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00019606-201006000-00003ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2010 Lippincott Williams & Wilkins, Inc.19June 2010 p 78-82Detection of Transplacental Melanoma Metastasis Using Quantitative PCROriginal ArticlesRaso, Alessandro PhD*; Mascelli, Samantha PhD*; Nozza, Paolo MD†; Biassoni, Roberto PhD‡; Negri, Francesca PhD†; Garaventa, Alberto MD§; Tarantino, Vincenzo MD∥; Garrè, Maria Luisa MD¶; Cama, Armando MD*; Capra, Valeria MD**Neurosurgery Unit†Pathology Unit‡Molecular Medicine Unit¶Neuro-Oncology UnitDepartments of §Haematology-Oncology∥Otolaryngology, Giannina Gaslini Children's Research Hospital, Genoa, ItalyReprints: Alessandro Raso, PhD, Neurosurgery Unit, Giannina Gaslini Children's Research Hospital, Genoa, Italy. Largo Gerolamo Gaslini 5, Genoa 16147, Italy (e-mail: [email protected]).Research was performed at the Neurosurgery Unit, Giannina Gaslini Children's Research Hospital, Genoa, Italy.The authors declare no conflict of interest.AbstractTo choose the most appropriate treatment for children affected by a transplacental metastasis, it is crucial to ascertain the maternal origin of the tumor. Up-to-date conclusive diagnosis is generally achieved through fluorescence in situ hybridization or karyotyping analysis. Herein, we report an alternative, reliable assay for rapidly defining vertical cancer transmission to the fetus by using quantitative polymerase chain reaction. Our assay indicates that quantification of the copy number of the sex chromosomes by specific short tandem repeats markers, in genomic DNA purified from the tumor biopsy cells, could be used to correctly evaluate transplacental metastasis events.Real-time quantitative polymerase chain reaction (qPCR) is used in a variety of fields, such as clinical diagnosis, molecular research, and forensics studies, to detect the presence of copy number changes in the genome or the viral/bacterial load in various body fluids, or to quantify the expression levels of specific genes.1 Regardless of its application, qPCR is currently used to detect either DNA or RNA molecules. It is based on a quantitative relationship between the amount of starting target sequence and the amount of amplified PCR product at any given cycle. To date, qPCR has been a sensitive, robust technique with a wide range of applications whose technology has been modified and optimized over the past decade.2 Recently, we developed a new qPCR application in an unusual clinical setting, that is, in a case of transplacental metastasis. In contrast, we had to conclusively show the maternal origin of a rare temporal bone tumor in an infant. Cancer transmission to the fetus is uncommon, and very few reports have ever shown such an event,3 even though cancer during pregnancy is not an exceptional event (1 case per 1000 live births).4 Biopsy can significantly contribute to the correct management of patients with transplacental metastases. Apart from a solid histologic diagnosis, this procedure allows us to obtain tissue for cytogenetic analysis [karyotyping or fluorescence in situ hybridization (FISH)], which will then allow us to correctly distinguish maternal cells from fetal cells. These methods require an adequate amount of good-quality tissue samples, and they must be performed by a skilled cytogeneticist. We therefore successfully developed a quick and simple screening method to show the maternal origin of tumors. This method is based on the evaluation of selected markers mapping on the sex chromosomes of the tumor cells, and may be used in cases of vertical transmission to a male fetus. To our knowledge, this is the first approach to correctly evaluate the maternal origin of tumor cells using qPCR. It may be added to the analyses that are commonly used for early sex determination by detection of fetal DNA in maternal plasma or for sex determination and genotyping both in genetic paleontology and forensic analysis.MATERIALS AND METHODSPathologic ExaminationA left middle ear temporal bone lesion was discovered in a 6-month-old boy whose mother had died of metastatic melanoma a few weeks after delivery. Histologic examination of biopsy