Nomogram for predicting survival of patients with diffuse large B-cell lymphoma.

The international prognostic index (IPI) system has been widely used to predict prognosis in diffuse large B-cell lymphoma (DLBCL). However, this system categorizes DLBCL patients into four risk groups, and cannot optimize individualized prognosis. In addition, other clinicopathological factors, such as molecular aberrations, are not incorporated into the system. To partly overcome these weak points, we developed nomograms to predict individual patient survival. We also incorporated MYD88L265P and CD79BY196 mutations into the nomograms since these mutations are associated with a worse prognosis and their signaling pathways have been highlighted as a therapeutic target. We analyzed 302 DLBCL cases for which multivariate analysis by Cox proportional hazard regression was performed. Nomograms for progression-free survival (PFS) and overall survival (OS) were constructed and assessed by a concordance index (C-index). The nomograms were also evaluated using an open external dataset (n = 187). The MYD88L265P and/or CD79BY196 (MYD88/CD79B) mutation was detected in 62/302 patients. The nomograms incorporating IPI factors exhibited a C-index of 0.738 for PFS and a C-index of 0.765 for OS. The nomograms incorporating IPI factors and the MYD88/CD79B mutation showed a C-index of 0.745 for PFS and a C-index of 0.769 for OS. The nomograms we created were evaluated using an external dataset and were well validated. The present nomograms incorporating IPI factors and the MYD88/CD79B mutation have sufficient discrimination ability, and may effectively predict prognosis in DLBCL patients. The prognostic models we have presented here may help clinicians personalize prognostic assessments and clinical decisions.

Fluorescent periodic acid Schiff-like staining combined with standard cytologic staining of the same cytologic specimen may facilitate cytopathologic diagnosis.

BACKGROUND: Periodic acid Schiff (PAS) staining which detects glycogen and mucosubstances is frequently used as an ancillary method for an accurate cytopathologic diagnosis. Unfortunately, cytologic slides for PAS stain are not routinely prepared. Aqueous 7-amino-4-methylcoumarin (AMC) is colorless and transparent under bright field illumination but exhibits strong fluorescence under ultraviolet (UV) light and can be used as a Schiff reagent. We recently reported that combining [author: Please define (H&E) in the first occurrence if necessary.]H&E and AMC is useful for histopathologic diagnosis of various disease conditions. In this study, we investigated whether standard cytologic staining (Papanicolaou [Pap] and Giemsa) combined with AMC was useful for cytopathologic analysis. METHODS: Specimens of non-neoplastic human tissues and archived cytologic specimens of various disease conditions were stained with a combination of Pap and AMC (Pap/AMC) or Giemsa and AMC (Giemsa/AMC). RESULTS: The addition of AMC had no significant effect on Pap or Giemsa staining, and the cytomorphology under bright field microscopy was perfectly preserved. The AMC fluorescent signals observed under UV light were intense and the staining pattern was identical to that obtained by PAS staining. Diastase digestion differentiated glycogen from other AMC-positive elements. The efficacy of using Pap/AMC and Giemsa/AMC for archived cytologic specimens was demonstrated in several diseases including cases of endometrial carcinoma, adenoid cystic carcinoma, metastatic signet-ring cell carcinoma, candidiasis, and trichomoniasis. CONCLUSION: Pap/AMC and Giemsa/AMC are useful in aiding cytopathologic diagnosis especially when the information gained from PAS staining is critical and cytologic specimens for PAS are not available.

7-Amino-4-methylcoumarin as a fluorescent substitute for Schiff's reagent: a new method that can be combined with hemalum and eosin staining on the same tissue section.

An aqueous 7-amino-4-methylcoumarin (AMC) solution exhibits strong fluorescence under ultraviolet (UV) light and can be used as a Schiff reagent to visualize aldehydes. We investigated hemalum and eosin (H & E) and AMC staining for histological and pathological analysis. Sections of normal and lesioned human tissues were stained with combined H & E/AMC staining. After H & E/AMC staining, the H & E morphology was preserved under bright field microscopy. The AMC fluorescent signals observed under UV light were intense and the staining pattern was identical to that obtained by periodic acid-Schiff (PAS) staining. AMC staining of archived H & E sections also was successful. Diastase digestion differentiated glycogen from other AMC positive elements. Using H & E/AMC staining, mucus-rich adenocarcinoma cells, amebic trophozoites and fungal hyphae were visualized clearly under UV excitation. Using H & E/AMC staining, H & E and PAS-like histological imaging can be obtained using a single tissue section. H & E/AMC is useful for pathologic diagnosis especially when information from PAS staining is critical, the number of tissue sections is limited and/or the lesion in question is small.

