Combining ALK Fluorescent In Situ Hybridization and Immunohistochemistry to Analyze Multiple Non–Small Cell Lung Carcinoma Samples per Patient Reveals Intermethod and Intersample-discrepant Results ALK inhibitors have improved the therapeutic management of patients with ALK-rearranged advanced non–small cell lung cancers (NSCLC). Several diagnostic methods, mainly fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), can be used as single or combined tests to detect the so-called “ALK-positive” NSCLC. Intersample and intermethod discrepancies could cause issues with therapeutic consequences in ALK testing. In this article, we report a case series of our real-life experience and issues in combining FISH and IHC in multiple tumor samples per patient to diagnose and treat a subset of “ALK-positive” patients with NSCLC. Among analyses conducted in 40 samples of 18 patients with advanced lung adenocarcinomas, we retrospectively encountered 10 patients with intersample-concordant ALK FISH and IHC results. Discrepant results about FISH and/or IHC were noted between different samples in 8 patients. Therapeutic responses were observed in 5 of 10 crizotinib-treated patients including 1 patient with ALK FISH+ IHC− status and 1 patient with ALK FISH− IHC+ status. Our data highlight the difficulty to predict the response/nonresponse to crizotinib therapy, in patients with advanced NSCLC, not only on the basis of single and multiple tumor samples, but also on the basis of single and combined diagnostic methods.

Lymphocyte-to-Monocyte Ratio May Serve as a Better Prognostic Indicator Than Tumor-associated Macrophages in DLBCL Treated With Rituximab There are multiple prognostic indicators for diffuse large B-cell lymphoma (DLBCL) including the international prognostic index (IPI), and gene expression profiling (GEP) to classify the disease into germinal center B-cell and activated B-cell subtypes, the latter harboring inferior prognosis. More recently, tumor-associated macrophages (TAM) and lymphocyte-to-monocyte ratio (LMR) were found to have prognostic implications in DLBCL. However, consensus is yet to be reached in terms of the significance of each. In this study, we evaluated the prognostic value of TAM as assessed by CD163 or CD68 positivity by immunohistochemistry on tissue biopsies and LMR was calculated from peripheral blood differential, with focus on the inclusion of rituximab as a treatment modality. The number of CD68-positive cells in the tumor microenvironment did not exhibit significant prognostic value, whereas higher number of CD163-positive cells was associated with inferior overall survival in patients treated with chemotherapy alone. This effect was no longer evident in patients treated with rituximab containing chemoimmunotherapy. In contrast, the prognostic significance of LMR on survival was more persistent regardless of treatment. There was no association between LMR and the number of CD163-positive cells. Our results suggest that LMR is the more easily and widely available prognostic marker in this era of chemoimmunotherapy. Our finding supports previous literature that the effect of TAM can vary according to treatment. Interaction between rituximab and TAM warrant further scientific investigation for mechanistic insights into targeted therapeutics.

Immunohistochemical Detection of γ/δ T Lymphocytes in Formalin-fixed Paraffin-embedded Tissues T lymphocytes can be distinguished based on the composition of the T-cell receptor (TCR) chain in α/β T cells and γ/δ T cells. Correspondingly, α/β lymphomas can be distinguished from γ/δ lymphomas. The latter are rare neoplasms, which are usually confined to particular organs and tissues and carry a dismal prognosis. Until recently, monoclonal antibody (mAb) clone g3.20 to the TCR γ-chain was the reagent of choice for the immunohistochemical detection of γ/δ T cells and lymphomas in standard formalin-fixed paraffin-embedded tissues. However, due to technical problems, mAb g3.20 became recently unavailable. Our attempts to identify another commercially available clone to the TCR γ-chain were unsuccessful. However, we were able to identify a mAb (clone H-41, SC-100289; Santa Cruz, Dallas, TX) to the TCR δ-chain. H-41 works well in immunohistochemistry on paraffin-embedded tissue and comparison with previously stained cases, shows superior immunolabeling to mAb g3.20. H-41 to the TCR δ-chain appears to be a suitable reagent for the replacement of mAb g3.20.

Utility of Ber-EP4 and MOC-31 in Basaloid Skin Tumor Detection Ber-EP4 has been the traditional immunostain used for the detection of basaloid skin tumors. Recently, MOC-31 has shown be superior to Ber-EP4 in the detection of basosquamous basal cell carcinoma (BCC) and many centers are now using both Ber-EP4 and MOC-31 antibodies together to detect these lesions. The objective of this study was to compare the utility of using both Ber-EP4 and MOC-31 immunostains in the detection of basaloid skin tumors and to better characterize the previously unknown staining properties of MOC-31 in cutaneous lesions. To do this, 76 basaloid skin tumors stained with both Ber-EP4 and MOC-31 were obtained. Diagnoses included basosquamous BCC, Merkel cell carcinoma, adenoid cystic carcinoma, microcystic adnexal carcinoma, sebaceous carcinoma, trichoepithelioma, trichoblastoma, sebaceous adenoma, sebaceoma, and follicular induction overlying dermatofibroma. The distribution and intensity of Ber-EP4 and MOC-31 staining in these lesions was scored. These scores were analyzed using a truth table, χ2 test, and Pearson correlation tests. The overall mean and SD of the scores were also obtained. Overall, we found Ber-EP4 and MOC-31 to be statistically equivalent immunostains for the diagnosis of basaloid skin tumors. We recommend the use of only one of these antibodies and favor MOC-31 for the detection of basaloid skin tumors. We also describe MOC-31 staining properties in different cutaneous lesions.

