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00019606-201006000-00005ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2010 Lippincott Williams & Wilkins, Inc.19June 2010 p 92-98Diagnosis and Origin Determination of Malignant Pleural Effusions Through the Use of the Breast Cancer Marker Human MammaglobinOriginal ArticlesRoncella, Silvio PhD*; Ferro, Paola PhD*; Franceschini, Maria Cristiana PhD*; Bacigalupo, Bartolomeo MD*; Dessanti, Paolo MD*; Sivori, Massimiliano MD†; Carletti, Anna Maria MD†; Fontana, Vincenzo PhD‡; Canessa, Pier Aldo MD†; Pistillo, Maria Pia PhD§; Fedeli, Franco MD**Division of Histopathology and Cytopathology, Sant'Andrea Hospital, La Spezia†Division of Pneumology, San Bartolomeo Hospital, Sarzana§Breast Cancer Laboratory of Tumor Genetics Unit‡Unit of Epidemiology and Biostatistics, National Institute for Cancer Research, Genova, ItalySupported by grant from AIL (Sezione Francesca Lanzone), La Spezia (Italy), from Comitato Assistenza Malati e Lotta Contro i Tumori, Sarzana (La Spezia) and from Regione Liguria 2009. Reprints: Silvio Roncella, PhD, Servizio di Anatomia ed Istologia Patologica, Ospedale Sant'Andrea, Via Mario Asso n. 2, 19124 La Spezia, Italy (e-mail: [email protected]).AbstractAs was reported that human mammaglobin (hMAM) may be expressed in malignant pleural effusions (PEs), we investigated the relevance of hMAM reverse-transcriptase polymerase chain reaction (RT-PCR) for their diagnosis and determination of primary origin. Two hundred and twenty-eight malignant (132 male, 96 female) and 185 benign (132 male, 53 female) PEs were investigated. Statistical analyses evaluated the diagnostic performance parameters in all PEs and in cytologically negative malignant PEs, the association between hMAM and benign or malignant status by the direct index of correlation [diagnostic odds ratio (DOR)], χ2 test, and P value (P). In addition, the discriminative diagnostic power of hMAM expression, independently in breast cancer, lung cancer (LC), malignant mesothelioma (MM), and other cancers was evaluated. In the entire patient population, hMAM was detected in 45.6% and 5.4% of malignant and benign PEs, respectively, in the male group in 41.7% and 4.5% and in the female group in 51.0% and 7.5% of malignant and benign PEs, respectively. A statistically significant correlation between hMAM and malignancy was found in the entire population (DOR=14.68, P<0.001) and in the male (DOR=15.00, P<0.001) or female (DOR=12.77, P<0.001) groups. hMAM RT-PCR increased the diagnostic rate of malignant PEs as it allowed us to detect as malignant 32.1% of cytologically negative PEs. In female patients the positivity of hMAM indicated with higher probability (50.8%) the origin of PEs from breast cancer but lower probability from LC (17%), MM (9.4%), or other cancers (15.1%), whereas in male patients it indicated with similar probability (about 40%) the origin from LC or MM. Our results suggest that hMAM RT-PCR may provide information both in the diagnosis of PE and in the search for the primary site of neoplasia, either in male or female patients.Many different diseases may cause pleural effusions (PEs), which may be subdivided into malignant and benign according to their origin. It has been reported that about 75% of PEs are of non-neoplastic origin whereas the residual ones are caused by tumors. However, the incidence of malignant PEs varies according to the study population.1–3Knowledge of the exact nature of effusions is a clinically crucial issue. In fact, the presence of neoplastic cells in PE indicates advanced stage of the tumor; it is related to limited survival and is associated with appropriate therapeutic choice.4,5Although it is important to clarify their precise cause, the diagnosis of PEs is sometimes difficult to establish, especially to differentiate between benign and malignant PEs. The detection of malignant cells by cytology (Cyt) is the most informative laboratory test for the diagnosis of PEs. It has been reported that Cyt is simple and specific but not sensitive, allowing diagnosis of only about 50% of malignant PEs.6–8To find better methods of identifying the causes of PEs numerous procedures, including immunocytochemistry, determination of tumor markers, telomerase activity, electron microscopy, flow cytometry, chromosome analysis, and fluorescent in situ hybridization, have been