BIVA results and abnormal NT-pro BNP levels; type 3, abnormal BIVA results and normal NT-pro BNP levels; and type 4, both abnormal. The associations between fluid status and outcome were analyzed.P <0.05). There were significant differences between patients with different fluid status before CRRT start on hospital mortality—type 1, 26.3% (5 out of 19); type 2, 33.3% (5 out of 15); type 3, 40% (4 out of 10); and type 4, 64.4% (29 out of 45) (P = 0.019)—as well as renal function recovery rates: type 1, 57.1% (4 out of 7); type 2, 67.7% (6 out of 9); type 3, 50% (3 out of 6); and type 4, 23.7% (9 out of 38) (P = 0.051).Eighty-nine patients were enrolled, 58 were males, and the mean age was 49.0 ± 17.2 years. The mean score of Acute Physiology and Chronic Health Evaluation II (APACHE II) was 18.8 ± 8.6. The fluid status before CRRT start was as follows: type 1, 21.3% (19 out of 89); type 2, 16.9% (15 out of 89); type 3, 11.2% (10 out of 89); and type 4, 50.6% (45 out of 89). There were significant differences between fluid status types before starting CRRT on baseline values for APACHE II scores, serum creatinine, hemoglobin, platelet count, urine volume, and incidences of oliguria and acute kidney injury  contains supplementary material, which is available to authorized users. Fluid overload is a risk factor for mortality among critically ill patients. As reported in an observational study using data generated by the RENAL  trial, a negative mean daily fluid balance was consistently associated with improved clinical outcomes . For patThe most commonly used methods to calculate daily fluid balance, such as monitoring changes in body weight, are obviously imprecise with pronounced diversity between different methods . BioimpeIn the present study, we assessed fluid status among patients receiving CRRT by using BIVA combined with serum NT-pro BNP and identified correlations between outcome and fluid status, as determined by the combination of these two parameters.From April 2013 to March 2014, all patients requiring CRRT in Jinling Hospital  were screened for enrollment. The inclusion criteria were age of more than 18 years, admission to the intensive care unit, and the requirement of CRRT, and the exclusion criteria were the presence of malignant disease, pregnancy, history of end-stage renal disease, and burn injury. The study protocol was previously approved by the Ethics Committee of Jinling Hospital, and informed consent was obtained from each patient or next of kin before participation. The treatment protocol, including CRRT and prescribed medications to maintain volume balance, remained unchanged after enrollment.A central venous catheter was used for blood access at a blood flow rate of 160 to 200 mL/minute. Pre-dilution continuous hemofiltration (CVVH) was performed by using a polysulphone filter , with a replacement fluid infusion rate of 2,000 to 3,000 mL/hour. Regional citrate anticoagulation combined with low-dose heparin was used to maintain activated clotting time within the extracorporeal circuit in a desired range (200 to 250 seconds). Daily net ultrafiltration volume was determined by the attending physician.BIVA evaluation and serum NT-pro BNP measurement were performed for all participants just before the start of CRRT and 3 days later. The clinical status, incidence of acute kidney injury (AKI), hospital mortality, and renal function recovery were recorded. Right-sided whole-body impedance, including resistance and reactance, was measured by using a Bodystat QuadScan 4000 device  at a frequency of 50 kHz. In accordance with the instructions of the manufacturer, a tetrapolar wrist-to-ankle method was used, in which a pair of electrodes was placed approximately 5 mm apart on the dorsal surface of the right wrist and ipsilateral ankle, with the patient in the supine position. The hydration status was determined by plotting the point vector on the reference bivariate tolerance ellipses (RXc point graph). Vector displacements parallel to the major axis of tolerance ellipses indicate progressive changes in tissue hydration, in which short vectors out of the lower pole  indicate overhydration , and renAccording to the results of BIVA and serum NT-pro BNP measurements, fluid status was divided into four types: type 1, normal status, neither overhydration nor elevation of NT-pro BNP; type 2, no overhydration with abnormal NT-pro BNP levels; type 3, overhydration with normal NT-pro BNP levels; and type 4, overhydration with abnormal NT-pro BNP levels.t test. Continuous variables with a skewed distribution are presented as the median (range) and compared between groups by using the rank-sum test. Categorical variables are expressed as number (percentage) and compared between groups by using the chi-squared test. Survival curves were generated by using the Kaplan-Meier method and compared by using the log-rank test. Univariate and multivariate adjusted logistic regression analyses were performed to determine whether NT-ProBNP, BIVA, or both was an independent risk factor for mortality. Data were analyzed by using SAS version 9.2 statistical software . A two-sided P value of less than 0.05 was considered statistically significant.Continuous variables with a normal distribution are presented as the mean (± standard deviation) and compared between groups by using the Student’s In total, 89 patients, including 58 males, with a mean age of 49.0 ± 17.2 years were enrolled in this study. The primary diseases were as follows (Table P <0.05). Although there were no differences between fluid status types on mortality on day 3, there were significant differences in hospital mortality as well as renal function recovery (Table P = 0.0252).The fluid status prior