Genetic Alterations of Epidermal Growth Factor Receptor in Glioblastoma: The Usefulness of Immunohistochemistry Epidermal growth factor receptor (EGFR) amplification is one of the common alterations in IDH-wildtype glioblastoma. It is frequently associated with EGFRvIII mutation. To evaluate the correlation between EGFR overexpression, gene amplification, and EGFRvIII mutation, we performed immunohistochemical (IHC) analysis, fluorescence in situ hybridization by Vysis LSI EGFR/CEP7 dual color probe, and polymerase chain reaction studies in 76 patients diagnosed with glioblastomas (67 IDH-wildtype and 9 IDH-mutant). EGFR expression was scored ranging from 0 to 3+. Using formalin-fixed paraffin-embedded sections, real-time reverse transcription-polymerase chain reaction was carried out with primers specific for EGFRvIII and EGFR wildtype. In addition, we evaluated the impact of EGFR status on prognosis. EGFR gene amplifications and EGFRvIII mutations were identified in 30.3% and 15.5% of all cases, respectively. All the EGFR-amplified or EGFRvIII mutant cases were IDH-wildtype glioblastomas and tested positive with IHC. The sensitivity and specificity of EGFR IHC predicting EGFR gene amplification status were 100.0% and 46.5%, respectively. The EGFR-amplified cases tended to show more intense immunostaining (3+) in a considerable number of tumor cells (≥50%). Survival analyses of 37 IDH-wildtype glioblastoma patients revealed that none of the EGFR alterations significantly affected prognosis. EGFR IHC displayed high sensitivity and low specificity in predicting EGFR gene amplification, and interpretation of IHC results is a challenge. Therefore, EGFR IHC represents a possible screening tool for evaluation of EGFR gene amplification in clinical neuropathology, and both the intensity and proportion score facilitate interpretation of EGFR IHC.

APOBEC3B High Expression in Gastroenteropancreatic Neuroendocrine Neoplasms and Association With Lymph Metastasis Purpose: Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3B (APOBEC3B) is known as a source of mutations in multiple cancers. Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a group of heterogeneous tumors. However, the expression and significance of APOBEC3B in GEP-NENs remains unclear. Materials and Methods: A total of 158 cases of GEP-NENs, including 78 cases of biopsy or endoscopic submucosal dissection resection specimens and 83 cases of surgical resection specimens were collected in this study. The cases were grouped according to tumor classification grade, including 42 cases of neuroendocrine tumors G1 (NET G1), 36 cases of NET G2, 36 cases of NET G3, 44 cases of neuroendocrine carcinoma (NEC). All of the 158 tumors were immunohistochemically studied using a polyclonal antibody against APOBEC3B. We evaluated APOBEC3B expression in GEP-NENs and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathologic features, such as age, sex, tumor site, Ki67 cell proliferation index, and lymph metastasis. Results: A total of 33 cases (78.6%) of NET G1 showed high expression of APOBEC3B. A total of 28 cases (77.8%) of NET G2 demonstrated high expression of APOBEC3B. In NET G3 and NEC cases, the positive rates were 52.8% and 2.3%, respectively. The expression of APOBEC3B in NETs was significantly higher than that in NECs, NET G1 and NET G2 were higher than NET G3, and the difference was statistically significant. APOBEC3B high expression cases have lower lymph node metastasis rate, lower Ki67 cell proliferation index. Conclusions: In this study, APOBEC3B is highly expressed in GEP-NETs and is a predictor of lymph node metastasis in NET G3 and NEC cases. These findings might provide new insights into the biological mechanisms of GEP-NENs tumorigenesis and progression.

