Assessment of RT-PCR Detection of Human Mammaglobin for the Diagnosis of Breast Cancer Derived Pleural Effusions : Diagnostic Molecular Pathology

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00019606-200803000-00005ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008 p 28-33Assessment of RT-PCR Detection of Human Mammaglobin for the Diagnosis of Breast Cancer Derived Pleural EffusionsOriginal ArticlesRoncella, Silvio ScB*; Ferro, Paola ScB*; Bacigalupo, Bartolomeo MD*; Dessanti, Paolo MD*; Pronzato, Paolo MD†; Franceschini, Maria Cristiana ScB*; Pratticò, Luca MD‡; Carletti, Anna Maria MD‡; Canessa, Pier Aldo MD‡; Fontana, Vincenzo ScB§; Fais, Franco ScB∥; Pistillo, Maria Pia ScB¶; Fedeli, Franco MD**Division of Histopathology and Cytopathology†Division of Oncology, Sant'Andrea Hospital, La Spezia‡Division of Pneumology, San Bartolomeo Hospital, Sarzana§Unit of Epidemiology and Biostatistics¶Unit of Translational Research A, National Institute for Cancer Research∥Human Anatomy Section, Department of Experimental Medicine, University of Genova, Genova, ItalySupported in part by grant from AIL (Sez. Francesca Lanzone, La Spezia), Fondazione CARISPE, (La Spezia), Fondazione CARIGE, Genova, and Comitato Assistenza Malati e Lotta contro i Tumori, Sarzana, Italy.Reprints: Dr Silvio Roncella, ScB, Servizio di Anatomia ed Istologia Patologica, Ospedale Sant'Andrea, Via Mario Asso n. 2, 19124 La Spezia, Italy (e-mail: [email protected]).AbstractThe present study investigates the diagnostic significance of human mammaglobin (hMAM) mRNA expression in pleural effusions (PE) from breast cancer (BC) patients. Two hundred and fifty PE samples, including 32 from patients who had diagnosis of BC, 116 from patients with other cancers, and 102 from patients with benign diseases, were subjected to nested reverse-transcription polymerase chain reaction (RT-PCR) for hMAM, and the results were compared with conventional cytology. hMAM was found expressed in 76/250 (30.4%) total PE and in 23/28 (sensitivity of 82.1%) of the PE subgroup owing to metastasis from BC. The specificity for hMAM detection method was 75.7%, whereas accuracy, positive predictive value, and negative predictive value were 76.4%, 30.3%, and 97.1%, respectively. hMAM was also detected in 46/116 (39.6%) PE specimens from other types of cancer and in 7/102 (6.8%) from benign diseases. Comparative analysis of RT-PCR and cytology showed that 14 PE samples from metastatic BC (50%) were positive by both PCR and cytology, 9 (32.1%) were positive only by PCR and 5 (17.9%) were negative by both tests, whereas no cases were found of positive cytology with negative PCR. RT-PCR increased sensitivity of BC effusion detection to 32.1% (McNemar test, P=0.004). We demonstrated that RT-PCR for hMAM test was more sensitive than cytomorphology suggesting that, although hMAM is not BC specific, it may be useful in adjunct to cytology for the routine screening of malignant BC effusions.Pleural effusions (PE) are commonly observed in patients with breast cancer (BC).1 They may occur many years after the initial diagnosis, may represent the only symptom of metastatic disease and predict an extremely poor prognosis.2–4 Approximately 20% of total malignant PE and 37% of PE occurring in females are due to BC metastasis.5Detection of BC cells in PE is usually achieved by routine cytomorphology. However, the diagnosis by this methodology is often difficult owing to the small number of malignant cells and their dispersion among reactive mesothelial cells, monocytes, or red blood cells.6,7 In addition, it may be very difficult to discriminate BC cells from other metastatic carcinoma cells showing a similar morphology.8 In this regard, immunohistochemistry (IHC) can be helpful to ascertain the origin of a primary tumor, but in some cases it may not be suitable for discriminating tumors sharing the same immunophenotype.9 Therefore, more sensitive and specific techniques for the diagnosis of PE would be required.Recently, polymerase chain reaction (PCR) has been developed for the detection of BC cells micrometastasis in peripheral blood, lymph nodes, and bone marrow, and it has been proven to be highly sensitive (reviewed in Ref. 10). In addition, PCR has also been proposed to improve the diagnostic accuracy of PE.11,12The ideal target gene used to recognize BC cells by PCR amplification should be tumor specific and not expressed in normal epithelial cells or in hematopoietic tissues. In this regard, different target