AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

Clinical Utility and Reimbursement of Next-Generation Sequencing-Based Testing for Myeloid Malignancies.

Next-generation sequencing is becoming increasingly important for the diagnosis, risk stratification, and management of patients with established or suspected myeloid malignancies. These tests are being incorporated into clinical practice guidelines and many genetic alterations now constitute disease classification criteria. However, the reimbursement for these tests is uncertain. This study analyzed the clinical impact, ordering practices, prior authorization, and reimbursement outcomes of 505 samples from 477 patients sequenced with a 50-gene myeloid next-generation sequencing panel or a 15-gene myeloproliferative neoplasm subpanel. Overall, 98% (496 of 505) of tests provided clinically useful data. Eighty-nine percent of test results, including negative findings, informed or clarified potential diagnoses, 94% of results informed potential prognoses, and 19% of tests identified a potential therapeutic target. Sequencing results helped risk-stratify patients whose bone marrow biopsy specimens were inconclusive for dysplasia, monitor genetic evolution associated with disease progression, and delineate patients with mutation-defined diagnoses. Despite the clinical value, prior authorization from commercial payors or managed government payors was approved for less than half (45%) of requests. Only 51% of all cases were reimbursed, with lack of medical necessity frequently cited as a reason for denial. This study demonstrates the existence of a substantial gap between clinical utility and payor policies on test reimbursement.

Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression.

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.

Phenogenomic heterogeneity of post-transplant plasmablastic lymphomas.

Plasmablastic lymphoma (PBL) is a rare and clinically aggressive neoplasm that typically occurs in immunocompromised individuals, including those with HIV infection and solid organ allograft recipients. Most prior studies have focused on delineating the clinicopathologic features and genetic attributes of HIV-related PBLs, where MYC deregulation and EBV infection, and more recently, mutations in JAK/STAT, MAP kinase, and NOTCH pathway genes have been implicated in disease pathogenesis. The phenotypic spectrum of post-transplant (PT)-PBLs is not well characterized and data on underlying genetic alterations are limited. Hence, we performed comprehensive histopathologic and immunophenotypic evaluation and targeted sequencing of 18 samples from 11 patients (8 males, 3 females, age range 12-76 years) with PT-PBL; 8 de novo and 3 preceded by other types of PTLDs. PT-PBLs displayed morphologic and immunophenotypic heterogeneity and some features overlapped those of plasmablastic myeloma. Six (55%) cases were EBV+ and 5 (45%) showed MYC rearrangement by fluorescence in situ hybridization. Recurrent mutations in epigenetic regulators (KMT2/MLL family, TET2) and DNA damage repair and response (TP53, mismatch repair genes, FANCA, ATRX), MAP kinase (KRAS, NRAS, HRAS, BRAF), JAK/STAT (STAT3, STAT6, SOCS1), NOTCH (NOTCH1, NOTCH3, SPEN), and immune surveillance (FAS, CD58) pathway genes were observed, with EBV+ and EBV- cases exhibiting similarities and differences in their mutational profiles. Clinical outcomes also varied, with survival ranging from 0-15.9 years postdiagnosis. Besides uncovering the biological heterogeneity of PT-PBL, our study highlights similarities and distinctions between PT-PBLs and PBLs occurring in other settings and reveals potentially targetable oncogenic pathways in disease subsets.

The clinical and pathological features of plasma cell myeloma post solid organ transplantation.

