Cytology material is equivalent to tumor tissue in determining mutations of BRCA 1/2 genes in patients with tubo-ovarian high grade serous carcinoma.

BACKGROUND: High-grade serous ovarian cancer is a detrimental disease. Treatment options in patients with a recurrent disease are dependent on BRCA1/2 mutation status since only patients with known BRCA mutation are eligible for treatment with poly(ADP-ribose) polymerase inhibitors (PARPi). The aim of this study was to compare concordance of BRCA mutation analyses from cytological samples (CS) with BRCA mutation analyses from histological formalin fixed paraffin embedded (FFPE) samples. METHODS: Mutation analysis of BRCA1 and BRCA2 genes was performed in 44 women diagnosed with primary or recurrent high-grade ovarian cancer from three different samples: blood, cytological sample (ascites, pleural effusion and enlarged lymph nodes) and tumor tissue. Results from all three samples were compared. RESULTS: Among 44 patients, there were 15 germline mutations and two somatic mutations. A 100% concordance was found between cytological and histologic samples. CONCLUSION: There is a 100% concordance in BRCA mutation testing between cytological and histologic samples. BRCA mutation testing from CS could replace testing from FFPE tissue in clinical decision making in ovarian cancer patients. TRIAL REGISTRATION: The study was retrospectively registered at ISRCTN registry on 24/11/2015 - ISRCTN42408038 .

Usefulness of Bcl-2 Expression and the Expression of Cytoplasmic Immunoglobulin Light Chains in the Differentiation Between B-Cell Lymphoma and Reactive Lymphocytic Proliferations in FNA.

Flow cytometry is helpful in differentiating between B-cell lymphoma (BCL) and reactive lymphocytic proliferation (RLP) in FNA biopsies. However; the presence of inconclusive surface immunoglobulin light chains (sIg LC) poses a problem. We investigated the usefulness of additional tests; namely Bcl-2 expression and expression of cytoplasmic Ig LC (cIg LC), mainly on samples with inconclusive sIg LC. Both tests were performed on 232 FNA samples from lymph nodes. Bcl-2 alone was determined qualitatively and quantitatively on 315 samples. The quantitative test was correctly positive in 76% of cases and falsely negative in 24%. The correctly positive results of the qualitative test were 11% points lower. cIg LC correctly identified 65% of BCL with dual positive sIg LC; 36% of BCL with difficult to interpret sIg LC and only 7% of BCL with negative sIg LC. The best results in differentiating between BCL and RLP were obtained when all three tests were used together. In samples with inconclusive sIg LC and additional monoclonal or polyclonal populations the κ:λ ratios did not differentiate between RLP and BCL. We propose that in case of inconclusive sIg LC Bcl-2 test is used first. The addition of cIg LC test is sensible only in cases with dual positive and difficult to interpret sIg LC.

Assessment of Ki-67 Proliferative Index in Cytological Samples of Nodal B-Cell Lymphomas.

BACKGROUND: The Ki-67 proliferative index (PI) is part of the diagnosis of nodal B-cell lymphoma (nBCL), but its determination in cytological samples is not standardized. We aimed to establish an approach for the accurate determination of the Ki-67 PI in cytological slides to differentiate between indolent and aggressive nBCLs. METHODS: Patients diagnosed with nBCL by fine-needle aspiration biopsy and subsequent excision biopsy were included. Cell suspensions were prepared from biopsy samples for CD3/Ki-67 double immunocytochemical staining and flow-cytometric verification of lymphoma B-cell counts. The Ki-67 PI was assessed by manual counting and eyeballing in cytology and eyeballing in histology. The cut-off values for the differentiation between aggressive and indolent lymphomas were determined for each method. RESULTS: A strong correlation between manual and flow-cytometric counting of lymphoma B cells was confirmed (interclass correlation coefficient (IC coef.) = 0.78). The correlation of the Ki-67 PI determined in cytological and histological slides was also strong (IC coef. > 0.80). Histologically, 55 cases were classified as indolent and 31 as aggressive nBCLs. KI-67 PI cut-off values of 28.5%, 27.5%, and 35.5% were established for manual counting and eyeballing in cytology and eyeballing in histology, respectively, with high sensitivity and specificity. CONCLUSIONS: The Ki-67 PI, assessed by manual counting and eyeballing in cytological samples, accurately differentiates between indolent and aggressive nBCLs.