mRNA markers have been evaluated.13In 1996, the cDNA of a novel gene termed human mammaglobin (hMAM) was cloned and characterized by Watson and Fleming.14 hMAM gene is a member of the uteroglobin gene family and is located on chromosome 11q13. Its expression was originally described as restricted to normal and malignant breast tissue with overexpression in about 23% of the BC cases.14 However, hMAM mRNA has rarely been detected at various levels of expression, in various types of normal and/or malignant epithelial tissues, including lung, ovary, cervix, prostate, uterus, and kidney.15,16 Nevertheless, hMAM has been confirmed to be a suitable marker for detecting micrometastasis in BC patients (reviewed in Ref. 17).On the basis of these observations, few studies have evaluated hMAM expression by reverse-transcription PCR (RT-PCR) in PE. Its expression has been found in fluids from BC, various other neoplasms, and, more rarely, in benign effusions.18–21The aim of this study was to investigate the possible application of a nested RT-PCR methodology for hMAM mRNA detection as an adjunctive test to routine cytologic examination in the diagnostic evaluation of PE.MATERIALS AND METHODSPatientsA total of 250 patients diagnosed between February 2002 and August 2005 at the Divisions of Oncology, Sant'Andrea Hospital, La Spezia, and of Pulmonary Medicine, San Bartolomeo Hospital, Sarzana, Italy, were enrolled in this study after giving their informed consent according to institutional procedures. All patients, from whom PE fluids were obtained, were diagnosed on the basis of clinical signs, imaging data, cytology of effusion, and/or histologic examination of pleural biopsies obtained by medical thoracoscopy.21 This study included 32 patients who had diagnosis of BC (median age, 72 y; range, 41 to 86 y), 116 patients with carcinomas unlike from breast (median age, 72 y; range, 30 to 94 y), and 102 with benign disease (median age, 74 y; range, 24 to 97 y).In the subgroup of 32 PE specimens from patients with BC, 28 were due to metastatic BC whereas, 3 PE were related to primary lung cancer and 1 PE to infectious disease (Table 1). These 4 cases were neither included in the metastatic BC group (n=28) nor in the groups of lung cancer (n=49) and benign effusions (n=102), but were analyzed separately.JOURNAL/dimp/04.03/00019606-200803000-00005/table1-5/v/2021-02-17T195944Z/r/image-tiff Characteristics of BC Patients, hMAM Expression, and Cytologic Features of PEBiologic SamplesCells from 10 mL of freshly aspirated and nonfixed PE were isolated by centrifugation at 1500g for 10 minutes at room temperature. Pellets were washed twice in phosphate-buffered saline, dissolved in RLT solution (RNeasy Mini Kits, Qiagen GmbH, Hilden, Germany) and snap-frozen at −30°C, ready for RNA extraction.RNA Extraction and Reverse-transcriptionTotal RNA was extracted using RNeasy Mini Kit (Qiagen GmbH) following the manufacturer's instructions. One microgram of total RNA was reverse transcribed into cDNA. These reactions were carried out in 50 μL using 300 units of M-MLV Reverse Transcriptase (Invitrogen, San Diego, CA), 0.8 μM 18-mer oligo dT as primer, 0.4 mM dNTPs, 10 mM DTT, and the buffer provided with the enzyme. The reaction mixture was incubated at 37°C for 1 hour. The enzyme was then inactivated by incubation at 70°C for 15 minutes.PCRA 10-μL sample of cDNA was amplified by nested PCR as previously described.27 The primers of our test amplify both known hMAM mRNA isoforms 1 (GenBank accession number U33147) and 2 (GenBank accession number AY217100). Briefly, the first PCR reaction was performed in a volume of 50 μL containing 0.4 μM PCR primers, 1.5 mM of MgCl2, 0.2 mM of dNTPs, 1.5 units of Platinum Taq DNA Polymerase (Invitrogen), and the buffer provided by the supplier. After an initial denaturation at 94°C for 2 minutes, 40 cycles were performed for 30 seconds at 94°C, 30 seconds at 64°C, and 5 seconds at 72°C followed by a final elongation of 2 minutes at 72°C. The sequence of the PCR primers was: forward, 5′-GCGGCTTCCTTGATCCTTG-3′ and reverse, 5′-GAAAGAGAAGGTGTGGTTTGC-3′ (transcript 423 bp).Nested PCR was carried out using 5 μL of the first PCR product in a final volume of 50 μL with conditions identical to those of the first PCR but using the following PCR primers: forward, 5′-CACCGACAGCAGCAGCCT-3′ and reverse, 5′-AGTTCTGTGAGCCAAAGGTCT-3′ (transcript 334 bp). Thirty cycles of amplification were performed.As internal control, GAPDH gene amplification was carried out for