tissue showed a tumor composed of small and medium-sized cells, with oval-to-round nuclei and poorly defined eosinophilic cytoplasms (Fig. 1). Some cells formed nests. Tumor cells were reactive for S100 protein and HMB-45. Their nuclei were positive for the BAF47 molecule. Labeling index was 15% CD3, CD20, CD99, epithelial membrane antigen, chromogranin A, cytokeratins, desmin, smooth muscle actin, specific muscle actin, synaptophysin, tirosin-hydroxilase were negative. The histologic findings were in keeping with melanoma. Complete dermatological assessment failed to reveal any lesions on the skin or in the mucosae.JOURNAL/dimp/04.03/00019606-201006000-00003/figure1-3/v/2021-02-17T200020Z/r/image-jpeg Hematoxylin and eosin staining of the tumor biopsy.Biopsy and Genomic DNA ExtractionThe frozen tumor biopsy was processed by drawing 6 μm sections that were stained with hematoxylin and eosin to be sure that the tumor cell content was above 80%. Genomic DNA samples were obtained from peripheral lymphocytes and from frozen tumor slides by using PureLink mini columns (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. The DNA samples were quantified by Nanodrop spectrophotometric analysis (Celbio, Milan, Italy). Written informed consent was obtained from the patient's father.Quantitative Real-time PCR, SC Construction, and Assay ConditionsAmplified sex chromosome-specific regions, such as short tandem repeats (STR) loci, were used to design a system that could detect the possible presence of maternal cells within the biopsy sample of the infant's tumor. Among the possible sex chromosome STR markers, we selected the most often used and efficient ones reported in the literature: DYS14 mapping on chromosome Y, DXS6803 and GATA165B12 on choromosome X, and telomerase loci as the control.5–7 To obtain the greatest efficiency of the systems, the published primer sequences were modified using Primer Express (PE Applied Biosystem, Foster City, CA), Oligo 4.1 (National Biosciences Inc, Plymouth, MN), and PrimerPy v0.97 (a GUI utility for Q-PCR primer design software). The best thermodynamically performing amplification was reached by varying the reaction conditions and the primer concentrations (Table 1). Both the sensitivity and reproducibility of each assay were ensured by generating standard curves (SCs) using several peripheral blood DNA samples at various concentrations (Fig. 2). Finally, robustness was evaluated by using a mixture of male and female DNA, ranging from 100% XY DNA to 100% XX DNA, stepping increasing by 25% fractions (Fig. 3A). The normalized fluorescent signal (ΔRn) was automatically calculated by an algorithm that normalizes the reporter emission signal. The threshold value that was applied to the algorithm generating the threshold cycle (Ct) was set at 0.05 in all the experiments. The relative quantification of each STR was performed according to the comparative method (ΔCt, Applied Biosystems User Bulletin no. 2P/N4303859). Amplifications were carried out in singleplex runs on 25 μL 20 ng of DNA and using Platinum QPCR-SYBR-GREEN SuperMix-UDG (Invitrogen) on the ABI PRISM 7500 HT Sequence Detection System (Applied Biosystems). Cycling conditions included degradation of preamplified templates for 2 minutes at 50°C, followed by 2 minutes of denaturation at 95°C, 32 cycles of denaturation at 95°C for 20 seconds, and annealing/extension at 55°C for 35 seconds, followed by the dissociation stage.JOURNAL/dimp/04.03/00019606-201006000-00003/table1-3/v/2021-02-17T200020Z/r/image-tiff PrimersJOURNAL/dimp/04.03/00019606-201006000-00003/figure2-3/v/2021-02-17T200020Z/r/image-jpeg Standard curves for the selected STR markers using several samples of peripheral male blood DNA: DXS14 and DXS6803 quantitative polymerase chain reaction assays run in singleplex. DYS14 slope=−3.3354 with R2=0.90; DXS6803 slope=−3.1233 with R2=0.99. GATA165B12 slope=−2.951 with R2=0.95. ΔCt (DYS14−DXS6803)=0.4.JOURNAL/dimp/04.03/00019606-201006000-00003/figure3-3/v/2021-02-17T200020Z/r/image-jpeg Evaluation of DYS14 and DXS6803 on a mixture of male and female DNA, ranging from 100% XY DNA to 100% XX DNA stepping with 25% fractions. A, Cts only show a correct