investigated but at present no ideal techniques have yet been found.9–12In recent years, to enhance diagnostic sensitivity (Se) by Cyt, reverse transcriptase-polymerase chain reaction (RT-PCR) assays for the amplification of specific tumor cell mRNA transcripts have been proposed.13,14 Among the different markers tested in RT-PCR the human mammaglobin (hMAM) gene appears to be, at present, one of the most promising markers for such an application.15–19The hMAM gene was cloned and characterized by Watson and Fleming20 in 1996 and was initially considered as specific for breast cancer (BC) cells, but later hMAM expression was reported in tissues of other carcinomas besides BC.21,22 Moreover, hMAM amplification has been used by many investigators to detect micrometastasis in peripheral blood, lymph node, and bone marrow of BC patients resulting as a specific and highly sensitive marker for the detection of malignant cells in these tissues.23,24The hMAM transcript was found expressed in about 80% of PEs from BC specimens, but also in about 21% to 74% of PEs from patients affected with other tumors including lung, ovarian, prostatic, renal, and pancreatic cancers, in addition to malignant mesothelioma (MM) and, less frequently, in about 0% to 13% of benign effusions.15–19 The hMAM expression in PEs from various human tumors opens new perspectives for its application in characterizing PEs and suggests further studies to confirm its value.The aim of this study was to assess the value of hMAM detection by RT-PCR as a marker for diagnosis and primary tumor origin determination of malignant PEs. As hMAM was preferentially expressed in typical female tumors (BC and ovarian cancer), with respect to other types of neoplasia,20–23 we investigated the suitability of the test in the entire patient population and according to sex.Statistical analyses were carried out to evaluate the correlation between hMAM expression versus disease status, the diagnostic performance parameters (DPPs), and the discriminative diagnostic power (DDP) of hMAM marker. DPPs were evaluated for all PEs and for Cyt-negative effusions in the entire patient population and according to sex.The DDP was intended as the predicted probabilities of being affected by a malignant or benign illness among hMAM-positive or hMAM-negative patients. Finally, DDP were estimated according to malignancy subgroups, that is, BC, lung cancer (LC), MM, and other cancers (OC).Although other studies using hMAM as a marker to distinguish malignant PEs have been published, most of them analyze relatively small numbers of samples. To our knowledge, this is the first study that analyzes hMAM in a large number of patients and, most importantly, provides its discriminating power separately in male or female patient populations.MATERIALS AND METHODSPatientsThe study was performed on 413 Italian patients diagnosed between February 2002 and October 2007 at the Division of Pulmonary Medicine, San Bartolomeo Hospital, Sarzana (Italy). The study protocol was approved by the ethics committee of the ASL5 of La Spezia (Italy), protocol number 57 25/11/2008. The patients were enrolled in the study after informed consent was given. All specimens were analyzed by Cyt and, when necessary for diagnosis, histology of the PEs taken during medical thoracoscopy was performed.This study included 228 PEs from patients with malignant tumors (132 males and 96 females) and 185 PEs from patients with non-neoplastic diseases (132 males and 53 females). One hundred and six of the 228 malignant PEs were Cyt negative (70 males and 36 females). The median age of the malignant group was 72.7 years (males: median age 73.8 years, range: 20 to 92 years and females: median age 71.6, range: 34 to 88 years), whereas the median age of the benign group was 72.9 years (males: median age 72.3 years, range: 35 to 97 years and females: median age 73.5 years, range: 24 to 96 years).Biologic Samples and Molecular AnalysisCells from 10 mL of freshly aspirated and nonfixed PEs 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 Kit, QIAGEN GmbH, Hilden, Germany) and snap-frozen at −30°C, ready for RNA extraction.19cDNA Preparation and RT-PCRTotal RNA was extracted using RNeasy Mini Kit (QIAGEN GmbH) following the manufacturer's instructions and 1 μg of total RNA was reverse transcribed into cDNA as previously described.19A 10 μL sample of cDNA was