Application of PCR in the diagnosis of B-type non-Hodgkin's lymphomas in cytological specimens from fine-needle aspiration

The application of PCR to detect monoclonality at the immunoglobulin heavy chain gene locus in FNA cytologic specimens was evaluated. MATERIALS AND METHODS: Immunoglobulin heavy chain gene rearrangement analysis was performed by a nested PCR for the VDJ region on 27 lymph nodes FNA paraffin embedded by cell blocking technique specimens (1 suspicious of NOS lymphoma, 1 Hodgkin's disease, 6 B cell non Hodgkin's lymphomas, 10 reactive hyperplasias). RESULTS: A monoclonal band was seen in 13 of 17 suspicious or positive malignant cases. The other 4 specimens gave a policlonal pattern. Specimens from reactive lymph nodes produced policlonal bands in 8 cases, no product in 1 case and 1 specimen gave a monoclonal band. When needed, the results of PCR examinations were compared with the subsequent histologic evaluation of the same lymph-node. CONCLUSIONS: From our experience we can see that clonal analysis by PCR based IgH gene rearrangement analysis can be applied to FNA material and can be useful in diagnosis.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

The use of the polymerase chain reaction in the diagnosis of B-cell lymphomas from formalin-fixed paraffin-embedded tissue

In this study the use of the polymerase chain reaction (PCR) to detect monoclonality in B-cell lymphoid proliferations in archival formalin-fixed paraffin-embedded tissue was assessed. Using consensus primers against the framework 3 (FR 3) region of the immunoglobulin heavy chain gene (IgH), PCR analysis was performed on 29 low grade B-cell non-Hodgkin's lymphomas. Cases of benign lymphoid hyperplasia served as polyclonal controls. Sequenced cases of acute lymphoblastic leukaemia served as positive controls. In the lymphomas, monoclonality could be demonstrated in 18 of 29 (62%) cases. Only five of 11 (45%) follicle centre cell lymphomas were positive by this method whilst the success rate for the remainder was 13 of 18 (72%). None of the polyclonal controls gave false positive results although occasional non-specific dominant bands were present which disappeared on repeating the experiments. These results show that this method will identify monoclonality in 62% of low grade B-cell non-Hodgkin's lymphomas in archival material. The success rate is increased to 72% if follicle centre cell lymphomas are excluded. Thus, this method is a useful adjunctive test to aid diagnosis in lymphoid infiltrates when standard morphology and immunohistochemistry are equivocal.     

Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to develop a simple technique for detecting monoclonality at the DNA level in B lymphocyte populations in formalin fixed, paraffin wax embedded material. Sections were dewaxed and dehydrated, and the DNA was extracted by boiling in water for 45 minutes. A semi-nested PCR was performed to amplify the V-D-J region of the immunoglobulin heavy chain gene. The product was electrophoresed and viewed under ultraviolet light after ethidium bromide staining. Specimens from 26 B cell lymphomas produced a monoclonal band in 24 cases and no amplification in two cases; monoclonality was specific for this disorder. Specimens from seven T cell lymphomas produced no amplification; specimens from nine reactive nodes produced a broad smear of polyclonal material; and specimens from 12 cases of carcinoma produced either no amplification or polyclonal material. As detection of monoclonality is strongly suggestive of neoplastic disease, this technique is likely to be of value in routine diagnosis, because of its speed, simplicity, and applicability to fixed, embedded material.     

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction (PCR) in the diagnosis of B cell lymphoma. METHODS: Analyses were applied to formalin fixed, paraffin wax embedded, routine diagnostic specimens from cases with a provisional diagnosis of reactive lymph node (n = 23), B cell lymphoma (n = 21), and T cell lymphoma (n = 4). Nonisotopic ISH for kappa and lambda immunoglobulin light chain mRNA was performed using both fluorescein and digoxigenin labelled oligodeoxynucleotide probe cocktails. PCR was carried out on DNA extracted from sections using primers to framework 3 (Fr3) of the V segments and to conserved sequences from the J regions of the immunoglobulin heavy chain genes. RESULTS: All reactive lymph nodes showed a polyclonal pattern of light chain mRNA by ISH, although one showed an excess of kappa positive cells. Nineteen of 21 (90%) cases of B cell lymphoma showed light chain restriction, and a further case showed a vast excess of kappa positive cells. By PCR, 20 of 23 reactive nodes (87%) showed a polyclonal pattern. In 13 of 21 B cell lymphomas (62%) a clonal band was detected. CONCLUSION: In the diagnosis of B cell lymphoma in routinely processed diagnostic material ISH for light chain mRNA was more sensitive (90%) than PCR for heavy chain gene rearrangement using Fr3 and J region primers (62%).     

Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes.

AIMS: Clonality analysis using polymerase chain reaction (PCR) amplification of the immunoglobulin heavy chain (IgH) gene is an important aid to the diagnosis of B cell lymphoproliferative diseases. However, the method has a relatively high false negative rate. In an attempt to improve detection rates simple PCR strategies for clonality analysis of B cell populations using amplification of Ig light chain genes have been developed. METHODS: Novel PCR protocols, designed to amplify Ig kappa and Ig lambda light chain genes, were evaluated using high molecular weight DNA samples from 28 selected cases of B cell lymphoma with known light chain expression and 12 reactive lymphoid specimens. Products were run on 10% polyacrylamide minigels using heteroduplex analysis. Conventional IgH PCR analysis was also performed. Twelve randomly selected formalin fixed, paraffin wax processed samples from cases submitted for molecular genetic analysis were also studied. RESULTS: Polyclonal products were seen in all reactive lymphoid samples. Using Ig kappa PCR, 24 of 28 lymphomas, including four of five IgH negative cases, displayed monoclonal patterns. Using Ig lambda PCR, eight of 12 Ig lambda expressing tumours, including two of five IgH negative cases, showed monoclonal patterns. Standard IgH PCR demonstrated monoclonality in 23 of 28 B cell lymphomas. The detection rate was improved to 27 of 28 lymphomas using heavy and light chain PCR. Efficient amplification was achieved using paraffin wax processed samples, seven of which showed monoclonality compared with eight using IgH PCR. CONCLUSIONS: Ig light chain PCR, used in conjunction with heavy chain analysis, enables improved detection of B cell monoclonality using routine histological specimens and can provide additional clone specific markers for the study of the biology of B cell tumours.     

Molecular insight into the diagnosis of lymphoma

Malignant lymphoma may be very difficult to diagnose with routine histopathological methods because they may recapitulate benign architecture or contain benign infiltrates. The best method of diagnosis is to establish the clonal profile of the lymphocyte population, since a monoclonal proliferation is highly suggestive of neoplasia. By means of a PCR (polymerase chain reaction) method it is possible to detect the immunoglobulin heavy chain (IgH) gene rearrangement and therefore establish the lymphocyte clonality. PCR with primers complementary to relatively conserved regions called frameworks (FR1-FR3) laying among hyper variable regions (CDR1-CDR3) of IgH gene unable us to detect monoclonal versus polyclonal B-cell population. The length of the PCR product with these primers is unique if we deal with a monoclonal population. On the contrary, a polyclonal population gives PCR products of a different size. In this retrospective study we used a semi-nested PCR to analyse 37 paraffin-embedded specimens. All of them had been evaluated previously by pathohistological and immunophenotypic criteria. A number of polyclonal (PBL and tonsils from healthy donors) and monoclonal cells (PBL from CLL patients, Raji cell line) were analyzed as controls. Clonality was successfully determined in all specimens. Our results support the concept that molecular techniques such as PCR provide a helpful approach for detection of monoclonal immunoglobulin rearrangements in malignant lymphoma. This is especially true for border cases, but always in the combination with other pathohistological methods.     

Fine needle aspiration cytology diagnosis of malignant lymphoma and reactive lymphoid hyperplasia.