to the start of CRRT was as follows: type 1, 19 cases 21.3%); type 2, 15 cases (16.9%); type 3, 10 cases (11.2%); and type 4, 45 cases (50.6%). As shown in Table 1.3%; typP = 0.051). Similar to the results of primary fluid status, the highest mortality was among patients with type 4 fluid status on day 3. For patients with type 4 fluid status before CRRT, those with continued type 4 fluid status on day 3 had high mortality (14 out of 20), whereas those with improvement in one parameter of fluid status on day 3 had the lowest mortality (1 out of 5 and1 out of 4) and those with improvements in both two parameters of fluid status (change to type 1 fluid status) had the highest mortality (3 out of 4).A second fluid status evaluation was performed for 70 patients 3 days after beginning CRRT. Patient mortality according to fluid status on day 3 was as follows: type 1, 25% (4 out of 16), type 2, 25% (3 out of 12), type 3, 40% (4 out of 10), and type 4, 60% (18 out of 30)  in patients receiving CRRT, and four types of fluid status corresponded to different clinical conditions and treatment outcomes. Although fluid status was not an independent risk factor for mortality in this study, changes in fluid status and mortality implied potential value of this method for fluid status evaluation in patients receiving CRRT. However, we have to acknowledge the major weakness of the study: the sample size was too small, and the sample size was not calculated, which made the statistics analysis less powerful.Even though the study included only 89 critically ill patients requiring CRRT and the statistical power of the multivariate analyses of independent risk factors for mortality was weak, the results implied that four classifications of fluid status according to two parameters (BIVA and NT-Pro BNP) correspond to respective clinical condition and treatment outcome. Therefore, the combination of BIVA and NT-pro BNP may have potential value for assessment of fluid status in critically ill patients. However, further studies with large sample sizes are needed to confirm our results.Previous studies have reported the independent use of BIVA and NT-pro BNP for assessment of fluid or volume status in critically ill patients. This report described that overhydration and abnormal NT-pro BNP levels corresponded to a poorer clinical condition and outcome, which confirmed the roles of these parameters in the assessment of fluid status as reported elsewhere -12. The Increasingly, more studies have challenged the role of single use of BNP for assessment of fluid status and prediction of treatment outcome -18. A reThe reason for these conflicting results may be due to the reflection of BNP level on only cardiac reaction to current fluid status, which is usually, but not always, consistent with fluid status per se. Therefore, the concept of ‘wet’ and ‘dry’ BNP was proposed ,22, althP = 0.001), experienced greater weight loss (P <0.001), had later transition to oral diuretics (P = 0.03), and were discharged earlier (P <0.001). Late hemoconcentration conferred a significant survival advantage (P = 0.009), whereas early hemoconcentration offered no significant mortality benefit over no hemoconcentration. Although BIVA was not performed in that study, it is expected that patients with late hemoconcentration would be type 4 fluid status, necessitating diuresis and removal of fluid to improve outcome, but that patients with early concentration likely have type 3 fluid status; thus, diuresis and removal of fluid may result in volume depletion but no benefit to outcome. Therefore, water removal via CRRT ultrafiltration should be considerably aggressive only in patients with type 4 fluid status.The subdivision of fluid status, as presented in this study, may provide a useful aid for the maintenance of fluid balance. Especially, type 3 fluid status  may indicate overhydration but without vascular volume expansion, a condition typically presented by patients with nephrotic syndrome and in some receiving peritoneal dialysis . In regaRepeated measurement of BIVA and NT-pro BNP in our study helped to elucidate the effect of change in fluid status on treatment outcome. Owing to the small sample size of this study, there is a lack of conclusive results, but some findings were still noteworthy: for patients with type 4 fluid status before CRRT, after intervention, improvement of either BIVA or NT-pro BNP versus no improvement conferred lower mortality; however, those with improvement of both BIVA and NT-pro BNP had the worst outcomes. A possible reason for this poor outcome may be that over ultrafiltration occurred in these patients, resulting in volume depletion. Therefore, the combined use of NT-Pro BNP and BIVA may avoid unnecessary overtreatment of patients with unrecognized normohydration or dehydration resulting in renal impairment.Fluid status abnormalities were common among patients receiving CRRT. Different types of fluid status distinguished by BIVA combined with serum NT-pro BNP measurements corresponded to different clinical conditions and treatment outcomes. For patients receiving CRRT, real-time monitoring of fluid status by using BIVA and NT-pro BNP may be useful in fluid management by aiding in the identification of an optimal net ultrafiltration rate during CRRT. However, owing to the small sample size and etiological heterogeneity of the enrolled patients, future studies are needed to confirm the value of the combined use of BIVA and NT-pro BNP.Different fluid status type prior to start of CRRT in critically ill patients requiring CRRT, as determined by the combined use of BIVA and serum NT-pro BNP measurement, is associated with different clinical conditions and treatment outcomes.