each cDNA sample in conditions identical to those for hMAM amplification with the primers forward, 5′-GGTCATCCC TGAGCTGAACG-3′ and reverse, 5′-TTCGTTGTCATACCAGGAAATG-3′ (transcript 295 bp).PCR products were electrophoresed on 1.5% agarose gel containing ethidium bromide, visualized on an UV light transilluminator, and photographed.The MDA-MB415 BC cell line, which expresses hMAM, was used as a positive control. The cells were obtained from the Interlab Cell Line Collection (National Institute for Cancer Research, Genova, Italy) and were grown in Dulbecco Modified Eagle Medium supplemented with 10% Fetal Calf Serum, 1% Glutamine, 1% Na Piruvate, and 2% Penicillin-Streptomycin (all from Biochrome AG, Berlin, Germany).22HistopathologyHistology, IHC, and cytology were performed by standard protocols used in the Laboratory of Histopathology and Cytopathology of Sant'Andrea Hospital, La Spezia, Italy. Histology was performed on 3-μm formalin-fixed, paraffin-embedded tissue sections, mounted on slides, deparaffinized, and rehydrated. The immunostaining with the appropriate antibodies was carried out with an automated immunostainer, NEX ES (Ventana Medical Systems, S.A Strasbourg, France), and the antigens were localized by means of a Ventana Medical System/view DAB detection Kit (Ventana Medical Systems).Cytologic examination of the samples was performed after conventional Papanicolaou staining.Statistical AnalysisDiagnostic performance of hMAM RT-PCR was assessed by computing sensitivity (Se), specificity (Sp), accuracy (Ac), positive predictive value (PPV), and negative predictive value (NPV), and corresponding exact (ie, based on binomial distribution) 95% confidence limits. χ2 test and, whenever necessary, Fisher exact test were used to evaluate the difference between independent proportions, whereas comparison between correlated proportions was assessed through pair-matched analyses using the nonparametric McNemar test.23 Two-tailed P value <0.05 was considered as statistically significant. All statistical analyses were performed using Stata (StataCorp, Stata Statistical Software, Release 8.0, Stata Corporation, College Station, TX, 2003).23RESULTShMAM mRNA Expression in PE From Patients With BC and Comparison With CytologyA nested RT-PCR assay for amplification of a cDNA fragment specific for hMAM was carried out in 250 total PE including 32 from patients with BC, 116 from patients with other cancers, and 102 from benign diseases. In the subgroup of 32 PE specimens from BC, 28 were indeed related to BC, whereas 3 PE were due to a second primary neoplasia of lung and 1 PE to an infectious disease (Table 1). As PE was not a manifestation of the BC disease in these 4 BC patients, these cases were analyzed separately and not included in other groups.A transcript of the expected size for hMAM (334 bp) was detected in 76/250 (30.4%) of total PE and in 23/28 (Se=82.1%) of BC cases (representative cases in Fig. 1). In the 28 BC samples, the malignant cells were evaluated either by RT-PCR or cytology, and the results were compared. All 14/28 samples were positive in cytology (Se=50%) and also positive in RT-PCR, whereas 9/28 (32.1%) were positive by RT-PCR alone and 5/28 (17.9%) were negative by both techniques. No cases were found of positive cytology with negative RT-PCR. The Se value of cytology was significantly lower than that of RT-PCR (McNamer test, P=0.004).JOURNAL/dimp/04.03/00019606-200803000-00005/figure1-5/v/2021-02-17T195944Z/r/image-jpeg Nested RT-PCR assay for hMAM. Ethidium bromide-stained 1.5% agarose gel of RT-PCR amplified hMAM and GAPDH mRNA. A transcript of the expected size for hMAM (334 bp) and for GAPDH (295) is shown in 1 sample of PE from BC patient and in BC cell line MDA-MB415 used as positive control. No transcript is shown for hMAM in PE derived from patient with pneumonia. MW indicates molecular weight marker.Statistical analysis demonstrated that the proportion of hMAM positive specimens in the BC subgroup (23/28) was significantly higher (χ2, P<0.001) than that of the other patients (53/218=24.3%). Moreover, the Sp rate for hMAM expression was 75.7%, whereas Ac, PPV, and NPV were 76.4%, 30.3%, and 97.1%, respectively (Table 2).JOURNAL/dimp/04.03/00019606-200803000-00005/table2-5/v/2021-02-17T195944Z/r/image-tiff Estimates of Diagnostic Performance Parameters of hMAM Expression in PE From BC Patients Versus Benign Diseases and Other CancersFinally, in the 3 BC sample cases (n=29 to 31) (Table 1) in which PE was due to a