AIMS: To assess the diagnostic accuracy of lymph node fine needle aspiration (FNA) cytology to distinguish reactive lymphoid hyperplasia from malignant lymphoma, and to evaluate the contribution of ancillary techniques applied to cytological material. METHODS: Two hundred and seventy seven consecutive lymph node FNA specimens reported to be consistent with reactive lymphoid hyperplasia (n = 213) or suggestive/diagnostic of malignant lymphoma (n = 64) were reviewed. Follow up data were obtained by case record review or by histological correlation. The value of immunocytochemistry, in situ hybridisation for immunoglobulin light chain mRNA, and polymerase chain reaction (PCR) towards the final clinicopathological diagnosis was assessed in 92, 61, and 45 cases, respectively. RESULTS: Sixty one of 67 lymphomas and 207 of 209 reactive lymph nodes were accurately diagnosed by FNA cytology. There were six false negative aspirates including three cases of follicular lymphoma, two cases of Hodgkin's disease, and one chronic lymphocytic leukaemia. Two FNA specimens considered suspicious of lymphoma proved reactive on histology or clinical follow up. One metastatic small cell carcinoma was wrongly diagnosed as lymphoma. Ancillary studies contributed to the correct diagnosis in most cases although occasional misleading results were obtained, particularly with PCR. CONCLUSIONS: FNA cytology accurately distinguished reactive lymphoid hyperplasia from malignant lymphoma in 97% of cases. However, occasional wrong diagnoses occurred owing to sampling error or misinterpretation. Ancillary studies can be applied to cytological samples and contribute to the diagnosis in most cases.     

Detection of concurrent/recurrent non-Hodgkin's lymphoma in effusions by PCR

A subset of patients with non-Hodgkin's lymphoma (NHL), present with or subsequently develop lymphocytic effusions. Differential diagnosis between reactive lymphocytosis and recurrent low-grade NHL is difficult by cytology alone. We studied the use of polymerase chain reaction (PCR)-based techniques to detect concurrent/recurrent NHL. Both primary tumors and atypical lymphocytic effusions of 12 low-grade B-NHL patients and 4 T-NHL patients were studied. Six pleural effusions (reactive/carcinomatous), in patients with no history of NHL, were included. Samples were amplified by PCR, using Fr3, Fr2, LJH, and VLJH primers specific for the immunoglobulin heavy chain (IgH) gene and Vgamma-8, Vgamma9, Vgamma10, Vgamma11 and Jgamma1/Jgamma2 consensus primers specific for the T-cell receptor gamma (TCR-gamma) gene. IgH gene PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). TCR-gamma gene PCR products were analyzed using a novel nonradioactive single-strand conformational polymorphism (SSCP) procedure. IgH gene rearrangement analysis demonstrated monoclonality in 11/12 primary low-grade B-NHLs. Identical monoclonal bands were found in both primary tumor and effusion in 9 patients. TCR-gamma gene rearrangement analysis demonstrated monoclonality in 4 of 4 primary T-NHLs. Identical monoclonal banded patterns were found in both primary tumor and effusion in 3 patients. Our results strongly support the diagnosis of concurrent/recurrent NHL in 13 of 16 (81%) cases of atypical lymphocytic effusions. IgH/PAGE and TCR-gamma/SSCP analyses are useful tools in the diagnoses of lymphocytic effusions in patients with NHL.     

Detection of monoclonality in low-grade B-cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type

Detection of B-cell monoclonality using the polymerase chain reaction (PCR) promises the quick and cost-effective separation of monoclonal from polyclonal B-cell disease. However, the efficiency of the method has yet to be fully assessed, particularly with regard to disease type and selection of PCR primers. We have evaluated two approaches based on amplification of the immunoglobulin heavy chain gene using frame work 2 (Fr2) and framework 3 (Fr3) region primers. Frozen tissue samples from 94 cases of low-grade B-cell lymphoma were investigated, all of which had previously been shown to be monoclonal by Southern blot analysis. Using a Fr2 primer, we were able to show monoclonality in 85 per cent of cases; with Fr3, 80 per cent of cases; and using both techniques in separate reactions, 90 per cent of cases. Thus, a significant false-negative rate exists with either primer which can be reduced by using both. We also found a difference in the efficiency of detection in different types of lymphoma; only 87 percent of mucosa-associated lymphomas and centroblastic/centrocytic lymphomas were shown to be monoclonal, whereas all of the other lymphoma types tested were positive using one or both methods. We conclude that PCR detection of B-cell monoclonality allows rapid analysis of tissue samples, including paraffin-processed material. False-negative results which occur in some types of lymphoma can be reduced by the use of two or more primer combinations.     