Pleomorphic adenoma: detection of PLAG1 rearrangement-positive tumor components using whole-slide fluorescence in situ hybridization.

Pleomorphic adenoma (PA) consists of heterogeneous histological architecture mixed with epithelioid and mesenchymal forms. Various types of epithelial or myoepithelial malignancies arise from PA, but sarcomas are extremely rare. A human androgen receptor gene (HUMARA) clonality assay has suggested that PA is clonal in nature. However, clonality of various tumor components of PA would be difficult to determine with this assay. In addition, the results obtained should be carefully interpreted. PLAG1 rearrangements are considered a good molecular marker for neoplasticity in PA. We aimed to clarify the neoplasticity of the various tumor components present in PA using whole-slide fluorescence in situ hybridization (FISH). Five PA cases positive for PLAG1 rearrangements were examined. Using an immunohistochemistry panel, cell components in PA were classified into eight cell types. To precisely localize PLAG1 rearrangement-positive cell components at the cellular level, sequential retrieval of whole-slide imaging (WSI) data of HE histology and FISH for PLAG1 rearrangement was carried out. PLAG1 rearrangements were detected in ductal cells, myoepithelial spindle cells, myoepithelial oncocytic cells, myoepithelial plasmacytoid cells, and mesenchymal chondroid cells, but not in mesenchymal lipid cells, mesenchymal fibrous cells, or vascular endothelial cells. Immunohistochemical PLAG1 expression was restricted to cell components harboring PLAG1 rearrangements.The results of the present study indicate that ductal and myoepithelial, chondroid cells are neoplastic but lipid, fibrous, and endothelial cells are not. PLAG1 immunohistochemistry is useful in discriminating neoplastic from non-neoplastic cell components. These findings may be important for elucidating tumorigenesis and the process of malignant transformation in PA.

Immunohistochemistry for CCR4 C-terminus predicts CCR4 mutations and mogamulizumab efficacy in adult T-cell leukemia/lymphoma.

Mogamulizumab targets extracellular N-terminal domain of CCR4, which is expressed in most adult T-cell leukemia/lymphoma (ATL) cases. Recently, we reported that CCR4 C-terminal gain-of-function mutations were frequent in ATL cases, and a subgroup with these mutations who were treated without allogenic hematopoietic stem cell transplantation (HSCT) and with mogamulizumab-containing [HSCT (-) and mogamulizumab (+)] regimens had a superior survival rate. Although these mutations are most likely a biomarker for predicting a strong response to mogamulizumab, their detection is time-consuming and costly. A more convenient screening tool may be necessary in the clinical setting. In this study, the clinicopathological importance of immunohistochemistry for the CCR4 N-terminus (CCR4-N-IHC) and C-terminus (CCR4-C-IHC) was examined in a large ATL cohort (n = 92). We found that CCR4-C-IHC, but not CCR4-N-IHC, was inversely correlated with the CCR4 mutation status. In ATL patients negative for CCR4-C-IHC, a subgroup treated with HSCT (-) and mogamulizumab (+) regimens showed a significantly better prognosis. In addition, CCR4-C-IHC was found to be a useful marker for high-sensitivity screening of the CCR4 mutational status (87%). The present study suggests that CCR4-C-IHC may be useful for identifying ATL patients harboring mutated CCR4 who may benefit from the superior efficacy of mogamulizumab-containing regimens and that CCR4-C-IHC may be a rapid and cost-efficient tool for screening for CCR4 mutation status.

Next-generation sequencing assay in salivary gland cytology: A pilot study.

