Validation of Linear Range HER2/Estrogen Receptor/Progesterone Receptor IHControls for Daily Quality Assurance.

OBJECTIVES: To evaluate a new US Food and Drug Administration (FDA)-cleared immunohistochemistry (IHC) control (IHControls [Boston Cell Standards]) comprising peptide epitopes for HER2, estrogen receptor (ER), and progesterone receptor (PR) attached to cell-sized microspheres and to compare its performance against conventional tissue controls. METHODS: IHControls and tissue/cell line controls for HER2, ER, and PR were compared side by side daily at 5 clinical IHC laboratories for 1 to 2 months. Separately, the sensitivity of the 2 types of controls was evaluated in simulated IHC assay failure experiments by diluting the primary antibody. Additional evaluations included lot-to-lot manufacturing reproducibility of 3 independent lots and specificity against 26 antigenically irrelevant IHC stains. RESULTS: Side-by-side testing revealed a 99.6% concordance between IHControls and tissue controls across 5 IHC laboratories and 766 individual evaluations. Three discordant quality control events were the result of operator error. Simulated assay failure data showed that both IHControls and tissue controls are similarly capable of detecting IHC staining errors. Manufacturing reproducibility of IHControls showed less than 10% variability (coefficient of variation). No cross-reactions were detected from 26 antigenically irrelevant IHC stains. CONCLUSIONS: IHControls, the first FDA-cleared IHC controls, can sensitively and accurately detect IHC assay problems, similar to tissue controls.

Postmortem study of organ-specific toxicity in glioblastoma patients treated with a combination of temozolomide, irinotecan and bevacizumab.

PURPOSE: Systemic chemotherapy including monotherapy with temozolomide (TMZ) or bevacizumab (BEV); two-drug combinations, such as irinotecan (IRI) and BEV, TMZ and BEV and a three-drug combination with TMZ, IRI and BEV (TIB) have been used in treating patients with progressive high-grade gliomas including glioblastoma (GBM). Most patients tolerated these regimens well with known side effects of hypertension, proteinuria, and reversible clinical myelosuppression (CM). However, organ- or system- specific toxicities from chemotherapy agents have never been examined by postmortem study. This is the largest cohort used to address this issue in glioma patients. METHODS: Postmortem tissues (from all major systems and organs) were prospectively collected and examined by standard institution autopsy and neuropathological procedures from 76 subjects, including gliomas (N = 68, 44/M, and 24/F) and brain metastases (N = 8, 5/M, and 3/F) between 2009 and 2019. Standard hematoxylin and eosin (H&E) were performed on all major organs including brain specimens. Electronic microscopic (EM) study was carried out on 14 selected subject's kidney samples per standard EM protocol. Medical records were reviewed with adverse events (AEs) analyzed and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. A swimmer plot was utilized to visualize the timelines of patient history by treatment group. The binary logistic regression models were performed to explore any associations between treatment strategies and incident myelosuppression. RESULTS: Twenty-four glioma subjects were treated with TIB [median: 5.5 (range: 1-25) cycles] at tumor recurrence. Exposure to IRI significantly increased the frequency of CM (p = 0.05). No unexpected adverse events clinically, or permanent end-organ damage during postmortem examination was identified in glioma subjects who had received standard or prolonged duration of BEV, TMZ or TIB regimen-based chemotherapies except rare events of bone marrow suppression. The most common causes of death (COD) were tumor progression (63.2%, N = 43) followed by aspiration pneumonia (48.5%, N = 33) in glioma subjects. No COD was attributed to acute toxicity from TIB. The study also demonstrated that postmortem kidney specimen is unsuitable for studying renal ultrastructural pathological changes due to autolysis. CONCLUSION: There is no organ or system toxicity by postmortem examinations among glioma subjects who received BEV, TMZ or TIB regimen-based chemotherapies regardless of durations except for occasional bone marrow suppression and reversible myelosuppression clinically. IRI, but not the extended use of TMZ, significantly increased CM in recurrent glioma patients. COD most commonly resulted from glioma tumor progression with infiltration to brain stem and aspiration pneumonia.

A Rare Case of Waldenstrom Macroglobulinemia of the Rectosigmoid Colon.

Waldenstrom macroglobulinemia is an uncommon mature B-cell lymphoma characterized by monoclonal immunoglobulin M protein in peripheral blood and lymphoplasmacytic cells in bone marrow and/or extramedullary sites. The gastrointestinal tract is a rare site of involvement. The diagnosis is based on clinicopathologic findings, although somatic mutations, such as MYD88, can aid in the diagnosis. We present a patient with irregular stools diagnosed with Waldenstrom macroglobulinemia involving the rectosigmoid colon by histopathology and immunohistochemistry on colonic biopsies, immunoglobulin M protein in serum, clonal plasma cells in bone marrow, and MYD88 mutation in colonic and bone marrow specimens.