second primary neoplasia of lung, we found 1 case positive by both PCR and cytology, 1 case negative by both tests, and 1 case negative by PCR but positive by cytology. In only 1 case (n=32) (Table 1) of PE from BC due to an infectious disease, molecular test and cytology gave negative results.hMAM mRNA Expression in PE From Patients With Other CancersWe evaluated a subgroup of PE derived from 116 patients with malignancies in organs different from the breast. The results showed that hMAM expression was observed in 46/116 (39.6%) samples (Table 3). In particular, hMAM was positive in 32/68 (47.1%) cases of epithelial origin (25/49 lung carcinomas, 4/6 ovarian carcinomas, 2/4 renal carcinomas, and 1/2 prostatic carcinomas), whereas in cases from nonepithelial origin hMAM was detected in 14/48 (29.2%) cases (13/43 mesotheliomas and 1/1 case of melanoma) (Table 3). Moreover, 2 pancreatic carcinomas, 3 gastric carcinomas, 1 colon carcinoma, 1 carcinoma of unknown origin, and 4 lymphomas were negative.JOURNAL/dimp/04.03/00019606-200803000-00005/table3-5/v/2021-02-17T195944Z/r/image-tiff hMAM Expression by Nested RT-PCR in PE From Patients With Other Cancers and Benign DiseasesThe estimates of diagnostic parameters related to these patients, in comparison with BC patients, were as follows: Sp=60.3%, Ac=64.6%, PPV=33.3%, and NPV=93.3% (Table 2).hMAM mRNA Expression in PE From Patients With Benign DiseasesTo further investigate the expression of hMAM mRNA in PE, we analyzed 102 specimens from patients with no history or evidence of cancer and who did not develop cancer during a 18-month follow-up. Nested RT-PCR for hMAM transcript was negative in 95/102 (93.1%) samples tested (Table 3). Among the 7 samples with hMAM expression, 1 derived from a patient with congestive heart failure (1/17), 2 from patients with pneumonia (2/51), and 4 from patients for whom the cause of PE remained unknown (4/18) (Table 3). The estimates of diagnostic parameters related to these patients, in comparison with BC patients, were as follows: Sp=93.1%, Ac=90.8%, PPV=76.7%, and NPV=95.0% (Table 2).DISCUSSIONhMAM gene has been reported to be highly and selectively expressed in normal and neoplastic breast epithelial cells,14 making it a potentially useful marker for BC.17,24 Some reports suggested that the analysis of hMAM mRNA by means of RT-PCR can be used to improve the detection of BC cells in the PE.18–20 However, as hMAM expression in PE has been investigated in a relatively small number of patients, its clinical utility must be further evaluated.In this study, we have assessed the expression of hMAM gene by nested RT-PCR in patients with BC with the aim of considering its possible use in routine analysis. The specificity of the assay was investigated in PE from tumors other than BC and from benign diseases. Moreover, we have evaluated the sensitivity of the test in comparison with conventional cytology in the BC subgroup.By comparing hMAM RT-PCR and cytology in PE from BC patients, the sensitivity of RT-PCR was significantly more elevated than that of cytology. This result is not surprising if we consider our previous observation that the test is able to amplify hMAM transcript from 1 tumor cell/106 normal cells,27 whereas the sensitivity of cytology is in the range of 1 tumor cell/103 to 105 normal cells.25 We found 5 patients (cases 24 to 28 in Table 1) in whom histopathology and IHC of pleural biopsies obtained by medical thoracoscopy revealed micrometastasis of BC that were negative for both hMAM expression and cytology. As none of the patients had either changed the therapy or started a new one before taking the PE samples, it is likely that these specimens contained malignant cells that the PCR technique was unable to detect. The negative results of cytologic examination support the hypothesis that the scarce number of BC cells may be one possible explanation. Nevertheless, we cannot exclude that other conditions of RT-PCR (different retrotranscriptase enzymes, primers, number, and cycle conditions) would give a positive result. In addition, the presence of Taq polymerase inhibitors in the fluid may have limited the efficiency of PCR reactions in some instances.It is also possible that BC cells have lost hMAM expression. In this regard, negativity of RT-PCR was previously reported for hMAM in about 7% of BC cases mainly of G3 grade.26,27 Therefore, we have analyzed the expression of hMAM in the original neoplastic tissue and demonstrated its positivity (data not shown). For