amplified by nested PCR as previously described.24 using primers that amplify hMAM gene mRNA (GenBank accession number U33147). In brief, the first PCR (total volume 50 μL) contained 1.5 mM/L MgCl2, 0.2 mM/l dNTPs, 1.5 U Platinum Taq DNA Polymerase (Invitrogen, Milano, Italy) with its buffer and 0.4 μM/L primers. The cycling conditions were 2 minutes at 94°C followed by 40 cycles of 94°C for 30 seconds, of 64°C for 20 seconds, 72°C for 5 seconds, and a final incubation of 72°C for 2 minutes. The nested PCR (total volume 50 μL) was carried out using 5 μL of the first PCR product with conditions identical to those of the first PCR but using the appropriate primers and 30 cycles of amplification. As internal control, GAPDH gene amplification was carried out for each cDNA sample in conditions identical to those for hMAM first amplification. PCR products were separated by standard electrophoresis on 1.5% agarose gel containing ethidium bromide. The MDA-MB415 BC cell line (ICLC, National Institute for Cancer Research, Genova, Italy), was used as hMAM-positive control as previously described.19CytologyCyt of the samples was performed after conventional Papanicolau staining, by standard protocols used in the Laboratory of Histopathology and Cytopathology of Sant'Andrea Hospital, La Spezia (Italy).Statistical AnalysisTo provide a measure of correlation between the biomarker under study and the disease status, the diagnostic odds ratio (DOR) was calculated along with corresponding likelihood-based 95% confidence limits (95% CL).25 In this research setting, DOR can be interpreted as the ratio of the probability of having a positive hMAM test result among cancer patients to the probability among non-cancer patients. Accordingly, DOR values can vary from zero to infinity. Values greater than 1 indicate a good biomarker performance, namely positive test results are correlated with malignant status. Values less than 1 reflect an improper test functioning: positive test results are correlated with benign status. Finally, a value of 1 shows that the test is unable to discriminate between the 2 study groups. Moreover, DPP of hMAM RT-PCR was assessed by computing Se, specificity (Sp), accuracy (Ac), positive predictive value (PPV), and negative predictive value (NPV) parameters and corresponding 95% CL.26 Finally, DDP of being cancer or non-cancer patients and independently for each subgroup of disease (BC, LC, MM, OC) among hMAM positive or negative individual were also computed.Chi-square test and, whenever necessary, Fisher exact test were used to evaluate the difference between independent proportions, whereas comparisons between correlated proportions was assessed through pair-matched analyses using the nonparametric McNemar test.27 Two-tailed P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using Stata (StataCorporation Stata Statistical Software. Release 10.0. Stata Corporation, College Station, TX, 2007).RESULTSComparative Analysis of hMAM Expression in PEs From Malignant Versus Benign Diseases in the Entire Patient PopulationBy the previously described RT-PCR method,24 we evaluated the expression of hMAM in a total of 413 PE specimens including 228 of malignant and 185 of benign origin. Amplification of the hMAM transcript was detected in 104 of 228 (Se=45.6%) malignant PEs and in 10 of 185 (1-Sp=5.4%) benign PEs (Table 1, Fig. 1 for a representative RT-PCR gel). As a consequence of this difference, a statistically significant correlation between hMAM expression and malignant status was found in the entire patient population (χ2 test P<0.001). This finding was further supported by the DOR value of 14.68 (95% CL=7.37 to 29.22) indicating that about a 15-fold increase in hMAM expression was observed among malignant PEs as compared with benign PEs (Table 2). It is noteworthy that the highest Se value was observed for BC patients (27/32, 84.4%) followed by LC (34/77, 44.1%), MM (29/80, 36.2%), and OC patients (14/39, 35.9%) (Table 1).JOURNAL/dimp/04.03/00019606-201006000-00005/table1-5/v/2021-02-17T200020Z/r/image-tiff Summary of RT-PCR Detection of Human Mammaglobin in Pleural EffusionsJOURNAL/dimp/04.03/00019606-201006000-00005/table2-5/v/2021-02-17T200020Z/r/image-tiff Correlation Between Human Mammaglobin Expression and Disease Status (Malignant vs. Benign Effusions)JOURNAL/dimp/04.03/00019606-201006000-00005/figure1-5/v/2021-02-17T200020Z/r/image-jpeg