Sensitivity of PCR in detecting monoclonal B cell proliferations.

AIMS--To evaluate the rapid detection of various forms of monoclonal B cell proliferations by using the polymerase chain reaction (PCR) to identify clonal immunoglobulin heavy chain genomic rearrangements. METHODS--Thirty four B cell lymphomas defined by morphology, immunophenotyping, and positive immunoglobulin heavy chain gene rearrangements detected by Southern blot analysis were examined. An additional 22 cases representing miscellaneous lymphoproliferative and non-lymphoproliferative disorders were also studied. RESULTS--Monoclonal rearrangements were identified in 19 (56%) cases of B cell lymphoma. The method was less sensitive in the detection of follicular centre cell lymphomas (15 of 28, or 54%) than non-follicular centre cell lesions (four of six, or 67%). Monoclonal rearrangement was not identified in 19 control cases, including T cell lymphomas, Hodgkin's disease, reactive lymphadenopathy and metastatic carcinoma. Three cases showed positive immunoglobulin gene rearrangement by PCR but were negative on Southern blotting. Two of these cases had definite clinical, morphological, and immunophenotypic evidence of monoclonal B cell proliferation suggesting that PCR could, on occasion, pick up cases missed by Southern blotting and that the two methods are complementary in clonal lymphoproliferative disease diagnosis. The third case represented a "false positive" PCR reaction involving a colonic adenocarcinoma. CONCLUSIONS--PCR analysis, using the primer sequences outlined in this study, will detect about 55% of clonal lymphoproliferative proliferations with increased sensitivity for non-follicular centre cell lesions. With these levels of detection in mind, this testing strategy can still be especially useful in cases which prove diagnostically problematic with standard morphological and immunophenotypic analysis, and in instances where the quantity and type of diagnostic material is limiting (needle aspirates and cellular fluids).     

PCR-based clonality analysis: a reliable method for the diagnosis and follow-up monitoring of conservatively treated gastric B-cell MALT lymphomas?

AIMS: We evaluated polymerase chain reaction (PCR) amplification of specific immunoglobulin heavy chain (IgH) gene rearrangements as a means of demonstrating monoclonality during follow-up of conservatively treated gastric MALT lymphoma, and compared the reproducibility of PCR on sequential frozen and paraffin-embedded endoscopic biopsies. We established an association between clonality detected by PCR and the histological observations. METHODS AND RESULTS: Sixty-nine pairs of sequential frozen and paraffin-embedded endoscopic biopsies from 21 conservatively treated patients were graded according to the Wotherspoon-Isaacson histological scoring system, which provides a measure of diagnostic confidence on a scale 0-5. PCR amplification of the IgH gene was performed using FR3/JH and FR2/JH primers. 68/69 paired samples (98.5%) showed identical mono- or polyclonal PCR amplification patterns. Forty-seven out of 48 pairs of samples sharing similar histological features produced identical amplification patterns in both fresh and paraffin-embedded tissues. In comparison with the histological grading, monoclonality was detected in 64.2% and 41.6% of samples scored 5 and 4, respectively. Conversely, among 64 samples scored 0-3, a monoclonal pattern was observed only in two samples, one of which was from a patient who relapsed 9 months later. CONCLUSIONS: PCR-based clonality analysis by demonstration of specific IgH gene rearrangement can be easily and reliably performed on both frozen and paraffin-embedded endoscopic biopsies. In conjunction with histological observation, this method can be used as a complementary tool to monitor MALT lymphoma regression during conservative treatment.     

Diagnosis of lymphoma in paraffin wax sections by nested PCR and immunohistochemistry.