Since its first description in 1999  concludiSeveral studies have quantified the proportion of patients in whom PPV can be used as a predictor of fluid responsiveness -7. LogicCannesson and colleagues , and morIn daily practice, it is at least as important to have a predictor of the amount of the increase in CO induced by fluid loading as knowing if CO will increase by more or less than 15%. What is the clinically relevant difference between two patients increasing their CO by 14 and 16%, respectively? Studies have repeatedly documented a linear and positive relationship between PPV before fluid administration and the percentage increase in CO in response to fluid loading ,15. ThisRecent studies about the applicability of PPV -7, or th


Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic treatment aims to eliminate (micro)metastatic cells. However decisions regarding systemic treatment depend largely on clinical and molecular characteristics of primary tumours. It remains, however, unclear to what extent metastases resemble the cognate primary breast tumours, especially on a genomic level, and as such will be eradicated by the systemic therapy chosen. In this study we used high-resolution aCGH to investigate DNA copy number differences between primary breast cancers and their paired LNMs. To date, no recurrent LNM-specific genomic aberrations have been identified using array comparative genomic hybridization (aCGH) analysis. In our study we employ a high-resolution platform and we stratify on different breast cancer subtypes, both aspects that might have underpowered previously performed studies.To test the possibility that genomic instability in triple-negative breast cancers (TNBCs) might cause increased random and potentially also recurrent copy number aberrations (CNAs) in their LNMs, we studied 10 primary TNBC–LNM pairs and 10 ER-positive (ER+) pairs and verified our findings adding additionally 5 TNBC-LNM and 22 ER+-LNM pairs. We found that all LNMs clustered nearest to their matched tumour except for two cases, of which one was due to the presence of two distinct histological components in one tumour. We found no significantly altered CNAs between tumour and their LNMs in the entire group or in the subgroups. Within the TNBC subgroup, no absolute increase in CNAs was found in the LNMs compared to their primary tumours, suggesting that increased genomic instability does not lead to more CNAs in LNMs. Our findings suggest a high clonal relationship between primary breast tumours and its LNMs, at least prior to treatment, and support the use of primary tumour characteristics to guide adjuvant systemic chemotherapy in breast cancer patients. Breast cancer is the result of a multistep process of genetic alterations in which normal breast cells are transformed into malignant cells and finally into cells with metastatic capacity Our study focuses on gross DNA copy number changes as genomic alterations in breast cancer. A fair number of studies have investigated genomic differences between primary breast tumours and lymph node or distant metastases by visualizing chromosomal losses and amplifications using either comparative genomic hybridization (CGH) or array-based CGH (aCGH) We hypothesized that using a high-resolution aCGH platform, and stratifying on different subtypes of breast cancer, we would be able to detect specific copy number changes between primary tumours and their LNMs if they occur. Furthermore, we speculated that LNMs of TNBCs would have more copy number aberrations than their primary tumours when compared to luminal tumours due to highest level of genomic instability. To explore these hypotheses, we analysed 10 TNBCs and 10 ER-positive tumours as well as their 20 paired LNMs on a 720 K Nimblegen aCGH platform and did an additional analysis by adding five TNBCs and 22 ER-positive tumour – LNM pairs on a 135 K Nimblegen aCGH platform. We found that regardless of subtype, metastases closely resembled their primary tumours, without specific recurrent copy number differences being present, suggesting that the potential to form metastases might not require specific recurrent CNAs.http://www.federa.org/code-goed-gebruik-van-lichaamsmateriaal-2011).All patients had given written informed consent to be included in the multicentre clinical trial described below, which had been approved by the institutional review committees at each of the participating centres , in which breast cancer patients with at least four tumour-positive axillary lymph nodes but no distant metastases participated Hematoxylin & Eosin (H&E) slides were scored for tumour percentages by a breast cancer pathologist (JW). ER, PR, P53, and HER2 were determined by immunohistochemistry as described previously Genomic DNA was extracted from FFPE primary tumours and their LNMs as previously published https://github.com/SeeLittle/LNCGH) unless otherwise noted. The aCGH data reported in this manuscript have been deposited in the GEO database (accession numbers GSE38888 and GSE56765).The primary first set of patients was analysed separately and results were verified in an analysis including the additional patients assayed on the low-resolution platform. For this second analysis the data of the 720 K arrays was downscaled to 135 K by using only the probes that are present on the 135 K arrays . Background corrected, non-segmented data output by the NimbleScan software were analysed using the R statistical programming language. All analyses were performed in this language . Segmentation parameters were standard, except that we used the option to undo a breakpoint call if the two segments adjacent to the breakpoint did not have mean probe values that were at least two standard deviations apart. When the breakpoint was undone the adjacent segments were merged. We tested whether the number of segments of the delta profiles per tumour – LNM pair was significant using the Wilcoxon rank sum test. To find recurrent pair-wise differential CNAs we searched for overlapping segments among all delta profiles. As a measure of genomic instability-induced change between the primary tumour and the LNM we counted the number of segments in the delta profiles that showed an absolute difference between the primary tumour and the LNM above 0.2 on a log2 scale. For these segments we also determined the copy number state in the primary tumour and the LNM sample. We called the regions in the individual samples ‘gain’ if the segmentation mean was above 0.2 and ‘loss’ if it was below -0.2. We did not perform these analyses on the whole group, since i) a lower resolution platform is less suitable for the identification of small or subtle changes, such as focal aberrations, the aim of this analysis and ii) quantile normalization requires a similar distribution of measurements. The low-resolution profiles demonstrated differential noise  in the measurements, which would be amplified in the quantile normalization process rendering them unusable for this approach.As any group-wise analysis might miss low frequency or specific pair-wise changes, we analysed each tumour-LNM pair separately for samples assayed on the high resolution platform. To allow for direct comparison of tumour and LNM samples we applied quantile normalization on the dataset. As the distributions of the data were not significantly different this makes the samples directly comparable by profile subtraction without losing information. After normalization we subtracted the tumour profile from the LNM profile, creating a so-called ‘delta-profile’. These delta profiles were then segmented by the We hybridized DNA from 20 tumours and paired LNM on the high-resolution 720 K Nimblegen platform, resulting in 40 aCGH profiles . PatientThe aCGH profiles of these two non-concordant pairs were evaluated to determine why the LNMs did not resemble their primary tumour. For patient 456 we found that although visually the profiles were similar, the hybridization was of such poor quality that the correlation distance was quite high . In contWe excluded both non-resembling pairs (patient 456 and 592) from further analyses, since they would introduce artificial differences .KC-smart