Ethidium bromide-stained 1.5% agarose gel of reverse-transcriptase polymerase chain reaction-amplified hMAM (A) and of internal control GAPDH (B). The hMAM bands are evident in the breast cancer cell line MDA-MB415 used as positive control (lane 1) and in the samples of effusion from patients with breast cancer, ovarian cancer, lung cancer, and malignant mesothelioma (lanes 3 to 6) but not in the patient with pneumonia (lane 2). GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase; hMAM, human mammaglobin; MW, molecular weight marker.The DPP of hMAM detection in benign versus malignant PEs were as follows: Sp=94.6%, Ac=67.6%, PPV=91.2%, and NPV=58.5% (Table 3).JOURNAL/dimp/04.03/00019606-201006000-00005/table3-5/v/2021-02-17T200020Z/r/image-tiff Estimates of Diagnostic Performance Parameters of Human Mammaglobin RT-PCR Detection in All Pleural EffusionsTo investigate whether the results can vary according to sex-related differences, we carried out separate analyses by sex.Comparative Analysis of hMAM Expression in PEs From Malignant Versus Benign Diseases in the Male Patient GroupThe PE specimens of the male patient group were 264 including 132 PEs diagnosed as malignant and 132 PEs diagnosed as benign (Table 1). The expression of hMAM was found in 55 of 132 (Se=41.7%) samples in the malignant group and in 6 of 132 (1-Sp=4.5%) samples in the benign group. A statistically significant correlation between higher hMAM expression levels and malignant disease status was detected (χ2 test; P<0.001) (Table 2) with a DOR value of 15.00 (95% CL=6.17 to 36.49) (Table 2). The DPP of hMAM detection in benign versus malignant PEs related to the male group were as follows: Sp=95.5%, Ac=68.6%, PPV=90.2%, and NPV=62.1% (Table 3).Comparative Analysis of hMAM Expression in PEs From Malignant Versus Benign Diseases in the Female Patient GroupThe PE specimens of the female patient group were 149 including 96 PEs diagnosed as malignant and 53 PEs diagnosed as benign (Table 1). The expression of hMAM was found in 49 of 96 (Se=51.0%) of the samples in the malignant group and in 4 of 53 (1-Sp=7.5%) samples in the benign group. As expected, in the female group as well, a statistically significant correlation between higher hMAM expression levels and malignant disease status was detected (χ2 test; P<0.001) with a DOR value of 12.77 (95% CL: 4.27 to 38.17) (Table 2). The DPP of hMAM detection in benign versus malignant PEs related to the female group were as follows: Sp=92.5%, Ac=65.8%, PPV=92.5%, and NPV=51.0% (Table 3).Gender-specific Relevance of hMAM Expression to Identify the Primary Tumor Originating the Malignant PEsWe evaluated by sex the DDP for each subgroup of disease. In the male patients the probability of being affected by a non-neoplatic disease (1-PPV), given the hMAM-positive result, was 9.8%. In contrast, the probability of being affected by a neoplastic disease (PPV) as LC, MM, and OC was 41.0%, 39.3%, and 9.8%, respectively (Table 4). In the same patients, NPV (the probability to have non-neoplastic diseases with negative result of the hMAM test) was 60.9%, whereas the probability of being affected by LC, MM, or OC, given the hMAM-negative expression, was 12.3%, 21.2%, and 4.4%, respectively (Table 4).JOURNAL/dimp/04.03/00019606-201006000-00005/table4-5/v/2021-02-17T200020Z/r/image-tiff Discriminative Diagnostic Power of Human Mammaglobin Expression According to the Origin of Pleural EffusionsIn the female patients, the probability of a benign disease given the hMAM-positive PE specimen (1-PPV) was 7.6%, whereas the PPV was 50.8% for BC, 17.0% for LC, 9.4% for MM, and 15.1% for OC (Table 4). Finally, among the female hMAM-negative patients, NPV was 51.0% whereas the probability of being affected by BC, LC, MM, or OC was 5.2%, 18.8%, 8.3%, and 16.7%, respectively (Table 4).hMAM Expression in the Cytologically Negative Malignant PEsTo evaluate the contribution of the hMAM RT-PCR assay to the diagnosis of PEs when Cyt is negative, we performed the analysis of DPP in Cyt-negative PEs from the entire patient population and by sex.One hundred and six of 228 (46.5%) malignant PEs were negative for tumor cells by Cyt and 34 cases of 106 were found positive for hMAM expression (Se=32.1%). In these samples, the DPP of hMAM detection, referred to as benign versus malignant PEs in the entire patient population, were as