Plasma cell neoplasms (PCNs), comprising plasma cell myelomas (PCMs) and plasmacytomas, which occur after solid organ transplantation, represent rare subtypes of monomorphic post-transplant lymphoproliferative disorders (M-PTLDs). Data regarding the clinical and pathological features of post-transplant (PT)-PCMs are limited. To gain a better understanding of disease biology, we performed comprehensive immunophenotypic analysis, reviewed cytogenetic analysis results and evaluated clinical outcomes of PT-PCMs diagnosed and treated at our institution. Fifteen PT-PCM (M: F - 4:1) and two PT-MGUS (two males) cases were identified. The median age of PT-PCM patients was 68 years (29-79 years) and PCMs presented at a median of 9.7 years (0.5-24.7 years) after transplantation. The PT-PCMs accounted for 11.6% of all M-PTLDs and the period prevalence was 9/3108 (0.29%), 3/1071 (0.28%), 2/1345 (0.15%) and 1/878 (0.11%) post kidney, heart, liver and lung transplantation. Lytic bone disease was observed in 1/11 (9%) patients. Marrow plasma cell infiltration ranged from 10%-70% (median 20%), with 10/15 (67%) and 5/15 (33%) cases manifesting immature and plasmablastic morphology. The immunophenotype of all cases and cytogenetic abnormalities, identified in 60% of cases, were similar to multiple myeloma (MM) of immunocompetent individuals. All PT-PCMs were EBER negative. Ten of 11 (91%) patients with active MM were treated, all with proteasome inhibitor-based therapy. Treatment response and 5-year overall survival (54.5%) was comparable to MM of immunocompetent individuals. However, the survival of patients with plasmablastic PCMs was inferior to those with immature PCMs. 0ur findings indicate PT-PCMs to be predominantly late onset PTLDs that have similar clinicopathologic characteristics as conventional MM.

Pathogenesis of Enteropathy-Associated T Cell Lymphoma.

PURPOSE OF REVIEW: To provide an update on the pathogenesis of enteropathy-associated T cell lymphoma (EATL) and its relationship with refractory celiac disease (RCD), in light of current knowledge of immune, genetic, and environmental factors that promote neoplastic transformation of intraepithelial lymphocytes (IELs). RECENT FINDINGS: EATL frequently evolves from RCD type II (RCD II) but can occur "de novo" in individuals with celiac disease. Recurrent activating mutations in members of the JAK/STAT pathway have been recently described in EATL and RCD II, which suggests deregulation of cytokine signaling to be an early event in lymphomagenesis. Intraepithelial T cells are presumed to be the cell of origin of EATL (and RCD II). Recent in vitro molecular and phenotypic analyses and in vivo murine studies, however, suggest an origin of RCD II from innate IELs (NK/T cell precursors), which could also be the cell of origin of RCD II-derived EATL. The immune microenvironment of the small intestinal mucosa in celiac disease fosters the development of EATL, often in a multistep pathway.

BCL6 as a therapeutic target for lymphoma.

INTRODUCTION: B cell lymphoma 6 (BCL6) is a transcriptional repressor critical for the development and maintenance of germinal centers (GCs), which are required for generation of an effective humoral immune response. Genomic aberrations of BCL6, including mutations and translocations that occur during the GC reaction, as well as alterations of genes that regulate BCL6 expression, lead to sustained activity of BCL6, which promotes the development of GC-derived lymphomas. Since many types of B cell non-Hodgkin lymphomas (B-NHL) arise from neoplastic transformation of GC B cells and a high proportion harbor genetic lesions that deregulate BCL6 expression, inhibition of BCL6 has emerged as an attractive therapeutic strategy for lymphomas. Areas covered: This review examines the rationale for and challenges in therapeutic targeting of BCL6 in lymphomas. We describe approaches that have been used and are currently being considered for inhibition of BCL6. Expert opinion: Several BCL6 inhibiting agents, including peptidomimetics, small molecules, and natural compounds, most of which target the BTB domain of the protein at the corepressor binding site, have been developed with demonstration of anti-lymphoma activity in preclinical models. Future clinical trials will be important to investigate the efficacy of targeting BCL6 in B-NHL (and other neoplasms), particularly in combination with other therapies.

Fatal Case of Primary Cutaneous Aggressive T-Cell Lymphoma Switching From a CD4+ to a CD8+ Phenotype: Progressive Disease With Bexarotene and Romidepsin Treatment.

