Immunohistochemical algorithms and gene expression profiling in primary cutaneous B-cell lymphoma.

OBJECTIVE: to assess whether immunohistochemical (IHC) algorithms used to classify the cell of origin (COO) of nodal Diffuse Large B-cell lymphoma (nDLBCL) in Germinal Center type (GCB) and non-GCB subtypes may be applied to Primary Cutaneous B-cell lymphoma (PCBCL) too, and which of these algorithms performs better on PCBCL. DESIGN: Retrospective case control study. SETTING: Pathology Department of the University Hospital "San Giovanni di Dio e Ruggi d'Aragona" Salerno, Italy. PARTICIPANTS: Fourteen PCBCL, including Primary Cutaneous follicle centre lymphoma (PCFCL) and primary cutaneous diffuse large B-cell lymphoma, Leg type (PCDLBCL-LT) and 14 nDLBCL were evaluated for 7-year period (January 2011 to December 2017). Primary cutaneous marginal zone cell lymphoma (PCMZL) cases were not included in the present study. INTERVENTION: Evaluation of immunohistochemical CD10, BCL6, MUM1/IRF4, BCL2, MYC and Ki-67 expression and classification according to three different algorithms. Gene expression profiling (GEP) was performed on the same series using Lymph2Cx assay (Nanostring). The data obtained were compared and analysed. RESULTS: All the IHC algorithms showed 13 GCB and 15 non-GCB. GEP showed 12 GCB, 12 activated B cell-type and 4 unclassified. CONCLUSIONS: The PCBCL were classifiable as GCB and non-GCB like the nDLBCL as IHC algorithms were concordant to GEP and produced the same results.

Peritumoral Immune Infiltrate as a Prognostic Biomarker in Thin Melanoma.

Thin melanomas are tumors less than 1 mm thick according to Breslow classification. Their prognosis is in most cases excellent. However, a small subset of these tumors relapses. These clinical findings emphasize the need of novel prognostic biomarkers to identify this subset of tumors. Characterization of tumor immune microenvironment (TIME) is currently investigated as a prognostic and predictive biomarker for cancer immunotherapy in several solid tumors including melanoma. Here, taking into account the limited availability of tumor tissues, by characterizing some of the characteristics of TIME such as number of infiltrating lymphocytes, HLA class I antigen and PD-L1 expression, we show that number of infiltrating CD8+ and FOXP3+ T cells as well as CD8+/FOXP3+ T cell ratio can represent a useful prognostic biomarker in thin melanoma. Although further investigations in a larger patient cohort are needed, these findings have potential clinical significance since they can be used to define subgroups of thin melanoma patients who have a worse prognosis and might need different treatment modalities.

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry.

Lymph node (LN) fine-needle cytology (FNC) coupled with flow cytometry immunophenotyping provides relevant information for the diagnosis of non-Hodgkin lymphoma (NHL). Numerous studies have shown FNC samples to be suitable for different molecular procedures; in this review, some of the molecular procedures most commonly employed for NHL are briefly described and evaluated in this perspective. Fluorescence in situ hybridization and chromogenic in situ hybridization are briefly described. Polymerase chain reaction (PCR)-based assays are used to identify and quantify mutations and translocations, namely immunoglobulin (IGH) and T-cell receptor rearrangements by clonality testing and IGVH somatic hypermutations either by Sanger sequencing, single-strand conformational polymorphisms or RT-PCR strategies. High-throughput technologies (HTT) encompass numerous and different diagnostic tools that share the capacity of multiple molecular investigation and sample processing in a fast and reproducible manner. HTT includes gene expression profiling, comparative genomic hybridization, single-nucleotide polymorphism arrays and next-generation sequencing technologies. A brief description of these tools and their potential application to LN FNC is reported. The challenge for FNC will be to achieve new knowledge and apply new technologies to FNC, exploiting its own basic qualities.

The diagnostic accuracy of fine-needle cytology of Hurthle cell lesions; A comprehensive cytological, clinical and ultrasonographic experience.

BACKGROUND: Fine-needle cytology (FNC) diagnosis and pre-operative classification of Hurthle cells (HC) lesions may be difficult. Rapid on-site evaluation (ROSE) enhances the efficiency of FNC, mainly when utilized in close combination to clinical and ultrasound (US) data. OBJECTIVE: to describe an experience on HC FNC with contextual clinical,US and ROSE evaluation and assess if this comprehensive approach improves the FNC accuracy of HC lesions. METHODS: FNC of 112 HC lesion were diagnosed and classified, according to the Bethesda system, by clinical, US and ROSE in one year. All the cases were controlled by follow-up and histology when performed. RESULTS: Eighty-five cases were diagnosed HC rich goiter or Hashimoto thyroiditis and were classified THY2; 5 cases were diagnosed hyperplastic nodular goiter and classified THY3A. Eight cases were diagnosed suspect neoplasia and classified THY3B. Two cases were diagnosed suspect HC papillary thyroid carcinoma (PTC) and classified THY4 and 2 cases were diagnosed HC-PTC and classified THY5. THY3B, THY4, THY5 and 1 THY3A were histologically controlled. FNC were confirmed in 14 out of the 17 THY3-THY5 cases. CONCLUSIONS: A comprehensive diagnostic approach that include FNC, clinical data, US and ROSE improves the diagnosis and classification of HC lesions.

Use of FTA cards for the storage of breast carcinoma nucleic acid on fine-needle aspiration samples.