follows: Sp=94.6%, Ac=71.8%, PPV=77.3%, and NPV=70.8% (Table 5).JOURNAL/dimp/04.03/00019606-201006000-00005/table5-5/v/2021-02-17T200020Z/r/image-tiff Estimates of Diagnostic Performance Parameters of Human Mammaglobin RT-PCR Detection in Cytologically Negative Pleural EffusionsWhen hMAM expression was analyzed according to sex, we observed that the Cyt-negative malignant samples from male patients were 70 of 106 and the DPPs of hMAM detection, referred to as benign versus malignant PEs in this subgroup, were as follows: Se=28.6, Sp=95.5%, Ac=72.3%, PPV=76.9%, and NPV=71.6% (Table 5). In contrast, the Cyt-negative malignant samples from female patients were 36 of 106 and the DPPs of hMAM detection, referred to as benign versus malignant PEs in this subgroup, were as follows: Se=38.9, Sp=92.5%, Ac=70.8%, PPV=77.8%, and NPV=69.0% (Table 5).DISCUSSIONWe previously developed a sensitive and specific nested RT-PCR assay for amplifying the hMAM mRNA transcript to detect BC tumor cells in PE and demonstrate that the addition of hMAM RT-PCR to the Cyt increased the diagnostic rate of BC by 32%.In this study, we investigated the usefulness of hMAM as a marker to identify all malignant PEs and their primary origin.We found the expression of hMAM in 45% of malignant PEs and in 5% of PEs collected from patients without a history or evidence of a tumor. This finding is in contrast with the results of other investigators who have analyzed the expression of hMAM in malignant15–17 and non-neoplastic effusions,15,17 but in agreement with our previous data19 and those from Grünewald et al16 on the hMAM expression in benign effusions.However, the discrepancy observed with the above studies may be explained by several reasons. The first common cause may be the variability of the efficiency of the RT-PCR methodology that may influence the performance of the test. The second cause may be the different sites of origin of the fluids. In fact, we analyzed samples from PEs alone at variance with other reports that also included ascitic effusions.16,17 It is also possible that differences in patient sex distribution, age, and disease stage may account for the discrepancy in addition to the different frequencies of the tumor type responsible for the effusions studied.Furthermore, we investigated whether the hMAM marker was suitable for distinguishing malignant versus benign diseases according to sex.As in our series of male malignant PEs the 2 most common cancers were LC and MM, whereas in the female malignant PEs a large number of cancers was represented by BC and the frequency of MM was lower, we found that hMAM was more expressed in female malignant PEs (Se=51%) than in male malignant PEs (Se=41.7%).However, the sex-based analysis between malignant versus benign PEs showed a highly significant correlation of hMAM expression with malignant PEs. In conclusion, hMAM RT-PCR could be used for differential diagnosis of PEs without sex limitation.Finally, based on the relative frequencies of tumors responsible for PEs and their different hMAM expression rates, we also analyzed whether the hMAM RT-PCR may provide a useful guide for distinguishing the origin of the PEs.In this context, subgroup analysis by sex pointed out a fairly high probability (probability=50.8%) of having BC among hMAM-positive female patients. In contrast, in the same subgroup the test failed to detect discernible differences between LC (probability=17.0%), MM (probability=9.4%), and OC (probability=15.1%) patients. In the male subgroup, as a positive test indicated with similar probabilities for LC (probability=41.0%) or MM (probability=39.3%) patients, hMAM expression was much less useful for discriminating between them.The major limit of hMAM RT-PCR was that the negativity of the test does not exclude the malignant origin of PEs in both patient groups (probability=39.1% in males and probability=49.0% in females). However, it was able to exclude, with high likelihood, the mammalian origin of PEs (probability=5.2% in females).It is important to point out that the usefulness of such predicted probabilities as measures of DDP is time and place-dependent. In fact, their magnitudes also reflect the frequencies by which the considered pathologic conditions are diagnosed in each hospital, which are, in turn, a function of the disease prevalence in the hospital catchment area. In other