Prognostic value of immunohistochemistry in the Ewing's sarcoma family of tumors.

BACKGROUND: The expressions of different markers have been immunohistochemically studied in various types of the Ewing's sarcoma family of tumors, especially for diagnostic purposes. However, little is known about their prognostic value in combination with the clinicopathological data of such patients. MATERIAL/METHODS: This retrospective study investigated the immunohistochemical expressions of NSE, TdT, EMA, S-100, CK MNF116, p53, bcl-2, CD99, and CD117 on formalin-fixed paraffin-embedded material of 72 patients (age range: 2-59 years) with various types of Ewing's sarcoma family of tumors using the tissue microarray method. The immunohistochemical results were compared with clinicopathological features using survival analysis (Cox regression). RESULTS: CD99 expression was detected in 90%, CD117 in 75.8%, bcl-2 in 70.1%, NSE in 66.6%, p53 in 66.6%, EMA in 26%, S-100 in 25.4%, TdT in 22.1%, and CK MNF116 in 10.2% of the cases. No immunoreactivity was observed in the normal tissue around the tumors. CONCLUSIONS: The expressions of EMA (p=0.107), NSE (p=0.126), CD99 (p=0.14), TdT (p=0.198), bcl-2 (p=0.382), p53 (p=0.54), CD117 (p=0.612), S-100 (p=0.867), and CK MNF116 (p=0.934) had no statistically significant impact on survival. Patient age at the time of diagnosis (p=0.008) and the presence of tumor necrosis (p=0.033) were the only significant prognostic factors in this study. Tumor location was an insignificant prognostic factor (p=0.38).

Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

BACKGROUND: p16/Ki-67 dual immunocytochemical staining (DS) has been proven as a sensitive and specific test for triage of HPV positive women with good reproducibility and accuracy. However, implementation of the test into an organized screening program (OSP) is not easy. The aims of this study were to compare the performance and agreement of DS results among three Slovenian cytopathological laboratories involved in the national OSP, and to define cases where staining results can be difficult to interpret. METHODS: Cervical smears were obtained for DS from 129 women referred to colposcopy. Smears were evaluated blindly in three laboratories by a cytotechnologist and a cytopathologist after initial training. Results were positive, suspicious, negative or inadequate. Five characteristics of DS staining were recorded. After primary evaluation, an extensive expert-led additional training was undertaken, including a discussion of difficult cases and a practical exam. Smears were re-evaluated and results compared to primary evaluation. RESULTS: After the additional training, the overall percentage of agreement among the three laboratories increased from 77.5 to 89.9% and kappa increased from 0.70 to 0.86. Sensitivity for CIN2+ increased in two laboratories, to 90.5 and 85.7%, without the loss of specificity (75.8%). In one laboratory, the sensitivity slightly decreased from 90.5 to 88.9%, but the specificity increased from 63.6 to 68.2%. Difficult cases had significantly less DS cells, weak intensity of p16 staining, suboptimal cell morphology and background staining compared to positive cases. CONCLUSION: Additional expert-led training and discussion of difficult cases are necessary for accurate interpretation of DS in laboratories involved in OSP. The most difficult cases were those with single stained cells and weak p16 staining. Training protocol for safe implementation of p16/Ki-67 DS in OSP is proposed.

Difficulties in diagnosing small round cell tumours of childhood from fine needle aspiration cytology samples.