Association between spinal stenosis and wild-type ATTR amyloidosis.

Age-related cardiac amyloidosis results from deposits of wild-type tranthyretin amyloid (ATTRwt) in cardiac tissue. ATTR may play a role in carpal tunnel syndrome (CTS) and in spinal stenosis (SS), indicating or presaging systemic amyloidosis. We investigated consecutive patients undergoing surgery for SS for ATTR deposition in the resected ligamentum flavum (LF) and concomitant risk of cardiac amyloidosis. Each surgical specimen (LF) was stained with Congo red, and if positive, the amyloid deposits were typed by mass spectrometry. Patients with positive specimens underwent standard of care evaluation with fat pad aspirates, serum and urine protein electrophoresis with immunofixation, free light-chain assay, TTR gene sequencing and technetium 99 m-pyrophosphate-scintigraphy. In 2018-2019, 324 patients underwent surgery for SS and 43 patients (13%) had ATTR in the LF with wild-type TTR gene sequences. Two cases of ATTRwt cardiac amyloidosis were diagnosed and received treatment. In this large series, ATTRwt was identified in 13% of the patients undergoing laminectomy for SS. Patients with amyloid in the ligamentum flavum were older and had a higher prevalence of CTS, suggesting a systemic form of ATTR amyloidosis involving connective tissue. Further prospective study of patients with SS at risk for systemic amyloidosis is warranted.

Use of Eculizumab in Transplant-Associated Thrombotic Microangiopathy in a Patient With Polycystic Kidney Disease Immediately Post-Kidney Transplant: A Case Report.

Transplant-associated thrombotic microangiopathy (TMA) in the post-organ transplantation setting occurs from a number of potential inciting factors, such as the use of calcineurin inhibitors, ischemic injury, infections, or antibody-mediated rejection leading to unchecked complement activation and end-organ damage. Delayed recognition of this condition can result in allograft loss. In this case description, we describe the first case of de novo TMA in a patient with polycystic kidney disease that occurred immediately after kidney transplantation. The diagnosis was made promptly on the basis of clinical and laboratory characteristics by a multidisciplinary team and confirmed through kidney biopsy, which showed acute TMA. The patient was successfully managed by replacing tacrolimus with belatacept, which targets cytotoxic T lymphocyte antigen 4, and use of eculizumab, a C5 inhibitor. Eculizumab treatment was discontinued after 3 months of complement inhibition on the patient's request, and relapse of TMA has not been encountered after more than 1 year of follow-up.

Immunohistochemical expression of vitamin D receptor and forkhead box P3 in classic Hodgkin lymphoma: correlation with clinical and pathologic findings.

BACKGROUND: Expression of forkhead box P3 (FOXP3), a key regulator of T-cell function, in the tumor immune microenvironment is related to survival in classic Hodgkin lymphoma (CHL). Vitamin D receptor (VDR), a transcription factor agonists have been shown to induce FOXP3 expression in T-cells and enhance recruitment of these cells to the inflammatory sites. VDR expression is CHL has been described. However, there is no data on expression of VDR in context of quantity of FOXP3 positive cells in CHL. METHODS: We examined and correlated immunohistochemical expression of VDR and FOXP3 along with clinical and pathology findings in 29 cases of CHL. RESULTS: VDR was expressed in Hodgkin Reed-Sternberg (HRS) cells and background lymphocytes and FOXP3 was expressed in background lymphocytes. 82% of CHL cases, regardless of the subtype, expressed VDR and in majority of the cases, VDR expression was directly proportional to the quantity of FOXP3 expressing lymphocytes in the tumor microenvironment. In cases with higher clinical stage (III/IV), only 28.5% of cases diffusely expressed VDR and FOXP3 compared to 71.4% showing focal positivity. Whereas in cases with lower clinical stages (I/II), the expression pattern of VDR and FOXP3 was almost similar (41.6% diffuse versus 33.3% focal). Interestingly, focal VDR and FOXP3 expression pattern was significantly higher among males. Mixed cellularity cases showed predilection for focal VDR and FOXP3 expression (80% cases); whereas nodular sclerosis subtype had focal and diffuse VDR and FOXP3 expression patterns in similar proportion. Cases with diffuse VDR and FOXP3 expression were less likely to have bone marrow involvement. Epstein Barr virus- encoded small RNA (EBER) positive cases were predominantly focally positive (80%) for VDR and FOXP3. CONCLUSIONS: In summary, quantity of FOXP3 positive T-cells in CHL microenvironment seems to correlate with VDR expression. Clinical stage show a trend of inverse correlation with expression of VDR and quantity of FOXP3 positive T-cells. These findings suggest that VDR could be a possible prognostic and therapeutic target in CHL.

Clinicopathologic characteristics of secondary squamous cell carcinoma of head and neck in survivors of allogeneic hematopoietic stem cell transplantation for hematologic malignancies.