identification of XY/XX ratio: 50% XY/50% XX DNA (the theoretical ratio is 1:4), 100% female DNA DYS14 is not amplified. B, Tumor ΔCt(DYS14) and ΔΔCt. Maternal origin of the tumor is assessed by ΔCt(DYS14)=3.54 and ΔΔCt (DYS14)=4.08 corresponding to at least 60% of XX cells.RESULTSSC Construction and Assay ValidationA preliminary thermodynamic analysis of the published primers of the selected markers was performed.5–7 Thus, both the primer concentrations and the sequence length were optimized until comparative SCs were reached using several different types of male genomic DNA (Table 1, Fig. 2). The greatest efficiency was reached by using DXS6803, which generated an SC that was comparable to that of the DYS14 marker, whereas the GATA165B12 did not achieve comparable amplification efficiency (Fig. 2). The relative quantifications of sex chromosomes were carried out by using DYS14 as the target, normalized to the DXS6803 as the calibrator using a range of male DNA concentrations ranging from 10 to 100 ng per reaction. The reproducibility of the calibration curve was analyzed by evaluating the slope and the correlation coefficient of the curve. DYS14 slope =−3.3354 with R2=0.90; DXS6803 slope =−3.1233 with R2=0.99. The qPCR efficiencies were calculated by the equation: E=10[−1/slope] and the difference between the efficiencies (EDYS14−EDXS6803) was <0.1, indicating that the data could be compared (Pfaffl, 2001). The ΔCt (Ct DYS14− CtDXS6803) indicated the same presence of sex chromosomes X and Y on male genomic DNA, as expected (Fig. 2). To determine the sensitivity of the method, samples containing various proportions of a mixture of male and female DNA were analyzed. The Ct target of DYS14 was normalized by the Ct of the DXS6803 by the equation ΔCt(DSY14)=Ct(DYS14)−Ct(DXS6803). The ΔΔCt(DSY14) for each DNA concentration mixture was calculated using the ΔCt(DSY14) at 100%XY as the calibrator by the equation ΔΔCtX=ΔCtX(DSY14)−ΔCt100%XY(DYS14). The results correctly identified the XY/XX ratio. In the absence of the Y chromosome (ie, 100% female DNA), DYS14 was not amplified, whereas with 50%XY/50%XX DNA (theoretical ratio 1:4), the ΔCt (DYS14) value was 2.71 and the ΔΔCt (DYS14) value was 3.02 (Fig. 3B).Detection of Chromosome Set in the Patient's BiopsyThe strategy for assessing the maternal origin of the tumor was founded on the difference in sex chromosomes between the mother and son. We searched for the presence of XX cells within the tumor mass using the peripheral blood DNA as the calibrator. By using qPCR on DYS14 and DXS6803, the same amount of the 2 markers (Y chromosome and X chromosome) was found in the DNA extracted from the peripheral blood, as expected, ΔCtblood(DYS14)=−0.4/−0.5, whereasthe DNA from the tumor biopsy showed ΔCttumor(DYS14)=3.54, indicating fewer Y chromosome markers than X chromosome markers (Fig. 3). The ΔΔCt calculated as ΔCttumor(DYS14)−ΔCtblood(DYS14) was 4.08 (Fig. 3B), thus indicating that at least 60% of cells had the XX chromosome set. The result was confirmed by FISH performed in the Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. The results did not change even when the Cts of telomerase loci were used as a control to generate ΔΔCtDSY14 by the equationJOURNAL/dimp/04.03/00019606-201006000-00003/math_3MMU1/v/2021-02-17T200020Z/r/image-tiffDISCUSSIONMaternal cancer during pregnancy is a rare event that is estimated to occur in approximately 1 case per 1000 gestations.4 Transplacental metastasis to the fetus is an even rarer event, and 58% of the reported cases involve melanoma.8 To choose the most appropriate therapeutic strategies for children affected by a possible metastasis from maternal disease, to inform the parents about the prognosis, and to offer proper genetic counselling, it is crucial to ascertain the origin of the tumor. Malignant primary tumors generally require tailored therapeutic protocols, posing a considerable risk of treatment-related toxicities, especially in newborns. However, vertical transmitted metastases can be considered as a “haploidentical transplant,” in which noninherited maternal antigens should theoretically lead to a rejection by an already competent fetal