profiles were highly similar and no significant differences were detected when all samples were analysed simultaneously  we found no significant difference between the TNBC and ER+ subtype .We also checked whether we could identify overlapping delta segments between patients, indicating a recurrent change between tumour and LNM. Although we found a number of overlapping delta segments between tumour and LNM  and five patients to the TNBC group  are largely based on histopathological and molecular characteristics of the primary tumour rather than the disseminated tumour cells. In this study we investigated the genomic differences between primary breast cancers and their paired LNMs by using a high-resolution aCGH platform. We hypothesized that metastasis-specific recurrent DNA alterations have not been identified in the past due to the use of low-resolution BAC-based platforms and the lack of stratification on breast cancer subtypes . Furthermore, we speculated that the genomic instability of TNBCs might result in an increase in CNAs in LNMs, simply due to random occurrence in this subgroup. Therefore, we studied a total of 15 primary breast tumour–LNM pairs of the TNBC subtype and 32 pairs of the ER+ tumours. We found that 1) most LNMs closely resembled their primary tumour with only small focal, non-recurrent CNA differences that were not statistically significant; 2) lack of resemblance was explained by poor quality hybridization or the presence of two histological components within one tumour; 3) no significantly regional differences occurred between primary tumours and LNMs overall or within the specific subtypes; and 4) the number of CNAs within the LNM compared to the primary tumour did not differ between TNBCs and ER+ tumours. These findings were confirmed in a larger dataset on a platform with a slightly lower resolution. Although TNBC tumours show more CNAs in total and are known to be more genomically unstable, the absolute amount of CNAs in the LNMs and their matched primary tumour are equal. The same holds for the ER+ tumours. Although one might expect additional random damage in a metastatic cell compared to its primary tumour simply due to high genomic instability, our results do not show this.This is the first study in which a high-resolution aCGH platform was used to detect differences between primary breast tumours and paired LNMs. Furthermore, this is the first study taking into account breast cancer subtypes. Although our study did not include all subtypes, we speculated that by stratifying on the two most extreme subtypes with regard to number of CNAs Recently, two studies have investigated the clonality within breast cancer by profiling genomes of cells sorted on ploidy of different subsections within the same tumour BRCA1-mutated breast cancers closely with regard to their genomic instability BRCA1-mutation carriers not only with respect to their level of genomic instability but also in terms of their gene expression patterns and their lack of expression of hormone receptors BRCA1-mutated breast cancer BRCA1-mutated breast cancers. This hypothesis was strengthened by the fact that some of these BRCA1-like tumours showed BRCA1-promoter methylation From previous studies it is known that TNBCs resemble In our study we found that biphasic tumours composed of two distinct molecular subtypes can cause discordance between primary tumours and lymph nodes. This finding is very interesting with regard to systemic treatment response, as it could explain the mixed responses  that are sometimes observed within breast cancer patients upon neoadjuvant systemic treatment. Our results suggest that it may be useful to histologically evaluate the primary tumour and (if possible) the LNMs for the presence of multiple components in order to select the optimal systemic treatment.Aside from this isolated, two-component case, there was little difference between primary tumours and their LNM. This apparent lack of difference suggests that intrinsic mechanisms of therapy response/resistance may be similar for both tumour and lymph node metastases, at least with regard to non-targeted therapies (i.e. most chemotherapeutics). Hence, neoadjuvant systemic therapy that leads to pathologic complete remission of the primary tumour might also be most effective for eradication of residual (disseminated) tumour cells in the adjuvant setting. The high level of similarity of genomic profiles further implicates that biomarkers based on genomic aberrations can also be tested on DNA from the LNMs instead of the primary tumour, which can be helpful in case insufficient primary tumour tissue is available.In summary we found a high level of similarity between primary breast tumours and their LNMs, suggesting a strong clonal relationship between both tumour cell compartments. This finding argues that, at least at the DNA copy number level, there is no early evolutionary split between primary tumour cells and  metastatic cells. Our findings argue against the notion that primary tumour cells need to accumulate many additional copy number changes to acquire local metastatic capacity and generally support the use of primary tumour characteristics for guiding the choice of adjuvant systemic therapy. However, our data also show that the existence of biphasic tumours composed of two distinct molecular subtypes may trigger differential responses to systemic therapy, which justifies molecular and histopathological characterization of both primary tumours and their LNMs.Figure S1Heatmap of a hierarchical clustering based on copy number profile. Samples are hierarchically clustered with correlation as distance on their smoothed copy number profiles. The intensity of correlation is shown from blue to yellow. The colored bar above the heatmaps denotes the origin of the sample, either a primary tumour or a lymph node metastasis. The numbers on the y-axis are the patient numbers. Patient numbers in red show primary tumours and lymph node samples that do not cluster together. Patient numbers in green show samples that were reassessed and visualized in (EPS)Click here for additional data file.Figure S2KC-smart package on either (B) the whole group, just the triple negative tumours (C) and just the ER+ tumours (D).Follow-up analyses. A. Hierarchical clustering of 135 K aCGH profiles. Samples are hierarchically clustered with correlation as distance on their smoothed copy number profiles. The intensity of correlation is shown from blue to yellow. The colored bar above the heatmaps denotes the origin of the sample, either a primary tumour or a lymph node metastasis. The numbers on the y-axis are the patient numbers. Patient numbers in red show primary tumours and lymph node samples that do not cluster together. (B-D) Group-wise comparison of primary tumour copy number profiles with lymph node metastasis copy number profiles. Overall profiles were generated using the comparative module of the (PDF)Click here for additional data file.File S1Visualizations of the 720 K copy number profile of all primary tumour – lymph node metastasis pairs that were included in all analyses. Each page shows the profiles for a single patient.(PDF)Click here for additional data file.File S2Visualizations of the delta segments that overlapped and exceeded an absolute log2 value of 0.1. Each page shows all delta segments found on a single chromosome. The upper row shows the raw data for a segment in the tumour sample. The middle row shows the raw data for a segment in the lymph node sample. The lower panel shows all delta segments and the values of the probes in the delta profile. Each patient is coloured differently.(PDF)Click here for additional data file.File S3Visualizations of the 135 K copy number profile of all primary tumour – lymph node metastasis pairs that were included in follow-up analyses. Each page shows the profiles for a single patient.(PDF)Click here for additional data file.File S4Table S1, Patient characteristics of patients with 720 K aCGH data available. Table S2, Overview of all overlapping delta segments. Table S3, Patient characteristics of all patients.Tables S1-S3. (DOCX)Click here for additional data file.