BACKGROUND: Preoperative diagnosis of salivary gland tumors (SGTs) by fine-needle aspiration (FNA) cytology is challenging. Next-generation sequencing (NGS)-based assays for somatic mutations have a great advantage in that a large number of genes can be analyzed simultaneously. Although NGS may have an enormous diagnostic potential in cytology, to our knowledge, the significance of NGS in SGT cytology remains to be clarified. METHODS: In this pilot study, we retrospectively examined 32 frozen SGT samples obtained at surgery (14 malignant and 18 benign). After the stored frozen tumor tissues were thawed, aspirate samples were obtained using 22-gauge needles and subjected to smear tumor samples and to DNA extraction for an NGS assay employing the Illumina AmpliSeq Cancer Hotspot Panel v2. The results were correlated to preoperative cytological diagnosis. RESULTS: The preoperative diagnoses obtained by FNA cytology included 23 negative lesions (no malignancy in 6 and benign tumor in 17) and nine positive lesions (suspicious for malignancy in 4 and malignancy in five), providing a sensitivity and a specificity of 9/14 (64%) and 18/18 (100%), respectively. The NGS assay detected somatic mutations in 10/14 malignant and 1/18 benign SGT cases, providing a sensitivity and a specificity of 71% and 94%, respectively. CONCLUSION: The NGS assay may be helpful for detecting the malignant potential in SGT cases and can be used as an ancillary test for SGT cytology.

Plasma cell myeloma positive for t(14;20) with relapse in the central nervous system.

t(14;20)(q32;q11)/IGH-MAFB is a rare chromosomal abnormality in plasma cell myeloma (PCM), accounting for 1-2% of PCM cases. Patients with this translocation may have a poor prognosis. However, the clinicopathological features and response to novel agents have not been well clarified. We present a 63-year-old Japanese female with PCM positive for t(14;20). The tumor responded well to a proteasome inhibitor, bortezomib, and the patient achieved complete remission. Six months after remission, tumor relapse was noted in the left cerebellum and the right frontal lobe of the cerebrum. After whole brain radiation therapy, the tumor masses decreased in size. The patient was followed up with best-care support, but died of the disease 29 months after the initial PCM diagnosis. t(14;20)-positive PCM responded well to bortezomib at the time of the initial treatment. The CNS tumor involvement, which is rare in PCM, may be associated with the clinical aggressiveness of the t(14;20)-positive form of this myeloma.

CCR4 is rarely expressed in CCR4-mutated T/NK-cell lymphomas other than adult T-cell leukemia/lymphoma.

CCR4 is expressed on tumor cells of most patients with adult T-cell leukemia/lymphoma (ATL). Gain-of-function mutations of the CCR4 gene in ATL patients may be associated with alterations at the carboxyl terminus, a finding which led to a high efficacy anti-CCR4 antibody, mogamulizumab. Only a few studies have reported CCR4 protein expression and genomic CCR4 mutations in non-ATL T/NK-cell lymphomas. Furthermore, an association between CCR4 protein expression, genomic CCR4 mutations, and transcript CCR4 mutations has not been well analyzed. The T/NK-cell lymphomas (n = 226) enrolled in this study were examined for CCR4 expression by immunohistochemistry. CCR4 mutations in the codons 322-348 were detected by direct sequencing and a SNaPshot Multiplex assay. CCR4 protein expression was positive in 48/52 (92%) and 58/174 (33%) of ATL and non-ATL cases, respectively, and genomic CCR4 mutations were detected in 17/52 (33%) and 6/174 (3.4%), respectively. While all 17 ATL cases with genomic CCR4 mutations were positive for CCR4 protein expression, five of six mutated non-ATL cases were negative for CCR4 protein expression and transcript CCR4 mutations. This study suggests that frequencies of CCR4 expression and genomic CCR4 mutations and an association between the two may be considerably different between ATL cases and non-ATL T/NK-cell lymphomas.

Immunohistochemistry for identification of CCND1, NSD2, and MAF gene rearrangements in plasma cell myeloma.