There are four basic reasons for the difficulties in diagnosing small round cell tumours (SRCT) in childhood from fine needle aspiration cytology (FNAC) samples. First, SRCTs are rare and it is difficult for cytopathologists to obtain enough experience for rendering reliable diagnoses. Second, SRCTs are morphologically very similar. Third, many SRCTs do not have specific antigens which could be demonstrated with immunocytochemistry (ICC) or they lose them when poorly differentiated. In addition, cross reactivity exists between some SRCTs. Unstandardized performance of ICC also contributes to the difficulties due to unreliable results. Fourth, suboptimal FNAC samples add additional pitfalls. The latter may be due to partly degenerate samples or to unrepresentative ones in cases where a SRCT is a heterologous component of another nosological entity. Lymphoma, neuroblastoma, nephroblastoma, Ewing's tumour/primitive neuroendocrine tumours and rhabdomyosarcoma are discussed in detail, while other less common SRCTs are mentioned as differential diagnoses when appropriate. The use of cytogenetic and molecular techniques for differentiating between certain SRCTs is helpful in some doubtful cases. However, there are still problems in the use of these techniques, especially their cost which may delay their being introduced in every cytopathology laboratory.

Inconclusive flow cytometric surface light chain results; can cytoplasmic light chains, Bcl-2 expression and PCR clonality analysis improve accuracy of cytological diagnoses in B-cell lymphomas?

BACKGROUND: Flow cytometric immunophenotyping (FCI), is widely used in cytology for distinguishing between B-cell lymphoma (BCL) and reactive lymphocytic proliferations (RLP), mainly by identifying monotypic B-cell populations. Since this cannot always be determined by ratios of surface immunoglobulin light chains (sIg LCs) we wanted to assess if cytoplasmic immunoglobulin (cIg) LCs, Bcl-2 and polymerase chain reaction (PCR) based clonality analysis can improve accuracy of cytological diagnoses of BCL. METHODS: Our study included 98 fine needle aspiration biopsies from lymph nodes suspicious for BCL with inconclusive sIg LCs. In all cases PCR clonality analysis was performed in order to determine immunoglobulin heavy chain (IGH) gene and T-cell receptor (TRC) gene rearrangement. In selected cases expression of Bcl-2 and cIg LC were determined by FC. RESULTS: Thirty patients had lymphoma and 68 had reactive lymphocytic proliferations. Three patterns of sIg LCs staining were found: negative, dual positive and difficult to interpret. Percentage of lymphomas was highest in the dual positive group (75 %). Morphology coupled with cIg LCs determination and/or Bcl-2 expression was able to give a correct diagnosis in 83 % of cases. Molecular tests would have been misleading in 15 % of cases because 7/30 BCL were polyclonal and 8/68 RLP were monoclonal. CONCLUSIONS: Determination of cIg LCs, Bcl-2 expression and PCR clonality analysis of B cells improved accuracy of cytological diagnoses in BCL with inconclusive sIg LC. However, clonality determined by PCR was misleading in some cases.

Vegetable cells in urinary samples of patients with bricker ileal conduit.

During routine cytopathological evaluation of urines for malignant cells we have occasionally noticed vegetable cells that were only present in patients with Bricker ileal conduit. We wanted to identify the means and sources of contamination of urinary samples from these patients. During the period between May and November 2010, 637 urinary samples were routinely evaluated for malignant cells. Among them were 13 urinary samples from Bricker ileal conduit which we rescreened. We prepared all urinary samples by membrane filtration and stained them according to Papanicolaou. Subsequently, we prepared samples from ostomy adhesives made by Coloplast and by ConvaTec which are used to secure the ostomy bag onto urostomy. We also took samples from different constituents (hydrocolloids) of ostomy adhesives. On the cytopathological review, we found vegetable cells along with intestinal mucosa cells in urinary samples of seven patients with Bricker ileal conduit. With the light microscopic examination of the samples prepared from different ostomy adhesives, we found vegetable cells only in Coloplast adhesives. In preparations of hydrocolloids, we found vegetable cells only in guar gum. They were morphologically identical to those found in urine samples of patients with Bricker ileal conduit and in Sensura and Sensura Xpro (Coloplast) ostomy adhesives. We determined that the origin of vegetable cells in urines from Bricker ileal conduit is the ostomy adhesive. The vegetable cells differ from human intestinal epithelial cells regarding size, shape, and color so it is difficult to misinterpret them as dysplastic cells.

Topical topic: value of fine needle aspiration biopsy in childhood rhabdomyosarcoma: twenty-six years of experience in Slovenia.