This paper studies the unsteady magnetohydrodynamics (MHD) thin film flow of an incompressible Oldroyd-B fluid over an oscillating inclined belt making a certain angle with the horizontal. The problem is modeled in terms of non-linear partial differential equations with some physical initial and boundary conditions. This problem is solved for the exact analytic solutions using two efficient techniques namely the Optimal Homotopy Asymptotic Method (OHAM) and Homotopy Perturbation Method (HPM). Both of these solutions are presented graphically and compared. This comparison is also shown in tabular form. An excellent agreement is observed. The effects of various physical parameters on velocity have also been studied graphically. In recent time, non-Newtonian fluids have become quite prevalent in industry and engineering. Some of their common examples are polymer solutions, paints, certain oils, exocitic lubricants, colloidal and suspension solutions, clay coatings and cosmetic products. As a consequence of diverse physical structures of these fluids, there is not even a single constitutive model which can predict all the salient features of non-Newtonian fluids. Generally there are three non-Newtonian fluids models. They are known as (i) the differential type, (ii) the rate type, and (iii) the integral type. But the most famous amongst them are the first two models. In this work, we will study the second model, the rate type fluid and consider its subclass known as Oldroyd-B fluid. The simplest subclass of rate type fluid is Maxwell fluid, however, this fluid model can only be described in terms of its relaxation time, while there are no information on its retardation time. The Oldroyd-B fluid model, on the other hand, has a measurable retardation time and can relate the viscoelastic manners of dilute polymeric solutions under general flow conditions. Fetecau et al.  obtainedBurdujan  studied Having such motivation in mind, Gul et al. –18 studiBased on the above motivation, the main goal of the present work is to venture further in the regime of Oldroyd-B fluid. More exactly, this article aims to study the unsteady MHD thin film flow of an Oldroyd-B fluid past an oscillating inclined belt using Optimal Homotopy Asymptotic Method (OHAM) and Homotopy Perturbation Method (HPM). These methods have been used successfully in the literature for the solutions of non-linear fluid problems. Marinca et al. –24 discuV is the velocity vector of the fluid, ρ is the fluid density, g is the external body force. Thus,the Lorentz force perunit volume isB =  is the uniform magnatic filed, B0 is theapplied magnetic field and σ is the electrical conductivity.Let us consider the unsteady MHD incompressible flow over an inclined belt defined by the following equationsJ isThe current density μ0 is the magnetic permeability, E is an electric field which is not considered in this study, andHere, λ1 (relaxation time) and λ2 (retardation time). In λ1 = λ2 the fluid becomes viscous. When λ2 = 0, it becomes a Maxwell fluid and reduced to Oldroyd-B fluid when 0<λ2<λ1<1.The above model can be reduced to different types of fluid depend on T isS is the extra tress tensor, pI is the isotropic stress, A1 is the Rivlin Ericksen stress tensor and μ is the viscosity cofficient.The cauchy stress tensor, δ, of the of liquid layer is considered to be uniform. A uniform magnetic field is applied to the belt in the direction perpendicular to the fluid motion. The external electric field is not considered and the magnetic Reynolds number is negligible, which implies that the current is totally dependent on the induced electric field and the electric current flowing in the fluid does not affect the magnetic field. The induced magnetic field created by the fluid motion is very small compared to the applied magnetic field. Therefore, the Lorentz force term in Let us consider a thin film flow of a non-Newtonian Oldroyd-B fluid on an oscillating inclined belt. The force of gravity will initiate the motion of a layer of liquid in the downward direction. The thickness, ω is the frequency of the oscillating belt.The velocity field is of the formThe momentum It follows from (7) and (9) thatB(y) is used as an arbitrary function. When t<0, then Syy is reduced to zero, which demonstrates that B(y) must also be zero. Therefore, from the Eqs (Here  the Eqs  and 15)B(y) is uω is the oscillating parameter, k1 is the relaxation paramter, k2 is the retardation parameter, m is the gravitational parameter and M is the magnetic parameter.Introducing non-dimensional variablesBy inserting the non-dimensional variables from v is the unknown function, Q is the known analytic function, B is the boundary operator, and D is the general differential operator which is expressed in linear part L) and non-linear part N) asTo illustrate the central concept of HPM to solve non-linear partial differential equation, we consider the following partial differential equationWe can also write qϵ is the embedding parameter and v0 is the initial approximation of Here From q from 0 to 1, v changes from v0 to v which is called deformation, L)-L) and D)-Q are called homotropic.By the variation of q asq = 1 in Approximation solution of L is the linear operator, N is the non-linear operator, G is the known function, v is the unknown function, y is the spatial independent variable, t is time independent variable and B is the boundary operator, and Ω is the domain of the problem.Here we will discuss the primary concept of OHAM, considering a general partial