AIMS: To investigate whether nested polymerase chain reaction (PCR) and immunohistochemistry can be used to diagnose malignant lymphoma. METHODS: Paraffin wax embedded tissue sections from 31 patients with malignant lymphoma were analysed by nested PCR and immunohistochemistry using standard protocols. RESULTS: Nested PCR amplification of 1 pg DNA confirmed monoclonality in B cell lymphoma; PCR amplification of 10 pg DNA confirmed monoclonality in T cell lymphoma. Twenty seven (87%) samples were diagnosed as malignant lymphoma by nested PCR, and 24 (77%) by immunohistochemistry. Seven samples were diagnosed as malignant lymphoma by nested PCR, but not by immunohistochemistry, whereas the use of both procedures gave a diagnosis of malignant lymphoma in all 31 samples. CONCLUSIONS: A combination of immunohistochemistry and nested PCR can be used to diagnose malignant lymphoma in routine paraffin wax embedded sections.     

PCR-based assays for the detection of monoclonality in non-Hodgkin's lymphoma: application to formalin-fixed,paraffin-embedded tissue and decalcified bone marrow samples

In crucial cases the diagnosis of non-Hodgkin's lymphoma (NHL) still represents a challenge to the pathologist since morphological criteria do not always help to distinguish between reactive and malignant lymphoproliferations. Clonality assays are a useful supplement since monoclonal cell proliferation is strong evidence for malignancy. The polymerase chain reaction (PCR) can be utilized to establish the clonal origin of B-or T-cell lymphocyte populations by amplification of rearranged immunoglobulin and T-cell receptor (TCR) genes. In the present study DNA was isolated from a variety of neoplastic and nonneoplastic formalin-fixed, paraffin-embedded lymph nodes (n=62), cutaneous tissue (n=9), samples of miscellaneous origin (n=11), and, reported here for the first time, decalcified bone marrow samples (n=35). These samples were submitted to PCR-based assays directed against the immunoglobulin heavy-chain (IgH), immunoglobulin light-chain (IgL), and TCR chain genes. The impact of various decalcifying agents on the ability to amplify DNA was investigated by PCR-based amplification of a single copy gene. Buffered and nonbuffered EDTA was found not to impede amplification of DNA fragments up to 300 bp in length. In lymph node and cutaneous specimens monoclonality was detected in 83% of B-NHL cases using a seminested PCR approach for the amplification of IgH, whereas the same approach gave rise to monoclonal bands in 80% of bone marrow samples. The subsequent amplification of IgL helped to raise the sensitivity of detection to 94%. Monoclonality was detected in seven of nine T-cell NHLs by amplification of TCR. Most of the false-negative results were related to DNA extracted from centroblastic-centrocytic lymphoma and lymphoplasmocytic immunocytoma (37% negative each). PCR-based rearrangement analysis of immunoglobulin and TCR chain genes should be used in diagnostic pathology for cases which are histopathologically and immunohistochemically questionable. The application of clonality assays to bone marrow samples previously decalcified with EDTA provides a new tool for the detection of minimal residual disease.Abbreviations BALT bronchus-associated lymphoid tissue - dNTP deoxynucleoside triphosphate - Ig immunoglobulin - IgH immunoglobulin heavy chain - IgL immunoglobulin light chain - MALT mucosa-associated lymphoid tissue - NHL non-Hodgkin's lymphoma - PCR polymerase chain reaction - TCR T-cell receptor     

Comparative analysis of immunoglobulin polymerase chain reaction and flow cytometry in fine needle aspiration biopsy differential diagnosis of non‐Hodgkin B‐cell lymphoid malignancies