words, the above-mentioned estimated figures can vary from area to area according to the frequency of diseases taken into consideration for analysis. In this regard, as La Spezia is a heavy industrialized Italian town, the likelihood of chronic exposure to industrial hazard, such as asbestos, is particularly high. Accordingly, MM is the second most common malignancy in the male population of this area, in contrast with other areas in which MM is a rare tumor.28In addition, the pretest probability of a disease significantly impacts PPV and NPV as well. In fact, given the estimates of Se and Sp pointed out in the Results section, and considering the observed prevalence of malignant PE in the patients under study, that is 55%, PPV-NPV estimates were 91.2% to 58.5%, 90.2% to 62.1%, and 92.5% to 51.0%, in the entire, male, and female populations, respectively. Different predicted probability estimates may be obtained when the same biomarker is applied to patients with a different malignant PE percentage. For instance, if we consider a malignant PE frequency of 25%, which can be regarded as a more common and typical prevalence in many clinical settings, as reported by Dollinger,1 we find the following PPV-NPV estimates: 73.8% to 83.9%, 75.5% to 69.4%, and 69.4% to 85.0% in the entire, male, and female populations, respectively.In conclusion, although other studies need to be performed to further assess the validity of the RT-PCR test for hMAM in other clinical settings, we believe that this test could be helpful in providing diagnostic information as its positivity is highly suggestive of malignancy for a PE. In addition, it will be important in future studies to identify those tumors that never express hMAM so that their involvement as events originating the PE may be excluded in case of hMAM RT-PCR positivity.Taken together, our results confirm that nested RT-PCR of a specific hMAM target gene provides an assay sensitive enough to alert and help the clinician when Cyt examination remains suspect or negative. In this contest, our results reveal that the Se of RT-PCR for hMAM in Cyt-negative malignant PEs was 38.9% and 28.6% in female and male subgroups, respectively.Besides hMAM, other molecular markers have been reported for the indirect detection of tumor cells in PEs, some of which seem to differentiate between benign and malignant PEs. They include EGP-2,29 GA733,30,31 MN/CA9,32 and Mucin33 whereas the markers, Lunx and TTF-1, were proposed to differentiate the origin of non-small cell LC34 and LC,35 respectively. Therefore, some investigators have used a panel of molecular markers for the differential diagnosis of PEs34,36,37 and we believe that hMAM is a suitable marker to be included in this type of approach. This hypothesis has been confirmed by Passebosc-Faure et al15 who provided evidence that the addition of hMAM to CEA and Ep-CAM markers could be a beneficial adjunct to Cyt in the diagnosis of malignant PEs.Moreover, the samples needed to perform the RT-PCR test may be extracted by thoracentesis together with the samples to perform Cyt and would represent a nonexcessive extra workload in terms of time, cost, and labor. 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The hMAM bands are evident in the breast cancer cell line MDA-MB415 used as positive control (lane 1) and in the samples of effusion from patients with breast cancer, ovarian cancer, lung cancer, and malignant mesothelioma (lanes 3 to 6) but not in the patient with pneumonia (lane 2). GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase; hMAM, human mammaglobin; MW, molecular weight marker. Estimates of Diagnostic Performance Parameters of Human Mammaglobin RT-PCR Detection in All Pleural Effusions Discriminative Diagnostic Power of Human Mammaglobin Expression According to the Origin of Pleural Effusions Estimates of Diagnostic Performance Parameters of Human Mammaglobin RT-PCR Detection in Cytologically Negative Pleural EffusionsDiagnosis and Origin Determination of Malignant Pleural Effusions Through the Use of the Breast Cancer Marker Human MammaglobinRoncella Silvio PhD; Ferro, Paola PhD; Franceschini, Maria Cristiana PhD; Bacigalupo, Bartolomeo MD; Dessanti, Paolo MD; Sivori, Massimiliano MD; Carletti, Anna Maria MD; Fontana, Vincenzo PhD; Canessa, Pier Aldo MD; Pistillo, Maria Pia PhD; Fedeli, Franco MDOriginal ArticlesOriginal Articles219p 92-98