differential equation of the formψ:ψ×→R, needs to satisfy the following equationp is the embedding parameter and pϵ, H(p) is the non-zero auxiliary function for p≠0, H(0) = 0. From p = 0,ψ = v0 and when p = 1 then ψ = v, We obtain v0 by inserting p = 0 in According to the basic theory of OHAM, the optimal homotopy c1,c2,c3 are called convergence controle parameters and will be determined accordingly.Here we select the auxiliary function as:ψasTo find the approximate solution, we expand the unknown function p, we obtain the zero, first and second order problem, v0, v1 and v2, so the governing equation is:By inserting uk are given byNm,v1….vm-1) is the coefficient of pm, in the expansion of Nψ.The general governing equations for c1,c2,… If it converges at p = 1, then the mth order approximation v isThe convergence of the series in ci,i = 1,2,3,4… We apply the Method of Least Squares in our problem as given below:a and b are the constant values taking from domain of the problem.Inserting c1,c2,…cn can be identified from:Auxiliary constants Finally, from these convergence controle parameters, the approximate solution is well-determined.By applying HPM method to The component problems of velocity profile areSolutions of Eqs –24 usingIn this section, we applied OHAM method to The component problems of velocity profile are)∂y2+m=0,p1:  ∂2∂2v0∂y2)]Solutionci for the velocity components are c1 = -1.093756464,c2 = 03004259427.The series solutions of velocity profile is obtained asy-coordinate only for a selected domain yϵ. The effect of first three periods, ω = 0.2,y = 0.4,M = 0.3,t = 1,k2 = 0.1. are used to study the thin layer near the belt as shown in m increases. Actually, it is due to friction which appears greater near the belt and smaller at the surface of the fluid. The effects of k1 (relaxation time parameter) and k2 (retardation time parameter) are shown Figs (Unsteady MHD thin film flow of an Oldroyd-B fluid over an oscillating inclined belt has been examined. The governing partial differential equations for velocity are analytically solved by using OHAM and HPM methods. Both of these results are compared. It is found that these results are in excellent agreement. In tables own Figs  and 8. IM, the fluid motion increases quickly as compared to small values. The influence of gravitational parameter can be seen from m, the speed of fluid layer increases. The effects of non-Newtonian parameters k1 and k2 on velocity profiles are shown in Figs (In case of oscillation, it can be observed in  in Figs  and 12. yϵ, thin film near the belt oscillates together with the belt in the same period and the velocity amplitude of the fluid layer increases gradually towards the free surface of the belt.It is found that for a specific region Due to no-slip condition, the force of friction reduces the gravitational effect near the belt and this effect seems to be greater at the fluid surface.Since magnetic field controls the growth of thin film thickness, therefore, the thickness of thin film remains the same for different frequencies.In this paper, the approximate solutions of unsteady MHD thin film flow of an Oldroyd-B fluid through oscillating inclined belt has been obtained using OHAM and HPM methods for velocity field. Both of these solutions are compared numerically and graphically. It is found that the solution obtained by OHAM and HPM are in excellent agreement. The concluded remarks have been precised as follows:


We firstly established the normal range of each of the 42 diagnostic probes using healthy donor samples. A total of 151 aberrations were detected in 59 patient samples, and 49/59 cases (83.1%) harbored at least one copy number variation. Overall, 0–7 aberrations were detected per case using MLPA, indicating the heterogeneity and complexity of MM cytogenetics. We showed the high efficiency of MLPA and the high congruency of the two methods to assess cytogenetic aberrations. Considering that MLPA analysis is not reliable when the aberration only exits in a small population of tumor cells, it is essential to use both MLPA and iFISH as complementary techniques for the diagnosis of MM.Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical characteristics and outcomes. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MM patients. In the present study, MLPA analysis was applied to analyze cytogenetics of CD138 tumor cells of 59 MM samples, and its result was compared, retrospectively, with the interphase fluorescence  Multiple myeloma (MM) is a terminally differentiated B cell neoplastic disorder, characterized by the presence of clonal proliferation of malignant plasma cells (PCs) in bone marrow and excessive monoclonal immunoglobulin associated with organ dysfunction . Furtherin situ hybridization (iFISH) is able to overcome this shortcoming, and approximately 90% of abnormalities are reported when iFISH is applied for CD138 tumor cells [Although conventional metaphase karyotyping offers a full view of chromosomes, alterations are only detected in 30% of MM cases due to the low mitotic activity of MM cells and the low resolution of the technique . InterphMultiplex ligation-dependent probe amplification (MLPA) is a multiplex polymerase chain reaction (PCR) method for detecting CNVs up to 50 different genomic sequences simultaneously. MLPA probes are able to recognize target sequences with 50–100 nucleotides in length, which makes it possible to be applied for highly fragmented DNA, and the detection of small deletion encompassing only a single exon . To dateIn this study, MLPA