Single primer pair polymerase chain reaction (PCR) assays for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements and immunophenotyping by flow cytometry have been proved as useful techniques in the diagnosis of lymphoid disorders in fine needle aspirates. However, a comparative analysis of both ancillary techniques in the same samples has not been previously performed. To compare the sensitivity of flow cytometry and PCR techniques, we made a wide prospective study of 77 fine needle aspiration biopsy (FNAB) samples from lymph nodes and extranodal lymphoid infiltrates. The adjunctive values of a single primer pair PCR amplification of IgH genes and of the immunophenotyping by flow cytometry were evaluated comparing their results with the final clinicopathological diagnosis of each patient supported by histological features and clinical follow up. Among the 24 B‐cell non‐Hodgkin lymphomas, monoclonal IgH bands were detected in 22 cases by PCR, and 21 cases were correctly considered B‐cell lymphoma by flow cytometry. A monoclonal IgH band was also detected in 1 of the 53 reactive lymphoid disorders. When both ancillary techniques were combined with morphological findings, 23 of the 24 B‐cell lymphomas were correctly diagnosed but one reactive lymphoid disorder was also considered a B‐cell lymphoma. We demonstrate a similar level of detection of B‐cell lymphomas by single round PCR and flow cytometry techniques, and a strong adjunctive value when combined with morphological findings to diagnose correctly lymphoproliferative disorders by FNAB. However, we must be cautious with PCR results since false‐positive cases can occur. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations

BACKGROUND: Fine-needle aspiration cytology (FNAC) is used as a screening test to evaluate lymphadenopathy. The combined use of genetic analysis and flow cytometry for immunophenotyping has increased the accuracy of diagnosis and correct categorisation of lymphomas on cytological preparations. AIM: To show the utility of immunocytochemistry and polymerase chain reaction (PCR) in the evaluation of cytological preparations of lymph nodes. METHODS: Fine needle aspirates were obtained from 33 patients (initial presentation, n = 27; recurrence, n = 6). Routine examination was undertaken using immunocytochemistry and DNA PCR to detect clonality and specific translocations. The cytodiagnosis and subclassification of lymphoma was correlated with histological diagnosis in the available follow-up biopsies. RESULTS: 14 patients had a cytological diagnosis of non-Hodgkin's lymphoma (NHL), 4 had suspected NHL, 2 had atypical lymphoid proliferation and 13 had reactive hyperplasia. A World Health Organization (WHO) subtype was suggested in 8 patients. Incorporating the results of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements enabled diagnosis of lymphoma in 17 patients, including 5 of the 6 patients suspected to have NHL or an atypical lymphoid proliferation. Identification of the translocations t (14;18) and t (2;5) helped WHO categorisation in 3 of the patients. The cytological findings were confirmed in 12 out of the 13 patients for whom histological follow-up was available. Seven of the 18 lymphoma patients were managed without a subsequent biopsy. We made one false-positive diagnosis of B-cell NHL on cytology. CONCLUSION: The use of immunocytochemistry and PCR is valuable in the definitive diagnosis and subtyping of malignant lymphomas on cytological preparations. The use of these techniques may avoid lymph node biopsies in some cases and allow definitive treatment based on aspirate findings alone.     

Fine-needle aspiration cytology of extranodal lymphoma

The assessment of lymphoproliferative disorders using fine-needle aspriation (FNA) cytology may be problematic particularly when organs other than lymph node are involved. In this report we have reviewed 26 consecutive FNA specimens from superficial extranodal sites which were reported as diagnostic or suggestive of malignant lymphoma. The aspirates were obtained from skin or subcutaneous tissue (ten cases), thyroid (five cases), salivary gland (five cases), breast (four cases), neck, and pharynx (one case each). Ancillary studies including immunocytochemistry, in situ hybridisation to detect immunoglobulin light chain mRNA expression, and polymerase chain reaction for analysis of immunoglobulin heavy chain gene rearrangement were performed in 20, 12, and 7 cases, respectively. Clinicopathologic correlation confirmed the diagnosis of lymphoma in 25/26 aspirates. Nine of the 14 patients whose initial presentation was with an extranodal mass were considered to have primary lymphomas of mucosa-associated lymphoid tissue (MALT) type. In contrast, ten of 11 patients with recurrent extranodal disease had primary nodal type lymphomas. There was one false-positive diagnosis, a neck mass misinterpreted cytologically as B-cell lymphoma which was ultimately shown to be a branchial cyst. FNA cytology supported by appropriate ancillary investigations provides accurate diagnosis in most cases of extranodal lymphoma. Diagn. Cytopathol. 1998;19:260–266. © 1998 Wiley-Liss, Inc.     