this reason, if any loss of hMAM expression occurred, this happened during the metastatic progression.In line with data from other reports, hMAM expression was not specific and was also detected in PE from carcinomas of the ovary, prostate, lung, kidney, and mesothelioma.18–21 In confirmation of that finding, the present study showed low Sp (60.3%) and PPV (33.3%) values of the hMAM expression versus PE derived from cancers other than BC. This finding may be important to establish the origin of the cancer causes of the PE. Of particular relevance are the cases of patients with BC who developed second primary cancers mainly lung and ovarian cancers. One example is shown in Table 1 where patient 29, who had a history of BC, developed a PE that was found positive for hMAM because of lung carcinoma. In this case, the positivity for hMAM could be misleading, suggesting that cytologic preparations with IHC markers are more appropriate to determine the primary site of cancer.To investigate further the Sp of our hMAM RT-PCR methodology, we also studied 102 cases of PE derived from benign diseases. In agreement with the nonmalignant nature of the group, about 93% of the samples scored as negative. Statistical analyses of the data in this subgroup confirmed that our methodology allowed us to distinguish the BC origin of PE with a high Sp and a high PPV.As to the 7 hMAM positive cases observed among the patients with benign diseases, we believe that 1 possible cause of hMAM expression may be because of the stimulation of non-neoplastic cells by inflammatory cytokines. Consistent with this hypothesis, inducible expression of hMAM in cell lines of different lineages, bone marrow, peripheral blood mononuclear cells, and stem cells after stimulation with several cytokines has been reported.28 However, we cannot exclude the presence of neoplastic cells, although a follow-up of 18 months did not show any clinical evidence of tumors in these patients. As our method to detect hMAM is not quantitative, it does not allow evaluation of different levels of mRNA in PE samples. Such levels may provide clues on the tumor origin and be a prognostic factor. Therefore, hMAM quantification study by real-time PCR will be carried out to clarify this aspect.In conclusion, the RT-PCR methodology developed for this study has provided a rapid, reproducible, and cost-effective hMAM analysis test for BC diagnosis of PE. Although the routine application of this test may still be difficult for many laboratories owing to the expensive technologies and expertise in molecular biology, we believe that these drawbacks will be overcome in the future. In fact, recent years have seen even small diagnostic laboratories equipped with adequate tools for molecular biology, and the number of molecular tests employed is increasing. We believe that the results of our study may find application in the near future.ACKNOWLEDGMENTSThe authors thank the doctors and technical staff of the Laboratory of Histopathology and Cytopathology of Sant'Andrea Hospital La Spezia (Italy) and Lucia Pietra for their cooperation. In addition, they also thank Dr Gaetano Leto for critically reading the manuscript.REFERENCES1. Raju RN, Kardinal CG. Pleural effusion in breast carcinoma: analysis of 122 cases. Cancer. 1981;48:2524–2527.[Context Link][CrossRef][Medline Link]2. Hausheer FH, Yarbro JW. 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Ethidium bromide-stained 1.5% agarose gel of RT-PCR amplified hMAM and GAPDH mRNA. A transcript of the expected size for hMAM (334 bp) and for GAPDH (295) is shown in 1 sample of PE from BC patient and in BC cell line MDA-MB415 used as positive control. No transcript is shown for hMAM in PE derived from patient with pneumonia. MW indicates molecular weight marker. Estimates of Diagnostic Performance Parameters of hMAM Expression in PE From BC Patients Versus Benign Diseases and Other Cancers hMAM Expression by Nested RT-PCR in PE From Patients With Other Cancers and Benign DiseasesAssessment of RT-PCR Detection of Human Mammaglobin for the Diagnosis of Breast Cancer Derived Pleural EffusionsRoncella Silvio ScB; Ferro, Paola ScB; Bacigalupo, Bartolomeo MD; Dessanti, Paolo MD; Pronzato, Paolo MD; Franceschini, Maria Cristiana ScB; Pratticò, Luca MD; Carletti, Anna Maria MD; Canessa, Pier Aldo MD; Fontana, Vincenzo ScB; Fais, Franco ScB; Pistillo, Maria Pia ScB; Fedeli, Franco MDOriginal ArticlesOriginal Articles117p 28-33

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