analysis was used to detect CNVs in MM using purified CD138 cells. The results were compared with iFISH data to determine the efficiency of MLPA. The combinatorial application of MLPA and iFISH in the routine diagnosis of MM was proposed.p = 0.05) and “Mean ± 3SD”  values for each individual probe were obtained, as listed in Table The X046-A1 MM probemix contained 53 MLPA probes (42 diagnostic probes and 11 reference probes) with amplified products between 122 and 499 nt, indicating that the PCR reaction efficiency was different among probes. As a result, it was inappropriate to use an arbitrary ratio range as the sole cutoff value for all probes, which was applied in most MLPA studies , 11. TheSixty-four MM samples were subjected to MLPA analysis, and 59/64 92.2%) cases passed the DNA quality control and were available for MLPA analysis. Overall, 151 aberrations were detected in 59 cases, and 49/59 cases (83.1%) harbored at least one CNV (Table .2% cases1p deletion 1p deletion was detected in 30/59 cases (50.8%). There were 9 probes specially designed for short arm of chromosome 1, including 5 probes for 1p32, 2 probes for 1p31 and 2 probes for 1p21. Eleven cases only had one probe abnormality; while 8 cases displayed 1p deletion covering more than 3 probes. The DPYD gene at 1p21.3 was the most frequently affected gene (18/30), followed by PPAP2B at 1p32.2 (17/30), and COL11A1 at 1p21.1 (12/30).1q amplification 1q amplification was detected in 32/59 cases (54.2%). Ten probes located in long arm of chromosome 1, including 2 probes for 1q21 and 8 probes for 1q23. Twenty-eight cases harbored large fragment amplification, which involved at least 4 probes, while only 4 cases showed amplification of less than 4 probes. All these 32 cases harbored 1q23 amplification, while 22 cases showed amplification of both 1q21 and 1q32. Contrary to 1p deletion, 1q amplification often represented large segment amplification, and 1q32 was more prone to amplification. In addition, 1q deletion was also detected in one case (#18) (* in Table 5q amplification 5q amplification was detected in 13/59 cases (22.0%). Six probes were included for the evaluation of 5q31.3. Eleven cases showed amplification covering more than 3 probes, while the other 2 cases showed amplification of only one single probe. However, 3 cases  were detected to possess 5q deletion (* in Table 59 cases 2.0%. Six12p deletion 12p deletion was detected in 12/59 cases (20.3%). Six probes hybridized to 12p13 were involved. Seven cases showed the loss of all six probes, indicating the large fragment deletion of 12p13. The other five cases showed only 1 or 2 probe abnormalities. The most frequently effected probes were CD27 (9/12), NCAPD2–2 (9/12) and CDH4–2 (9/12). However, we also found 3 cases  with NCAPD2–2 amplification (* in Table 13q deletion 13q deletion was detected in 36/59 cases (61.0%), which presented the most frequent aberration detected by MLPA. Two probes were designed for 13q14.2, while 2 probes were designed for 13q14.3. Thirty-one cases had the loss of large fragment, which involved at least 3 probes. The most frequently affected probe was RB1–7 (34/36), followed by DLEU1-down (33/36).16q deletion 16q deletion was detected in 14/59 cases (23.7%). CYLD-2/-19 probes were particularly designed for 16q14.2, while WWOX-1/-8 probes were designed for 16q23.1. Ten cases showed the loss of at least 2 probes, and the most frequently affected probe was CYLD-19 (12/14), followed by WWOX-7 (10/14).17p deletion 17p deletion was detected in 7/59 cases (11.9%). The 3 probes could target different exons of TP53, and were found lost in all 7 cases.p = 0.001) and OS  than those without del(12p)  and a median OS of 10.5 vs. 26.3 months (p = 0.023)  and OS of 29.8 vs. 22.2 months (p = 0.049) .These chromosomal aberrations detected by iFISH have already been found prognostically relevant. For instance, 5q31.3 amplification was reported to be a favorable prognostic factor of MM , while a) Figure . Meanwhi) Figure . Moreove) Figure . While tTo evaluate the performance of MLPA as a candidate diagnostic method for the identification of CNVs in MM, iFISH results of 59 cases were studied retrospectively and compared with MLPA results directly. The frequency of genetic lesions determined by iFISH and MLPA was listed in Table Three abnormalities, including 1q amplification, 13q deletion and 17p deletion were detected by both iFISH and MLPA. Out of 177 (3 × 59) comparisons, 156 results were concordant, and the whole consistency was 88.1%. The McNemar test was used for the comparison between iFISH and MLPA. As shown in Table In 13 cases, 13q deletion was only detected by MLPA rather than iFISH. As displayed in Table It is worth noting that there were still 8 aberrations only detected by iFISH but not by MLPA, although MLPA showed a higher resolution. The discordant results were listed in Table Although MM patients present common histological and morphological diagnosis, MM displays enormous cytogenetic complexity and marked variations in clinical characteristics and outcomes. Thereby, risk stratification and prognosis assessment based on cytogenetic changes are important for treatment of MM. It has been well established that del(17p), t and t confer high risk of MM , 12. HowMLPA can analyze up to 50 CNVs in a single PCR reaction. Owing to the high throughput capability of the technique, MLPA analysis kit can be updated rapidly according to the latest research progress in cytogenetic study. For example, compared with SALSA P425-A1 kit, P425-B1 