immune system.9 Therefore, the administration of modified and/or reduced therapeutic regimes could be justified, giving time in the new born to the development of efficient immune responses. Conclusive diagnosis requires proof of the presence of exogenous cells in the fetus' tumor, and classic histopathology may be insufficient. Therefore, thanks to the progress that has been made in cytogenetics, accurate assessment can be achieved through FISH and/or karyotyping analysis.10 However, these methods require an adequate amount of good-quality tissue samples, and must be performed by a skilled cytogeneticist. Therefore, we tried to develop an easier and more objective assay for the detection of exogenous cells within a tumor biopsy. A 6-month-old boy, whose mother had died of metastatic malignant melanoma a few weeks after delivery, presented with a left middle ear and temporal bone lesion. Although the temporal bone would have been a distinctly unusual site for a primary melanoma, the need for molecular biology testing was not merely of academic or forensic value, as thorough examination of the placenta performed in a specialized institution had failed to find any metastases. Furthermore, on these small biopsies, the possibility of a variant of another, less unusual pediatric tumor (such as a malignant progonoma) cannot be definitively ruled out. To design a truly molecular-based system that is able to detect the presence of maternal cells within the biopsy specimen of the child's tumor, quantification of the sex chromosomes may be a useful strategy. This can be carried out by the widely used specific STR loci that have been well characterized with respect to cross-species reactivity and DNA mutation rates.11 Our job was made easier because this type of metastasis is made up of both somatic fetal cells (ie, blood cells with XY karyotype) and parental tumor cells (with XX karyotype), resembling a cellular mosaicism. When developing the specific assay, several important qPCR properties were taken into consideration: (i) specificity for amplifying the sex chromosome markers (ie, no spurious cross-reactivity to human autosomes or to the homologous sex chromosome); (ii) comparable PCR efficiency (>90%); (iii) quality of SC (correlation coefficient); (iv) sensitivity and precision of quantification, especially at low copy numbers of template; and (v) discriminatory ability. We chose to use the most efficient and commonly used STR markers reported in the literature, that is, DYS14 mapping on chromosome Y, DXS6803 and GATA165B12 on choromosome X, and telomerase loci as the control (Table 1). The DYS14 was chosen over the more frequently used SRY, as reported earlier.6 The efficiency of the 2 selected X-chromosome markers was compared: the quantification sensitivity of the DXS6803 PCR system was greater than that of the GATA165B12 system (Fig. 2). The use of the STR marker as a qPCR target sequence may give rise to different resulting amplicons because of its variability within the population, and such an effect would be extremely detrimental in the case of SYBR Green assays. The dissociation curve of each amplicon did not reveal any allele-dependent effects (data not shown), and indicated good specificity of the PCR amplifications as assessed by any of the spurious melt peaks in the curve. Therefore, the well-optimized SYBR-Green assays allowed us to avoid using the more expensive, dye-labeled detection probes. We searched for the presence of XX cells within the tumor mass by using the patient's peripheral blood DNA sample (ie, XY alone) as the calibrator. By using qPCR on DYS14 and DXS6803, an equal amount of the 2 markers (Y chromosome and X chromosome) was found in the DNA that had been extracted from the peripheral blood: ΔCt(DYS14)=−0.4/−0.5 (theoretical value: 0). As the efficiency of the systems was <0.1, such deviation from the theoretical value may be considered negligible.12 Although a smaller amount of DYS14 was found in the biopsy DNA, favoring the X-chromosomes and indicating an imbalance in the sex chromosome ratio, ΔCttumor(DYS14)=3.54, and compared with the peripheral blood, the ΔΔCttumor(DYS14) was 4.08, thus indicating that