BACKGROUND: The preservation and storage of nucleic acids is important for DNA molecular techniques. The material obtained by fine-needle aspiration (FNA) is often scanty and can not be wasted. FTA cards are filter papers that immobilize and stabilize nucleic acids and can be stored at room temperature. The current study evaluated whether nucleic acids of breast carcinoma cells, obtained by FNA in a clinical setting, may be collected, stored, and preserved on FTA cards. METHODS: Thirty breast carcinoma, 5 non-Hodgkin lymphoma (NHL), and 5 benign reactive lymph node (RLN) cell samples obtained by FNA were stored at -80 °C and on FTA cards. DNA extraction and polymerase chain reaction were performed on cells at -80 °C and on 2 punched disks of FTA cards. Fifty nanograms of extracted DNA from both sample types were used to amplify the Janus Kinase 2 (JAK2) gene. RESULTS: The mean value of DNA extracted from breast carcinoma cells was 28.19 ng/µL for that stored at -80 °C and 3.28 ng/µL for that stored on FTA cards. Agarose gel analysis demonstrated expected bands of DNA in 29 cases (97%) with both methods. The mean value of DNA extracted from NHL and RLN samples was 37.54 ng/µL and 4.28 ng/µL, respectively, and agarose gel analysis demonstrated bands of high molecular weight DNA in both methods. Significant differences in DNA yield were found between storage at -80 °C and FTA cards (P<.0001), but no differences were detected between 260/280 nm ratios in breast carcinoma and NHL/RLN samples. CONCLUSIONS: FTA cards can be conveniently used for the storage of breast carcinoma cells obtained by FNA, thus providing a reliable alternative to traditional methods.

Lymph node fine needle cytology in the staging and follow-up of cutaneous lymphomas.

BACKGROUND: Lymph nodal involvement is an important clinical-pathological sign in primary cutaneous lymphoma (PCL), as it marks the transformation/evolution of the disease from localized to systemic; therefore the surveillance of lymph nodes is important in the staging and follow up of PCL. Fine needle cytology (FNC) is widely used in the diagnosis of lymphadenopathies but has rarely been reported in PCL staging and follow-up. In this study an experience on reactive and neoplastic lymphadenopathies arisen in PCL and investigated by FNC, combined to ancillary techniques, is reported. METHODS: Twenty-one lymph node FNC from as many PCL patients were retrieved; 17 patients had mycosis fungoides (MF) and 4 a primary cutaneous B-cell lymphoma (PBL). In all cases, rapid on site evaluation (ROSE) was performed and additional passes were used to perform flow cytometry (FC), immunocytochemistry (ICC) and/or polymerase chain reaction (PCR) to assess or rule out a possible clonality of the corresponding cell populations. RESULTS: FNC combined with FC, ICC, and PCR identified 12 cases of reactive, non specific, hyperplasia (BRH), 4 dermatopathic lymphadenopathy (DL), 4 lymph nodal involvement by MF and 1 lymph nodal involvement by cutaneous B-cell lymphoma. CONCLUSIONS: FNC coupled with ancillary techniques is an effective tool to evaluate lymph node status in PCL patients, provided that ROSE and a rational usage of ancillary techniques is performed according to the clinical context and the available material. The method can be reasonably used as first line procedure in PCL staging and follow up, avoiding expensive and often ill tolerated biopsies when not strictly needed.

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia.

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Cytologic, flow cytometry, and molecular assessment of lymphoid infiltrate in fine-needle cytology samples of Hashimoto thyroiditis.

BACKGROUND: The thyroidal lymphoid infiltrate (TLI) in Hashimoto thyroiditis (HT) represents the substrate from which thyroid lymphoma may arise. The objective of the current study was to classify the TLI in HT by comparing the cytologic features with flow cytometry (FC) data and evaluating the kappa/lambda light chain ratio and its molecular assessment. METHODS: Fine-needle aspiration cytology (FNAC) was performed in 34 patients with HT with nodular or diffuse palpable enlargement of the gland. Two or 3 passes were performed to prepare traditional smears, FC, and immunophenotyping, and RNAlater suspensions for molecular assessment. FC was performed using the following antibodies: CD3, CD5, CD4, CD8, CD10, CD19, and kappa and lambda light chains. In 4 cases, high molecular weight DNA was extracted and used for polymerase chain reaction (PCR) to amplify the variable diversity joining region of the heavy chain immunoglobulin (Ig) genes (IgH). Statistical analysis was performed to evaluate possible associations between clinical ultrasound presentation, cytologic pattern, and TLI phenotype. Light chain expression was evaluated as the percentage of the expressing cells (20%) and as the kappa/lambda ratio. RESULTS: Smears were classified as "lymphocytic," "lymph node-like," or "mixed." FC demonstrated T cells (CD3 positive [+], CD5+) in all cases, and T cells and B cell (CD19+, CD10+/-) lymphocytes in 22 cases. Light chains were expressed in 30 cases (in <20% of the gated cells in 13 cases and in >20% of the gated cells in 17 cases). Five cases demonstrated small kappa/lambda ratio imbalances and PCR analysis demonstrated diffuse bands in the gel and Gaussian curves at the heteroduplex. Statistical analysis indicated significant associations between the "lymphocytic" pattern and T-cell phenotype and between the "lymph node-like" pattern and B-cell phenotype. A significant association also was observed between light chain restriction and low light chain expression (P < .005). CONCLUSIONS: The cytologic pattern of TLI in HT is quite representative of the clinical presentation and phenotypic cell type. Small light chain imbalances are not sustained by heavy chain Ig gene (IgH) rearrangements. FNA coupled with FC may contribute to making the distinction between florid TLI and non-Hodgkin lymphoma.