A 77-year-old white male presented to the clinic with two isolated cutaneous tumors on his forehead. A cutaneous biopsy showed a focally folliculotropic CD4 cutaneous lymphoma. The tumors were irradiated with a complete response, and he was started on oral bexarotene. He experienced localized cutaneous relapse 3 months into treatment. These new tumors now revealed a surprisingly CD8 cytotoxic phenotype, but with the same clone. A systemic workup was negative. His regimen was switched to romidepsin, and he was treated with local radiation again. Another 3.5 months passed in remission until he developed widespread cutaneous tumors. Positron emission tomography/computed tomography revealed multifocal systemic disease involving his diaphragm, liver, distal duodenum, proximal jejunum, anterior chest wall including pectoral muscles, and lungs without significant adenopathy. He died a few days later. Given his full clinical and pathological course, he was given the diagnosis of an aggressive primary cutaneous T-cell lymphoma, unspecified.

Histopathological features of papillary thyroid carcinomas detected during four screening examinations of a Ukrainian-American cohort.

BACKGROUND: There are limited data on the histopathology of papillary thyroid carcinomas (PTCs) diagnosed in irradiated populations. We evaluated the associations between iodine-131 dose and the histopathological characteristics of post-Chernobyl PTCs, the changes in these characteristics over time, and their associations with selected somatic mutations. METHODS: This study included 115 PTCs diagnosed in a Ukrainian-American cohort (n=13,243) during prescreening and four successive thyroid screenings. Of these PTCs, 65 were subjected to somatic mutation profiling. All individuals were <18 years at the time of the Chernobyl accident and had direct thyroid radioactivity measurements. Statistical analyses included multivariate linear and logistic regression. RESULTS: We identified a borderline significant linear-quadratic association (P=0.063) between iodine-131 dose and overall tumour invasiveness (presence of extrathyroidal extension, lymphatic/vascular invasion, and regional or distant metastases). Irrespective of dose, tumours with chromosomal rearrangements were more likely to have lymphatic/vascular invasion than tumours without chromosomal rearrangements (P=0.020) or tumours with BRAF or RAS point mutations (P=0.008). Controlling for age, there were significant time trends in decreasing tumour size (P<0.001), the extent of lymphatic/vascular invasion (P=0.005), and overall invasiveness (P=0.026). CONCLUSIONS: We determined that the invasive properties of PTCs that develop in iodine-131-exposed children may be associated with radiation dose. In addition, based on a subset of cases, tumours with chromosomal rearrangements appear to have a more invasive phenotype. The increase in small, less invasive PTCs over time is a consequence of repeated screening examinations.

ETV6-NTRK3 is a common chromosomal rearrangement in radiation-associated thyroid cancer.

BACKGROUND: In their previous analysis of papillary thyroid carcinomas (PTCs) from an Ukrainian-American cohort that was exposed to iodine-131 ((131) I) from the Chernobyl accident, the authors identified RET/PTC rearrangements and other driver mutations in 60% of tumors. METHODS: In this study, the remaining mutation-negative tumors from that cohort were analyzed using RNA sequencing (RNA-Seq) and reverse transcriptase-polymerase chain reaction to identify novel chromosomal rearrangements and to characterize their relation with radiation dose. RESULTS: The ETS variant gene 6 (ETV6)-neurotrophin receptor 3 (NTRK3) rearrangement (ETV6-NTRK3) was identified by RNA-Seq in a tumor from a patient who received a high (131) I dose. Overall, the rearrangement was detected in 9 of 62 (14.5%) post-Chernobyl PTCs and in 3 of 151 (2%) sporadic PTCs (P = .019). The most common fusion type was between exon 4 of ETV6 and exon 14 of NTRK3. The prevalence of ETV6-NTRK3 rearrangement in post-Chernobyl PTCs was associated with increasing (131) I dose, albeit at borderline significance (P = .126). The group of rearrangement-positive PTCs (ETV6-NTRK3, RET/PTC, PAX8-PPARγ) was associated with significantly higher dose response compared with the group of PTCs with point mutations (BRAF, RAS; P < .001). In vitro exposure of human thyroid cells to 1 gray of (131) I and γ-radiation resulted in the formation of ETV6-NTRK3 rearrangement at a rate of 7.9 × 10(-6) cells and 3.0 × 10(-6) cells, respectively. CONCLUSIONS: The authors report the occurrence of ETV6-NTRK3 rearrangements in thyroid cancer and demonstrate that this rearrangement is significantly more common in tumors associated with exposure to (131) I and has a borderline significant dose response. Moreover, ETV6-NTRK3 rearrangement can be directly induced in thyroid cells by ionizing radiation in vitro and, thus, may represent a novel mechanism of radiation-induced carcinogenesis.