The risk of late complications including secondary malignancies is increased in long-term survivors of allogeneic hematopoietic stem cell transplants (HSCT). There is limited literature on the biological behavior and clinical features of squamous cell carcinoma (SCC) of head and neck post-HSCT. We present the clinical and pathologic characteristics on six patients who were diagnosed with SCC while in remission following an allogeneic HSCT. Median follow-up was 8 years. Five patients (83%) developed SCC of tongue and one developed esophageal SCC. Five patients had oral chronic graft-versus-host disease (cGvHD). The conventional risk factors of alcohol, tobacco, and human papillomavirus were absent. The most common presenting finding was the new-onset focal oral pain and ulcerated plaques clinically indistinguishable from a flare of their oral cGvHD lesions. We demonstrated that the SCC in three patients was of donor origin.

Correction: Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel.

[This corrects the article DOI: 10.18632/oncotarget.25209.].

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel.

T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Accelerated Systemic Autoimmunity in the Absence of Somatic Hypermutation in 564Igi: A Mouse Model of Systemic Lupus with Knocked-In Heavy and Light Chain Genes.

564Igi mice have knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an autoantibody recognizing RNA. Previously, we showed that these mice produce pathogenic IgG autoantibodies when activation-induced deaminase (AID) is expressed in pre-B and immature B cells but not when it is expressed only in mature B cells. AID has two functions; it is necessary for somatic hypermutation (SHM) and class switch recombination (CSR). To determine the role of each of these functions in the generation of pathogenic autoantibodies, we generated 564Igi mice that carry a mutant AID-encoding gene, Aicda (AicdaG23S), which is capable of promoting CSR but not SHM. We found that 564Igi AicdaG23S mice secreted class-switched antibodies (Abs) at levels approximately equal to 564Igi mice. However, compared to 564Igi mice, 564Igi Aicda G23S mice had increased pathogenic IgG Abs and severe systemic lupus erythematosus-like disease, including, glomerulonephritis, and early death. We suggest that in 564Igi mice SHM by AID changes Ig receptors away from self reactivity, thereby mitigating the production of autoantibody, providing a novel mechanism of tolerance.

The Importance of Epitope Density in Selecting a Sensitive Positive IHC Control.

Clinical Immunohistochemistry (IHC) laboratories face unique challenges in performing accurate and reproducible immunostains. Among these challenges is the use of homemade controls derived from pathological discard samples. Such positive controls have an unknown number of analyte molecules per cell (epitope density). It is unclear how the lack of defined analyte concentrations affects performance of the control. To address this question, we prepared positive IHC controls ( IHControls) for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), or progesterone receptor (PR) with well-defined, homogeneous, and reproducible analyte concentrations. Using the IHControls, we examined the effect of analyte concentration on IHC control sensitivity. IHControls and conventional tissue controls were evaluated in a series of simulated primary antibody reagent degradation experiments. The data demonstrate that the ability of a positive IHC control to reveal reagent degradation depends on (1) the analyte concentration in the control and (2) where that concentration falls on the immunostain's analytic response curve. The most sensitive positive IHC controls have analyte concentrations within or close to the immunostain's concentration-dependent response range. Strongly staining positive controls having analyte concentrations on the analytic response curve plateau are less sensitive. These findings emphasize the importance of selecting positive IHC controls that are of intermediate (rather than strong) stain intensity.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

The Genetic Basis of Hepatosplenic T-cell Lymphoma.

Hepatosplenic T-cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole-exome sequencing of 68 HSTLs, we define recurrently mutated driver genes and copy-number alterations in the disease. Chromatin-modifying genes, including SETD2, INO80, and ARID1B, were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in STAT5B (31%), STAT3 (9%), and PIK3CD (9%), for which there currently exist potential targeted therapies. In addition, we noted less frequent events in EZH2, KRAS, and TP53SETD2 was the most frequently silenced gene in HSTL. We experimentally demonstrated that SETD2 acts as a tumor suppressor gene. In addition, we found that mutations in STAT5B and PIK3CD activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates gene mutations linked to HSTL pathogenesis and potential treatment targets.Significance: We report the first systematic application of whole-exome sequencing to define the genetic basis of HSTL, a rare but lethal disease. Our work defines SETD2 as a tumor suppressor gene in HSTL and implicates genes including INO80 and PIK3CD in the disease. Cancer Discov; 7(4); 369-79. ©2017 AACR.See related commentary by Yoshida and Weinstock, p. 352This article is highlighted in the In This Issue feature, p. 339.

Primary Squamous Cell Carcinoma Arising From a Cutaneous Ureterovesical Stoma (Modified Mitrofanoff): Case Report and Review of Literature.

Squamous cell carcinoma arising from a urinary stoma is exceedingly rare, and none so far is reported from a cutaneous ureterovesical stoma. Squamous cell carcinoma usually occurs as a late complication of urinary diversion, and we report the first such case in a cutaneous ureterovesical stoma with a review of published literature.