Evaluation of lymphoid cell populations in cytology specimens using flow cytometry and polymerase chain reaction.

Differential diagnosis between lymphomas and reactive lymphoid proliferations often requires ancillary techniques and morphologic evaluation. Flow cytometry (FCM) and polymerase chain reaction (PCR) can aid the detection of monoclonal B-cell populations. In the present study, the sensitivity and specificity of these two methods in the study of cytology specimens were compared. Eighty-six cytologic specimens from 81 patients (lymph nodes, solid organs, and body cavities) were evaluated. These specimens were taken from three groups of patients: those who underwent an initial evaluation for suspected lymphoma; those who were previously diagnosed with B-cell lymphoma and were now evaluated for possible disease recurrence; and those who were diagnosed with a nonhematologic malignancy. Histologic diagnosis was available for 51 samples. All samples were tested by FCM for the detection of monoclonality using kappa:lambda ratio and for clonal immunoglobulin heavy chain (IgH) gene rearrangements using a single-round PCR after cytologic evaluation. Tissue morphology, FCM and PCR results, and clinical findings in specimens without histologic diagnosis were correlated. Histologic evaluation (N = 51) revealed 44 specimens with B-cell malignancy. Twenty of the 44 lymphoma specimens (45%) were accurately diagnosed in cytologic smears, 18 (41%) were classified as suspicious of lymphoma, and 6 (14%) were diagnosed as reactive. FCM had superior sensitivity compared with PCR (77% vs. 64%). Fifty-six percent of specimens with B-cell malignancy were FCM+/PCR+, 23% were FCM+/PCR-, 14% were FCM-/PCR+, and 7% were FCM-/PCR-. The combined use of FCM and PCR resulted in a diagnosis of B-cell lymphoma in 41 (93%) of 44 B-cell lymphoma specimens and increased the sensitivity of fine needle aspiration by 48%. Both FCM and PCR aid in the diagnosis of lymphoid lesions in cytology specimens, and both can detect monoclonal B-cell populations that may be interpreted in cytology smears as reactive, even by experienced cytologists. Although FCM had higher sensitivity than PCR test in the present study, their combined use should be considered because of a relatively large number of specimens that were detected as monoclonal only with PCR.     

Specificity of PCR-based clonality analysis of immunoglobulin heavy chain gene rearrangements for the detection of bone marrow involvement by low-grade B-cell lymphomas

A study was performed to investigate the utility of polymerase chain reaction (PCR)-based analysis of immunoglobulin heavy chain (IgH) gene rearrangements for the diagnosis of low-grade malignant B-cell lymphomas on formalin-fixed, EDTA-decalcified, and paraffin-embedded bone marrow trephine biopsies. On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in 32 patients with reactive lymphoid hyperplasias. In contrast, 5/14 patients with known low-grade B-cell lymphomas, but histomorphologically and immunohistochemically lymphoma-free bone marrow, showed a reproducible monoclonal IgH gene rearrangement. In three of these cases, sequence analysis revealed completely different amplification products on comparing bone marrow and lymph node infiltrations, while in the other two cases the products were identical. In one of the latter biopsies, an unequivocal lymphoma infiltrate was found after step sectioning of the biopsy, while the other case remained lymphoma-free according to conventional criteria. A third group of three patients with known lymphomas and bone marrow findings that were suggestive but not diagnostic of bone marrow involvement showed monoclonal PCR results in all three cases, with identical sequences in bone marrow and extramedullary lymphoma infiltrates. These data suggest that a reproducible monoclonal IgH gene rearrangement is highly specific for the presence of malignant B-cells in bone marrow. In staging procedures for low-grade B-cell lymphomas, PCR yields no additional information in cases that are morphologically and immunohistochemically lymphoma-free after evaluation of representative sections. PCR may be useful in equivocal cases, provided that IgH gene rearrangements of extramedullary lymphoma and bone marrow are sequenced and compared.