kit also contains probes related to amplification of chromosome 9 and 15 . As a reMLPA results were compared with iFISH data because iFISH is the most commonly used method. Although there is a great concordance between iFISH and MLPA, some discrepancies still exist. In our study, the highest discrepancy exists in 13q deletion, with especially low specificity of 60.6%. The discrepancy may result from different probes used in the two techniques. In our study, del(13q) abnormality detected by iFISH was analyzed with the probe specific for 13q14.1–13q14.3 locus (LSI RB-1), which is approximately 220 kb and contains sequences targeting the entire RB1 gene. While in MLPA analysis, the two probes located in 13q14.2 only target exon 7 and exon 27 of RB-1 gene. The different resolution of the two techniques may be a reason of the differences. Moreover, the genes DLEU-1 and DLEU-2, targeting 13q14.3 in MLPA, located about 2.0 Mb far from the probe LSI RB-1 [To conclude, discrepancies between the two methods may be related to three major reasons: different resolution, point mutation and subclone. Firstly, iFISH analysis is only able to detect deletion or amplification of sequences larger than 20–50 kb, while MLPA can recognize sequences of 50–100 nt in length. Consequently, the application by MLPA can achieve the detection of highly fragmented cryptic lesion. Secondly, mutation or polymorphism in the sequence detected by a probe can also result in reduction of the relative peak area in MLPA analysis, which cannot be picked up by iFISH. Usually, several different probes for a specific gene or different genes in the same chromosomal region are used in a MLPA kit. It is much more reliable when several related probes show the same aberration simultaneously. However, it is often ambiguous when only one single probe is used for the detection of abnormality. We cannot discriminate mutation or deletion without using other methods. Thirdly, with the development of novel technologies, such as CGH and NGS, clonal heterogeneity and clonal evolution have emerged as critical concepts in the field of oncology, especially in the pathogenesis of MM –23. It iFurthermore, MLPA failed to detect IgH translocation, which accounts for 40%–50% of primary cytogenetic events in MM and strongly influences disease phenotype and prognosis. This shortage of MLPA makes iFISH irreplaceable. Taken together, MLPA can detect CNVs in a high throughput fashion with higher resolution, while iFISH can detect both balanced and unbalanced rearrangements and are more reliable to detect aberrations existing in a small subclone. MLPA and iFISH analysis are mutually complementary for the detection of cytogenetic aberrations of myeloma.Bone marrow samples from 64 newly diagnosed MM patients were obtained at The Institute of Hematology and Blood Disease Hospital from August 2011 to March 2013. The male to female ratio was 40/24, and the median age of the patients was 58 years old (range: 25–75 years old) with the median follow-up time of 17 months (range: 0–36 months) from the time point of sample collection. Ten peripheral blood samples from healthy donors were also collected. All patients and healthy donors signed an informed consent form approved by institutional local ethics committee.Mononuclear cells (MNCs) from MM patients and healthy donors were isolated by gradient density centrifugation . Malignant PCs were purified from MM samples using the Miltenyi technology (anti-CD138-coated magnetic beads) as described previously to ensure plasma cell purity of higher than 90% .Interphase FISH analysis was used to detect cytogenetic aberrations in purified PCs of MM. In detail, Del (13q) abnormality was analyzed with the probe specific for the 13q14 locus . Del (17p13) was assessed using a probe specific for the 17p13.1 locus . In order to detect the amplification of 1q21, we used 1q21 (CKS1B) probe . The LSI IGH/FGFR3 dual-color probe (Abbott Laboratories), LSI IGH/CCND1 XT probe (Abbott Laboratories) and IGH/MAF DF probe (Abbott Laboratories) were used to detect t, t, and t, respectively.A total of 200 interphase nuclei were analyzed. Cut-off values recommended by the European Myeloma Network (EMN) were used. For the deletion and numerical aberration, the cut-off level was set at 20%; for translocation in IgH locus as well as other translocation, the cut-off level was set at 10% .http://Coffalyser.Net software  , and subjected to MLPA analysis using SALSA kit X046-A1 that was renamed as SALSA P425-A1 . The probe mix contained 42 probes for the following chromosomal regions such as 1p32-p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23 and 17p13. These probes could predict prognostic relevance in MM. In addition, 11 reference probes were included in this probe mix for detecting 11 different chromosomal locations with relative quietness in MM. MLPA reactions were performed according to the manufacturer's instructions. Finally, the PCR products were analyzed using an ABI 3130 capillary sequencer  and erlands) .p < 0.05.The McNemar test was used for the comparison between MLPA and iFISH for the aberration detection. Survival curves were plotted by the Kaplan-Meier method, and the difference was assessed by log-rank test. Progression free survival (PFS) was calculated from the initiation of therapy to the date of death, progression or last follow-up. Overall survival (OS) was measured from the initiation of treatment to the date of death or last follow-up according to the International uniform response criteria . The sta