The Role of CD44 and RHAMM in Endometrial (Endometrioid Type) Cancer: An Immunohistochemical Study Hyaluronan controls cell migration, differentiation, and proliferation, and it is involved in tumor invasion. The extracellular matrix containing hyaluronan regulates cell behavior via cell surface receptors such as CD44 and receptor for hyaluronan-mediated motility (RHAMM, CD168). We investigated the expression of CD44 and RHAMM in tissue samples of endometrial cancer and the relation of their expression with clinicopathologic parameters of patients. In order to evaluate the value of CD44 and RHAMM as prognostic factors, we investigated the relation of their expression with patients’ survival. Our results demonstrated a statistically significant correlation with the depth of myometrial invasion, lymphovascular invasion (LVSI), The International Federation of Gynecology and Obstetrics stage of disease, and, in the case of RHAMM expression, a significant correlation with histologic tumor grade as well. CD44 expression was present in the cell membrane in all cases, but in a proportion of tumors in the cytoplasm as well. In this group of patients, we noticed a significantly greater number of cases with deeper myometrial invasion and LVSI. Finally, we sorted out the group of tumors with simultaneous strong CD44 and strong RHAMM expression, and found a statistically significant correlation with the depth of myometrial invasion and LVSI. Using an univariate analysis, we demonstrated that, in our sample of patients, CD44 expression showed a statistically significant influence on patients’ 5-year survival. However, using a multivariate Cox regression analysis, neither CD44 nor RHAMM confirmed themselves as independent prognostic factors.

Endoglin is Highly Expressed in Human Mast Cells Endoglin, known to be expressed in proliferating vessels, is of worth when evaluating microvessel density as a prognostic factor in many types of malignancies, including some subtypes of leukemia cells. In childhood acute lymphoblastic leukemia, endoglin is associated with adverse outcome. In bone marrow, endoglin identifies the repopulating hematopoietic stem cells. Mast cells are a component of normal tissue and play an important role in the regulation of several processes, including inflammation and neoplasia. The aim of this study was to evaluate the use of endoglin as a biological marker of mast cells compared with the gold standard stains. We studied 15 specimens of neurofibroma, 9 of mastocytosis, and 6 of fibrous scar tissue through immunohistochemistry (for endoglin and mast cell tryptase) and histochemical staining using toluidine blue. Quantitative analysis of the cells was performed by counting 5 hotspots. The validity of endoglin as a mast cell marker was assessed by intraclass correlation coefficient. The Kruskal-Wallis test was used to compare mast cell count for each marker. A strong endoglin expression was found in the cytoplasmic granules of mast cells within the 3 groups. Similar results were observed with mast cell tryptase as well as toluidine blue. The intraclass correlation coefficient revealed that endoglin is a highly reliable biomarker of mast cells when compared with mast cell tryptase and toluidine blue. In conclusion, endoglin may assist in the diagnosis and pathogenesis study of various processes associated with mast cells. An endoglin-neutralizing treatment for solid cancers and leukemia could also affect mastocytes and the immunologic system.

The Expression of TMEM74 in Liver Cancer and Lung Cancer Correlating With Survival Outcomes Transmembrane 74 (TMEM74), a transmembrane protein as an autophagy inducer, has been proven to promote tumor cell (including cervical cancer cell line HeLa and hepatic carcinoma cell line HepG2) proliferation by triggering autophagy. To further determine the role of TMEM74 in cancer, we performed immunohistochemical staining on tissue array, and the results showed that TMEM74 exhibited significantly higher expression in several tumor types, especially in hepatocellular carcinoma, lung adenocarcinoma, and squamous carcinoma. Furthermore, higher expression level of TMEM74 in HepG2, A549, and H1299 cell lines were also detected compared with the corresponding normal cell lines, as detected by western blot. Meanwhile, further analysis showed that the levels of TMEM74 expression were closely correlated to survival period of patients—the higher expression of TMEM74 was correlated with shorter survival period. Moreover, the in vitro experiments showed that overexpression of TMEM74 led to accelerated proliferation of A549 and H1299 cells, while knockdown of TMEM74 reversed the outcomes. In conclusion, the results suggested that TMEM74 acts as an oncogene and a potential diagnostic marker and a therapeutic target for liver cancer and lung cancer.

Are Ski and SnoN Involved in the Tumorigenesis of Oral Squamous Cell Carcinoma Through Smad4? Transforming growth factor-β has been implicated in the tumorigenesis of oral squamous cell carcinoma (OSCC). Ski and SnoN are negative regulators of transforming growth factor-β/Smad pathway with both prooncogenic and antioncogenic functions in different cancers. The aim of this study was to assess the expression of Ski and SnoN in OSCC for the first time. Smad4 was also evaluated in these tumors. Clinical data on 61 primary OSCCs were gathered, and the specimens were subjected to immunohistochemical staining with monoclonal antibodies against SKI, SnoN, and Smad4 and scored semiquantitatively. Spearman rank, Fisher exact, and χ2 tests were used for statistical analysis, and P-value <0.05 was considered significant. Ski positivity and SnoN positivity were mostly cytoplasmic and found in 96.7% and 100% of the cases, respectively. Smad4 staining was low to negative in 65% of the specimens. No significant relationship was found either among the markers or between each of the proteins and the clinicopathologic data (P>0.05). According to our findings, Ski, SnoN, and Smad4 seem to play a role in OSCC oncogenesis, and we suggest that Ski and SnoN functions may take place independent of Smad4. Considering the dual and complex role of these proteins in tumorigenesis, further investigation to clarify the molecular pathways involved in their mode of action is suggested.