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post-Chernobyl thyroid cancer and their association with iodine-131 radiation dose and other characteristics.

BACKGROUND: Childhood exposure to iodine-131 from the 1986 nuclear accident in Chernobyl, Ukraine, led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. METHODS: Mutational analysis was performed on 62 PTCs diagnosed in a Ukrainian cohort of patients who were < 18 years old in 1986 and received 0.008 to 8.6 Gy of (131) I to the thyroid. Associations between mutation types and (131) I dose and other characteristics were explored. RESULTS: RET/PTC (ret proto-oncogene/papillary thyroid carcinoma) rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement were identified. A significant negative association with (131) I dose for BRAF and RAS point mutations and a significant concave association with (131) I dose, with an inflection point at 1.6 Gy and odds ratio of 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements were found. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared with point mutations, rearrangements were associated with residence in the relatively iodine-deficient Zhytomyr region, younger age at exposure or surgery, and male sex. CONCLUSIONS: These results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with (131) I dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and (131) I exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients.

Inhibition of EGFR-STAT3 signaling with erlotinib prevents carcinogenesis in a chemically-induced mouse model of oral squamous cell carcinoma.

Chemoprevention of head and neck squamous cell carcinoma (HNSCC), a disease associated with high mortality rates and frequent occurrence of second primary tumor (SPT), is an important clinical goal. The epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription (STAT)-3 signaling pathway is known to play a key role in HNSCC growth, survival, and prognosis, thereby serving as a potential therapeutic target in the treatment of HNSCC. In the current study, the 4-nitroquinoline-1-oxide (4-NQO)-induced murine model of oral carcinogenesis was utilized to investigate the chemopreventive activities of compounds that target the EGFR-STAT3 signaling pathway. This model mimics the process of oral carcinogenesis in humans. The drugs under investigation included erlotinib, a small molecule inhibitor of the EGFR, and guggulipid, the extract of an Ayurvedic medicinal plant, which contains guggulsterone, a compound known to inhibit STAT3. Dietary administration of guggulipid failed to confer protection against oral carcinogenesis. On the other hand, the mice placed on erlotinib-supplemented diet exhibited a 69% decrease (P < 0.001) in incidence of preneoplastic and neoplastic lesions compared with mice on the control diet. Immunostaining of dysplastic lesions demonstrated modest decreases in STAT3 levels, with both drug treatments, that were not statistically significant. The results of the present study provide the basis for exploring the efficacy of erlotinib for prevention of HNSCC in a clinical setting.

Honokiol inhibits epidermal growth factor receptor signaling and enhances the antitumor effects of epidermal growth factor receptor inhibitors.