at least 60% of the cells had an XX chromosome set (Fig. 3B). Our result was subsequently confirmed by FISH analysis carried out in an independent laboratory (Department of Pathology, Brigham and Women's Hospital, Boston, MA), which highlighted the presence of 54% of maternal cells in the biopsy. The discrepancy between the 2 percentage values of the XX cells may reasonably be attributed to the different levels of affinity of the XY probes used in the FISH, and, furthermore, it may depend on the sampling of the biopsy. Our assay indicates that quantification of the copy number of the sex chromosomes by specific STR markers in genomic DNA purified from the tumor biopsy cells can provide a rapid and robust method for the detection of transplacental metastasis by using qPCR. Although we used a comparative method (tumor vs. blood), the test we developed is able to quantify the presence of maternal cells in a tumor from a boy, even in the absence of such a comparison. We assume that by performing qPCR on specific STRs, and by looking for their highly polymorphic alleles within the population it would be possible to detect maternal cells even in the case of transplacental metastasis to a girl. In fact, the quantification of specific DNA sequences, on the basis of the principles of qPCR, is widely used for several purposes, such as early fetal sex determination,5,7 chimerism monitoring after allogeneic transplantation,13 prenatal diagnosis, and molecular diagnosis in general.14,15 The rapidity with which qPCR can be performed makes it an additional tool that may be used by the pathologist to define vertical cancer transmission to the fetus.16 This is the first approach that is able to unambiguously evaluate the maternal origin of tumor cells using a qPCR technique, and it may be added to those commonly used for clinical diagnosis, molecular research, and forensics studies.ACKNOWLEDGMENTSThe authors thank the “Associazione Italiana per la Ricerca sui Tumori Cerebrali del Bambino,” which funded this study. The authors also thank Prof Christopher D.M. Fletcher (Professor and Director of Surgical Pathology Brigham and Women's Hospital, Boston, MA) for FISH analysis. The authors are grateful to Valerie Perricone for her editorial assistance. They also thank Camusso Raffaella and Tedeschi Luca (photographic service, Giannina Gaslini Children's Research Hospital, Genoa, Italy) for their graphic assistance.REFERENCES1. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008;44:619–626.[Context Link][CrossRef][Medline Link]2. Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ. 2005;29:151–159.[Context Link][CrossRef][Medline Link]3. Alexander A, Samlowski WE, Grossman D, et al. Metastatic melanoma in pregnancy: risk of transplacental metastases in the infant. J Clin Oncol. 2003;21:2179–2186.[Context Link][Full Text][CrossRef][Medline Link]4. Potter JF, Schoeneman M. Metastasis of maternal cancer to the placenta and fetus. Cancer. 1970;25:380–388.[Context Link][CrossRef][Medline Link]5. Picchiassi E, Coata G, Fanetti A, et al. 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Primers Standard curves for the selected STR markers using several samples of peripheral male blood DNA: DXS14 and DXS6803 quantitative polymerase chain reaction assays run in singleplex. DYS14 slope=−3.3354 with R2=0.90; DXS6803 slope=−3.1233 with R2=0.99. GATA165B12 slope=−2.951 with R2=0.95. ΔCt (DYS14−DXS6803)=0.4. Evaluation of DYS14 and DXS6803 on a mixture of male and female DNA, ranging from 100% XY DNA to 100% XX DNA stepping with 25% fractions. A, Cts only show a correct identification of XY/XX ratio: 50% XY/50% XX DNA (the theoretical ratio is 1:4), 100% female DNA DYS14 is not amplified. B, Tumor ΔCt(DYS14) and ΔΔCt. Maternal origin of the tumor is assessed by ΔCt(DYS14)=3.54 and ΔΔCt (DYS14)=4.08 corresponding to at least 60% of XX cells.Detection of Transplacental Melanoma Metastasis Using Quantitative PCRRaso Alessandro PhD; Mascelli, Samantha PhD; Nozza, Paolo MD; Biassoni, Roberto PhD; Negri, Francesca PhD; Garaventa, Alberto MD; Tarantino, Vincenzo MD; Garrè, Maria Luisa MD; Cama, Armando MD; Capra, Valeria MDOriginal ArticlesOriginal Articles219p 78-82