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The current study examined demographic, psychosocial, and substance use factors associated with distinct patterns of past 12-month suicide thoughts, plans, and attempts among adolescents drawn from a nationally representative sample of high schoolers. Data were from the 2015, 2017, and 2019 National Youth Risk Behavior Survey. Four mutually exclusive 12-month suicidal behavior patterns were identified: suicide thoughts only (pattern 1), suicide thoughts and plans without suicide attempt (pattern 2), suicide attempt with thoughts and/or plans (pattern 3), and suicide attempt without thoughts or plans (pattern 4). Multinomial logistic regression analyses were conducted to examine factors correlated with these distinct patterns. Psychosocial and substance use factors were modeled as independent predictors, controlling for demographic characteristics, as well as simultaneously to represent the potential for co-occurrence. The analytic sample included 7491 respondents. About 24% (n = 1734) of youth endorsed pattern 1, 38% (n = 2779) pattern 2, 35% (n = 2716) pattern 3, and 3% (n = 262) pattern 4. All psychosocial and substance use factors measured were individually associated with greater odds of suicide attempts with thoughts or plans (pattern 3) than patterns 1 or 2. Black and male youth were at greater odds of suicide attempts without thoughts or plans (pattern 4) than all other patterns. When modeled simultaneously, respondents who were bullied online, sad or hopeless, had a history of sexual violence, used cigarettes, and misused prescription opiates retained greater odds of suicide attempts with thoughts or plans (pattern 3) than patterns 1 or 2. Findings suggest screening for suicidal behaviors should include factors that differentiate between varying suicidal expressions and that may cue providers to intervene in the absence of suicide thoughts and plans., (© 2021. Society for Prevention Research.)

Context: End-stage renal disease (ESRD) is more common among Blacks, but Blacks are less likely to receive a live donor kidney transplant (LDKT)., Objective: The objective of this study is to identify barriers and coping mechanisms that Black LDKT recipients and donors experienced while receiving or donating a kidney., Design: A qualitative study was conducted using structured interviews. Thematic analysis was used for data interpretation., Participants: All 20 participants identified as Black, with two participants identifying themselves as multiracial. The mean age for the 14 recipients was 60, and the average age for the 6 living donors was 47., Results: Themes emerging from the data suggest both recipients and donors faced barriers in the LDKT experience. Recipients faced barriers associated with their denial and avoidance of the severity of their ESRD, their desire to maintain the privacy of their health status, and their refusal to approach potential donors. Donors encountered negative responses from others about the donors' desire to donate and the initial refusal of recipients to accept a LDKT offer. Recipients identified faith as a coping mechanism, while donors identified normalization of donation as their method of coping. Various types of social support helped donors and recipients navigate the transplant process., Conclusion: Black LDKT recipients and donors must overcome barriers prior to receiving or donating a kidney. Most of these barriers arise from communication and interactions with others that are either lacking or undesirable. Future interventions to promote LDKT among Blacks may benefit by specifically targeting these barriers.

Humanized monoclonal anti-human growth factor receptor 2 (HER2) antibody trastuzumab was approved for HER2 positive breast cancer patient treatment 11 years after the demonstration of HER2 gene amplification associated with the HER2 protein overexpression in breast cancer in 1987. HER2 positive status of breast cancer patients is assessed by HER2 gene amplification with in situ hybridization (ISH) and/or HER2 protein overexpression with immunohistochemistry (IHC). Because the discordance between quantitative HER2 ISH and subjective, semi-quantitative HER2 IHC assay results is a well-recognized issue of HER2 testing, we developed an assay combining HER2 ISH and HER2 IHC assays (HER2 gene-protein assay; HER2 GPA) as one test on the same tissue section. HER2 GPA allows pathologists to score the HER2 gene and HER2 protein status simultaneously at the individual cell level. The possibility that HER2 GPA may become the next generation of HER2 testing is discussed, particularly for cases in which it is difficult to assess the HER2 status of breast cancer patients due to the HER2 heterogeneity., (© 2016 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Aims: Immunoglobulin light-chain expression is used routinely as an indirect marker of clonality for recognizing B cell lymphoproliferative disorders., Methods and Results: Here we describe four floral follicular hyperplasia cases in the gastrointestinal tract (appendix and rectum) of children (4 to 6 years). Immunohistochemical studies revealed lambda light-chain restriction that was associated with polyclonal IgH pattern. Clinical features and follow-up of the patients did not reveal any other systemic symptoms, laboratory abnormalities or organ alterations., Conclusions: Recognition of this phenomenon is useful in the diagnosis of nodular lymphoid hyperplasia of the gastrointestinal tract, for avoiding overdiagnosis of lymphoid malignancies, and raises concerns that the identification of light-chain restriction is not necessarily a marker of monoclonality., (© 2014 John Wiley & Sons Ltd.)

Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC) results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR), but shorter ischemic intervals (less than 17 minutes) facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT) compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

Patients with aggressive, BCL2 protein-positive (+) diffuse large B-cell lymphoma (DLBCL) often experience rapid disease progression that is refractory to standard therapy. However, there is potential for false-negative staining of BCL2 using the standard monoclonal mouse 124 antibody that hinders the identification of these high-risk DLBCL patients. Herein, we compare 2 alternative rabbit monoclonal antibodies (E17 and SP66) to the 124 clone in staining for BCL2 in formalin-fixed, paraffin-embedded DLBCL tissues. Overall, in 2 independent DLBCL cohorts, E17 and SP66 detected BCL2 expression more frequently than 124. In the context of MYC expression, cases identified as BCL2 (+) with SP66 demonstrated the strongest correlation with worse overall survival. The 124 clone failed to detect BCL2 expression in the majority of translocation (+), amplification (+), and activated B-cell DLBCL cases in which high levels of BCL2 protein are expected. Using dual in situ hybridization as a new tool to detect BCL2 translocation and amplification, we observed similar results as previously reported for fluorescence in situ hybridization for translocation but a higher amplification frequency, indicating that BCL2 amplification may be underreported in DLBCL. Among the discrepant cases, phosphorylation of BCL2 at T69 and/or S70 was more common than in the concordant cases and may contribute to the 124 false negatives, in addition to previously associated mutations within the epitope region. The accurate detection of BCL2 expression is important in the prognosis and treatment of DLBCL particularly with new anti-BCL2 therapies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies., Methods: The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator., Results: 39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant., Conclusions: Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856.

Introduction: The demonstration of anaplastic lymphoma kinase (ALK) positivity in non-small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement., Methods: We developed a horseradish peroxidase-based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization., Results: The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole-based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay., Conclusion: The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens.

Background: A point mutation (V600E) in the BRAF oncogene is a prognostic biomarker and may predict for nonresponse to anti-EGFR antibody therapy in patients with colorectal carcinoma. BRAFV600E mutations are frequently detected in tumors with microsatellite instability and indicate a sporadic origin. We used a mutation-specific antibody to examine mutant BRAFV600E protein expression and its concordance with BRAFV600E mutation data., Methods: Primary stage III colon carcinomas were analyzed for BRAFV600E mutations in exon 15, and 50 BRAFV600E mutation carriers and 25 wild-type tumors were selected for analysis of BRAF proteins by immunohistochemistry (IHC). IHC was performed in archival tissue specimens using a pan-BRAF antibody and a mutation-specific antibody against BRAFV600E proteins. Staining was scored by 2 pathologists who were blinded to clinical and mutation data., Results: Using a pan-BRAF antibody, total BRAF protein expression was observed in the tumor cell cytoplasm in 74 of 75 colon carcinomas. A mutation-specific antibody identified diffuse cytoplasmic staining of mutant BRAFV600E proteins in 49 of 74 cancers. Analysis using a polymerase chain reaction-based assay revealed that all 49 of these cancers carried BRAFV600E mutations. In contrast, BRAFV600E staining was absent in all 25 tumors that carried wild-type copies of BRAF., Conclusions: A BRAF mutation-specific (V600E) antibody detected tumors with BRAFV600E mutations and exhibited complete concordance with a DNA-based method. These results support the use of IHC as a simplified strategy to screen colorectal cancers for BRAFV600E mutations in clinical practice., (© 2013 American Cancer Society.)

Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. In this study, we included 379 patients who underwent surgical resection (179 diagnosed as having adenocarcinoma [ADC]; 150, squamous cell carcinoma [SCC]; 41, sarcomatoid carcinoma and 9, large cell carcinoma). IGF-1R expression and gene copy number were assessed by immunohistochemistry and bright-field in situ hybridization (BISH), respectively. IGF-1R expression in non-small cell lung carcinoma was observed in 41.4% of samples and was more prevalent in SCC (69.3%) than in ADC (25.1%), large cell carcinoma (33.3%), and sarcomatoid carcinoma (12.2%) (P < .001). Among ADCs, most mucinous ADCs (75%) showed strong membranous staining with the IGF-1R antibody. Compared with protein expression, IGF-1R gene alteration was rare (8.4%). A statistically significant correlation between IGF-1R expression and positive IGF-1R BISH was observed (γ = 0.762, P < .001). IGF-1R-positive tumors were more common in smokers (P = .004), and these tumors were larger (P = .006) than the IGF-1R-negative tumors. IGF-1R BISH positivity was not correlated with any clinicopathologic factor. IGF-1R expression and IGF-1R BISH positivity were not correlated with overall survival. IGF-1R is highly expressed in SCC and mucinous ADC, although copy number alterations in the IGF-1R gene were rare. These findings may have important implications for future anti-IGF-1R therapeutic approaches., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl-2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B-cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype., Methods and Results: Two hundred and five follicular lymphomas were investigated with immunohistochemistry and fluorescence in-situ hybridization (FISH). The use of three distinct anti-bcl-2 antibodies together with CD10 expression data and FISH analysis for bcl-2 and bcl-6 rearrangements allowed subclassification of follicular lymphoma into four distinct subgroups: (i) CD10-positive/bcl-2-positive, (ii) CD10-positive/bcl-2-negative, (iii) CD10-negative/bcl-2-positive, and (iv) CD10-negative/bcl-2-negative. All cases were bcl-6-positive. STMN1 (stathmin) was shown to be helpful in diagnosing bcl-2-negative and/or CD10-negative follicular lymphomas, and in their distinction from marginal zone B-cell lymphoma., Conclusions: Combined immunohistological and molecular analyses reveal that follicular lymphomas showing an atypical immunophenotypic and molecular profile exist, and we demonstrate that STMN1 represents a novel useful diagnostic marker for these., (© 2013 Blackwell Publishing Ltd.)

Brightfield in situ hybridization (BISH) applications have significant advantages over traditional fluorescence in situ hybridization (FISH). BISH slides can be analyzed using a regular microscope while FISH slides require the use of a specialized fluorescence microscope. BISH slides allow observers for correlating the gene status (gene amplifications, gene rearrangements, and gene deletions) and tissue morphology better than FISH slides. Also, BISH slides are ideal for the permanent preservation of gene signals. Furthermore, BISH applications can be optimized using an automated tissue slide processing system. BISH applications are becoming a popular method for clinical examination of gene status for selecting cancer treatments.

Background: Insulin-like growth factor-1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-type 2 (HER2), and c-Met are members of the receptor tyrosine kinases (RTKs). The associations between the RTK status [protein expression and gene copy number (GCN)] and patient characteristics and between the RTK status and prognosis remain undetermined., Materials and Methods: The study included 140 patients who underwent surgery for thymic tumors. Protein expression was evaluated by immunohistochemistry (IHC) and GCN was evaluated by bright-field in situ hybridization (BISH). The correlations between the RTK status and clinicopathological findings were examined., Results: IGF-1R protein was frequently detected in thymic carcinoma (83.8%) and EGFR in thymic tumors (91.4%). Thirty-six and 39 tumors were BISH high for IGF-1R and EGFR, respectively: 28 and 25 exhibited high polysomy; 8 and 14 exhibited gene amplification. No tumor was positive for HER2 or c-Met by IHC and BISH. Multivariate analysis revealed that IGF-1R gene amplification (P = 0.027), thymic carcinoma histology, and higher tumor stage were significantly correlated with an adverse prognosis., Conclusions: Thymic epithelial tumors frequently express IGF-1R and/or EGFR proteins. IGF-1R gene amplification is suggested to define an unfavorable subset for thymic epithelial tumors.

Background: The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section., Methods: The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays., Results: HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively., Conclusions: We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays., Virtual Slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297.

Loss of major histocompatibility complex class II (MHC II) expression is associated with poor patient outcome in diffuse large B-cell lymphoma (DLBCL). As MHC II molecules are lost with plasmacytic differentiation in normal cells, we asked whether MHC II loss in DLBCL is associated with an altered differentiation state. We used gene expression profiling, quantum dots, and immunohistochemistry to study the relationship between MHC II and plasma cell markers in DLBCL and plasmablastic lymphoma (PBL). Results demonstrate that MHC II(-) DLBCL immunophenotypically overlap with PBL and demonstrate an inverse correlation between MHC II and plasma cell markers MUM1, PRDM1/Blimp1, and XBP1s. In addition, MHC II expression is significantly higher in germinal center-DLBCL than activated B cell-DLBCL. A minor subset of cases with an unusual pattern of mislocalized punctate MHC II staining and intermediate levels of mRNA is also described. Finally, we show that PBL is negative for MHC II. The results imply a spectrum of MHC II expression that is more frequently diminished in tumors derived from B cells at the later stages of differentiation (with complete loss in PBL). Our observations provide a possible unifying concept that may contribute to the poor outcome reported in all MHC II(-) B-cell tumors.

Background: During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of μ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma., Design and Methods: Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas., Results: We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%)., Conclusions: We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.