BACKGROUND: Chemotherapy (Cht) for rhabdomyosarcoma (RMS) given before local treatment can prevent mutilating surgery and high-dose irradiation (RT). Fine needle aspiration biopsy (FNAB) can confirm the diagnosis and neoadjuvant treatment can start without delay. The purpose of our study was to assess the role of FNAB in the management of childhood RMS in Slovenia. PROCEDURE: A total of 78 children and young adults were included. FNAB provided the pre-treatment diagnosis in 37 and surgical biopsy in 41 patients. In 61 cases recurrent/metastatic disease was aspirated. Cytological diagnoses were compared to the original histological diagnoses. All case histories, cytological and histological material were reviewed and immunocytochemical staining performed when necessary. RESULTS: FNAB provided a correct diagnosis of malignancy in all 37 primary tumours, a specific diagnosis of RMS was given in 29 (78%). With the use of immunocytochemistry during the last 15 years, the accuracy has risen to 87%. FNAB provided the diagnosis of recurrence/metastasis in 57/61 cases. No complications of FNAB were noted. Review of histology reclassified five original diagnoses of RMS into one malignant rhabdoid tumour and four sarcomas NOS. In review of cytology we were able to sub classify 80% of RMS. CONCLUSIONS: FNAB is a safe method, which enables us to establish the pre-treatment diagnosis of RMS, and to some extent even its type, without delay. In our study, FNAB successfully replaced surgical biopsy in 87% of RMS patients during the last 15 years. Neoadjuvant Cht was started immediately, surgery was delayed and more conservative. Consequently, the risk for treatment sequelae was considerably reduced.

Infantile myofibroma in a prematurely born twin: a case report.

Infantile myofibromatosis is a rare benign tumor of infancy and childhood that occurs in solitary, multiple, and generalized forms with similar histology but different clinicopathologic and prognostic implications. Even solitary tumors need follow-up, as the type of presentation will be determined over time. It is necessary to differentiate this entity from other more aggressive tumors, especially rhabdomyosarcoma, which is treated by chemotherapy prior to excision. We describe a prematurely born twin girl who had at birth a solitary tumor of the cervicoscapular region, involving the dermis and subcutis. A fine-needle aspiration biopsy (FNAB) specimen obtained soon after her birth suggested a diagnosis of benign neoplasm. The tumor was excised 1 month later, at which time it was significantly enlarged, ulcerated, and also exhibited worrisome histologic features including mitoses and infiltrative growth. It had the characteristic histologic pattern of infantile myofibromatosis, and myofibroblastic features of tumor cells were confirmed immunohistochemically and ultrastructurally. During the follow-up period of 39 months, there was no sign of recurrence or new tumors.

DNA ploidy as a prognostic factor in rhabdomyosarcoma. Analysis of 35 cases with image cytometry.

OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies. STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS. Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry. DNA ploidy was assessed on the basis of DNA index values. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival. RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors. We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not. The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms. CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal. Larger, cooperative studies could give statistically more reliable results.

Value of image cytometry in the subclassification of rhabdomyosarcoma.

OBJECTIVE: To test the discriminatory capability of nuclear features in the subclassification of rhabdomyosarcoma (RMS) and especially to differentiate embryonal from alveolar RMS. STUDY DESIGN: The study included 42 patients with RMS. We performed the analysis on Feulgen-stained filtrates of cell suspensions prepared from deparaffinized tissue sections. Image analysis was performed by an automated, high-resolution image cytometer on at least 200 nuclear images. Photometric, morphometric and nuclear texture features were analyzed. Probability density distributions were calculated for each nuclear feature of individual RMS subgroups and compared in order to detect possible differences. RESULTS: There were significant differences between embryonal and alveolar RMS in five nuclear features: DNA index, sphericity, elongation, low_DNA_area and fractall_area. We were able to differentiate between the two main RMS subgroups in 82% of cases on the basis of either sphericity or elongation alone, while the power of differentiation for texture features was 72-79%. CONCLUSION: Differentiation between embryonal and alveolar RMS using one nuclear feature is not an important adjunct to light microscopy. However, the possibility of using a combination of nuclear features would probably increase the discriminatory ability.