PURPOSE: This study aimed to investigate the utility of honokiol, a naturally occurring compound, in the treatment of head and neck squamous cell carcinoma (HNSCC) as well as its ability to target the epidermal growth factor receptor (EGFR), a critical therapeutic target in HNSCC, and to enhance the effects of other EGFR-targeting therapies. EXPERIMENTAL DESIGN: Human HNSCC cell lines and the xenograft animal model of HNSCC were used to test the effects of honokiol treatment. RESULTS: Honokiol was found to inhibit growth in human HNSCC cell lines, with 50% effective concentration (EC(50)) values ranging from 3.3 to 7.4 micromol/L, and to induce apoptosis, as shown through Annexin V staining. These effects were associated with inhibition of EGFR signaling, including downstream inhibition of mitogen-activated protein kinase, Akt, and signal transducer and activator of transcription 3 (STAT3), and expression of STAT3 target genes, Bcl-X(L) and cyclin D1. Furthermore, honokiol enhanced the growth inhibitory and anti-invasion activity of the EGFR-targeting agent erlotinib. Although HNSCC xenograft models did not show significant inhibition of in vivo tumor growth with honokiol treatment alone, the combination of honokiol plus cetuximab, a Food and Drug Administration-approved EGFR inhibitor for this malignancy, significantly enhanced growth inhibition. Finally, HNSCC cells rendered resistant to erlotinib retained sensitivity to the growth inhibitory effects of honokiol. CONCLUSIONS: These results suggest that honokiol may be an effective therapeutic agent in HNSCC, in which it can augment the effects of EGFR inhibitors and overcome drug resistance.

Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3.

Treatment of human head and neck squamous cell carcinoma (HNSCC) cell lines with guggulsterone, a widely available, well-tolerated nutraceutical, demonstrated dose-dependent decreases in cell viability with EC(50)s ranging from 5 to 8 microM. Guggulsterone induced apoptosis and cell cycle arrest, inhibited invasion and enhanced the efficacy of erlotinib, cetuximab and cisplatin in HNSCC cell lines. Guggulsterone induced decreased expression of both phosphotyrosine and total signal transducer and activator of transcription (STAT)-3, which contributed to guggulsterone's growth inhibitory effect. Hypoxia-inducible factor (HIF)-1alpha was also decreased in response to guggulsterone treatment. In a xenograft model of HNSCC, guggulsterone treatment resulted in increased apoptosis and decreased expression of STAT3. In vivo treatment with a guggulsterone-containing natural product, Guggulipid, resulted in decreased rates of tumor growth and enhancement of cetuximab's activity. Our results suggest that guggulsterone-mediated inhibition of STAT3 and HIF-1alpha provide a biologic rationale for further clinical investigation of this compound in the treatment of HNSCC.

Bortezomib up-regulates activated signal transducer and activator of transcription-3 and synergizes with inhibitors of signal transducer and activator of transcription-3 to promote head and neck squamous cell carcinoma cell death.

Head and neck squamous cell carcinomas (HNSCC) are commonly resistant to conventional chemotherapy drugs and exhibit overexpression of signal transducer and activator of transcription 3 (STAT3). STAT3 promotes both the proliferation and survival of HNSCC cells. Recent studies have shown that the proteasome inhibitor bortezomib shows cytotoxic activity against HNSCC in vitro and in vivo. We report that treatment of HNSCC cells with bortezomib led to up-regulation of total STAT3 protein and the phosphorylated/activated form of STAT3, as well as an increase in cellular STAT3 activity. This suggested that the ability of bortezomib to kill HNSCC cells may be blunted due to induction of STAT3, and inhibition of STAT3 may be a useful means for improving bortezomib efficacy. Indeed, forced expression of dominant-active STAT3 inhibited bortezomib-induced cell death, whereas expression of dominant-negative STAT3 served to enhance killing by this compound. In addition, specific inhibition of STAT3 with the use of a STAT3 decoy oligonucleotide resulted in enhancement of bortezomib-induced apoptosis signaling and loss of clonogenic survival. Cotreatment of HNSCC cells with bortezomib and guggulsterone, a naturally occurring compound known to inhibit STAT3 activation, led to synergistic activation of cell death and loss of clonogenic survival. In summary, these studies show that bortezomib induces the expression of active STAT3, a key growth- promoting protein in HNSCC cells. Furthermore, our findings suggest that the therapeutic activity of bortezomib against HNSCC may be markedly improved by cotreatment with molecular targeting agents against STAT3.