The t(11;14)/CCND1-IGH, t(4;14)/NSD2(MMSET)-IGH, and t(14;16)/IGH-MAF gene rearrangements detected by fluorescence in situ hybridization (FISH) are used for risk stratification in patients with multiple myeloma (MM). Compared with conventional FISH techniques using fresh cells, immunohistochemistry (IHC) is much more cost- and time-efficient, and can be readily applied to routinely prepared formalin-fixed, paraffin-embedded (FFPE) materials. In this study, we performed tissue FISH and IHC employing FFPE specimens, and examined the usefulness of IHC as a tool for detecting CCND1, NSD2, and MAF gene rearrangements. CD138 signals were used to identify plasma cells in tissue FISH and IHC analyses. With cohort 1 (n = 70), we performed tissue FISH and subsequently IHC, and determined IHC cut-off points. In this cohort, the sensitivity and specificity for the 3 molecules were ≥.90 and ≥.96, respectively. With cohort 2, using MM cases with an unknown gene status (n = 120), we performed IHC, and the gene status was estimated using the cut-off points determined with cohort 1. The subsequent FISH analysis showed that the sensitivity and specificity for the 3 molecules were ≥.92 and ≥.98, respectively. CCND1, NSD2, and MAF gene rearrangements were estimated accurately by IHC, suggesting that conventional FISH assays can be replaced by IHC.

Cellular-level characterization of B cells infiltrating pulmonary MALT lymphoma tissues.

Mucosa-associated lymphoid tissue (MALT) lymphoma mainly consists of three types of tumor B cells, small (centrocyte-like), scattered large transformed, and intraepithelial. However, it is difficult to differentiate tumor B cells from reactive B cells at the cellular level. We examined five cases of API2-MALT1 fusion-positive MALT lymphoma of the lung. A single paraffin section for each case was subjected to sequential retrieval of whole-slide imaging (WSI) data of hematoxylin and eosin (HE) staining, immunofluorescence staining for CD79a, and fluorescence in situ hybridization (FISH) for the MALT1 split. We counted the number of MALT1 split-positive or MALT1 split-negative cells among CD79a-positive cells. The MALT1 split was detected in 59, 46, and 76 % of small, large, and intraepithelial B cells, respectively. A review of the HE-WSI data showed that cytomorphological distinction between the MALT1 split-positive and MALT1 split-negative B cells was virtually impossible. None of CD79a-negative lymphoid cells, epithelial cells, and microvascular endothelial cells was positive for MALT1 splits. As API2-MALT1 fusion is an early and critical event in the lymphomatogenesis, our findings are best interpreted as that a considerable number of B cells, either small, large, or intraepithelial, are reactive cells and that it is difficult to distinguish cytomorphologically between tumor B cells and reactive B cells. These findings suggest that the tumor architecture may be the central factor for making a correct histopathological diagnosis of MALT lymphoma. The sequential WSI of HE staining, immunofluorescence staining, and FISH as described here is a useful tool for pathological analysis at the cellular level.

Quantitative PCR for HTLV-1 provirus in adult T-cell leukemia/lymphoma using paraffin tumor sections.

Detection of HTLV-1 provirus using paraffin tumor sections may assist the diagnosis of adult T-cell leukemia/lymphoma (ATLL). For the detection, non-quantitative PCR assay has been reported, but its usefulness and limitations remain unclear. To our knowledge, quantitative PCR assay using paraffin tumor sections has not been reported. Using paraffin sections from ATLLs and non-ATLL T-cell lymphomas, we first performed non-quantitative PCR for HTLV-1 provirus. Next, we determined tumor ratios and carried out quantitative PCR to obtain provirus copy numbers. The results were analyzed with a simple regression model and a novel criterion, cut-off using 95 % rejection limits. Our quantitative PCR assay showed an excellent association between tumor ratios and the copy numbers (r = 0.89, P < 0.0001). The 95 % rejection limits provided a statistical basis for the range for the determination of HTLV-1 involvement. Its application suggested that results of non-quantitative PCR assay should be interpreted very carefully and that our quantitative PCR assay is useful to estimate the status of HTLV-1 involvement in the tumor cases. In conclusion, our quantitative PCR assay using paraffin tumor sections may be useful for the screening of ATLL cases, especially in HTLV-1 non-endemic areas where easy access to serological testing for HTLV-1 infection is limited.