Nemanja Rodić

Long interspersed element-1 (LINE-1): passenger or driver in human neoplasms?

LINE-1 (L1) retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the “mobilome” have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis.

Long Interspersed Element-1 Protein Expression Is a Hallmark of Many Human Cancers

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

LINE-1 expression and retrotransposition in Barrett's esophagus and esophageal carcinoma.

Significance By studying premalignant conditions, we can gain a better understanding of the sources of genomic instability and improve cancer prevention and treatment. Because retrotransposition has been observed in many gastrointestinal epithelial cancer types, we focused on L1 mobilization as a source of instability in cancer. Here, we demonstrate that L1 retrotransposition is active in esophageal adenocarcinoma and its precursor, Barrett’s esophagus (BE). We detected clonal populations with precursor cells marked by L1 retrotransposition events either in the normal esophagus or BE. These clones expanded in the BE or esophageal adenocarcinoma (EAC), indicating that somatic L1 insertions are not only potential mutagens in the development of EAC, but also useful markers of tumor clones as well. Barrett’s esophagus (BE) is a common disease in which the lining of the esophagus transitions from stratified squamous epithelium to metaplastic columnar epithelium that predisposes individuals to developing esophageal adenocarcinoma (EAC). We hypothesized that BE provides a unique environment for increased long-interspersed element 1 (LINE-1 or L1) retrotransposition. To this end, we evaluated 5 patients with benign BE, 5 patients with BE and concomitant EAC, and 10 additional patients with EAC to determine L1 activity in this progressive disease. After L1-seq, we confirmed 118 somatic insertions by PCR in 10 of 20 individuals. We observed clonal amplification of several insertions which appeared to originate in normal esophagus (NE) or BE and were later clonally expanded in BE or in EAC. Additionally, we observed evidence of clonality within the EAC cases; specifically, 22 of 25 EAC-only insertions were present identically in distinct regions available from the same tumor, suggesting that these insertions occurred in the founding tumor cell of these lesions. L1 proteins must be expressed for retrotransposition to occur; therefore, we evaluated the expression of open reading frame 1 protein (ORF1p), a protein encoded by L1, in eight of the EAC cases for which formalin-fixed paraffin embedded tissue was available. With immunohistochemistry, we detected ORF1p in all tumors evaluated. Interestingly, we also observed dim ORF1p immunoreactivity in histologically NE of all patients. In summary, our data show that somatic retrotransposition occurs early in many patients with BE and EAC and indicate that early events occurring even in histologically NE cells may be clonally expanded in esophageal adenocarcinogenesis.

Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

Clinical and Histologic Features of Lichenoid Mucocutaneous Eruptions Due to Anti–Programmed Cell Death 1 and Anti–Programmed Cell Death Ligand 1 Immunotherapy

Importance Antagonist antibodies to programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have shown remarkable activity in multiple tumor types. Recent US Food and Drug Administration approval of such agents for advanced melanoma, non-small cell lung cancer, and renal cell carcinoma has hastened the need to better characterize their unique toxicity profiles. Objective To provide a clinical and pathologic description of the lichenoid mucocutaneous adverse effects seen in patients receiving anti-PD-1/PD-L1 treatment. Design, Setting, and Participants Patients with advanced cancer who were referred to dermatology at Yale-New Haven Hospital, a tertiary care hospital, after developing cutaneous adverse effects while receiving an anti-PD-1 or PD-L1 antibody therapy either as monotherapy or in combination with another agent were identified. Medical records from 2010 to 2015 and available skin biopsy specimens were retrospectively reviewed. Main Outcomes and Measures Patient demographic characteristics, concurrent medications, therapeutic regimen, type of disease, previous oncologic therapies, clinical morphology of cutaneous lesions, treatment of rash, peripheral blood eosinophil count, tumor response, and skin histologic characteristics if biopsies were available. Results Patients were 13 men and 7 women, with a mean (range) age of 64 (46-86) years. The majority of cases (16 [80%]) had a clinical morphology consisting of erythematous papules with scale in a variety of distributions. Biopsies were available from 17 patients; 16 (94%) showed features of lichenoid interface dermatitis. Eighteen patients were treated with topical corticosteroids, and only 1 patient required discontinuation of anti-PD-1/PD-L1 therapy. Only 4 of 20 patients (20%) developed peripheral eosinophilia. Sixteen patients (80%) were concurrently taking medications that have been previously reported to cause lichenoid drug eruptions. Conclusions and Relevance Papular and nodular eruptions with scale, as well as mucosal erosions, with lichenoid features on histologic analysis were a distinct finding seen with anti-PD-1/PD-L1 therapies and were generally manageable with topical steroids. Concurrent medications may play a role in the development of this cutaneous adverse effect.

PD-L1 Expression in Melanocytic Lesions Does Not Correlate with the BRAF V600E Mutation

Rodić and colleagues analyzed archival melanocytic lesions and cultured melanomas and found no correlation between melanocyte PD-L1 expression and BRAF V600E mutation, indicating that distinct biomarkers should be used to select patients for BRAF inhibitor and PD-1/PD-L1 checkpoint blockade therapies. PD-L1 expression in melanoma correlates with response to PD-1 pathway–blocking antibodies. Aberrant tumor-cell PD-L1 expression may be oncogene driven and/or induced by IFNγ. Melanomas express PD-L1 in association with tumor-infiltrating lymphocytes (TIL), but the potential contribution of the BRAF V600E mutation (BRAFmut) to induced PD-L1 expression has not been determined. Fifty-two archival melanocytic lesions were assessed for PD-L1 expression, TIL infiltration, and BRAFmut simultaneously. IFNγ-induced PD-L1 expression in cultured melanomas was assessed in parallel according to BRAF status. Melanocyte PD-L1 expression was observed in 40% of specimens, and BRAFmut was observed in 42% of specimens, but no significant concordance was found between these variables. Almost all melanocytes displaying PD-L1 expression were observed to be adjacent to TILs, irrespective of BRAF status. TIL− lesions were not more likely to be associated with BRAFmut, when compared with TIL+ lesions. Baseline expression of PD-L1 by melanoma cell lines was virtually nil, regardless of BRAFmut status, and the intensity of IFN-induced PD-L1 expression in melanoma cell lines likewise did not correlate with BRAF mutational status. PD-L1 expression in melanocytic lesions does not correlate with the BRAFmut. Thus, distinct populations of melanoma patients will likely benefit from BRAF inhibitors versus PD-1 pathway blockade. Cancer Immunol Res; 3(2); 110–5. ©2014 AACR.

Somatically Acquired LINE-1 Insertions in Normal Esophagus Undergo Clonal Expansion in Esophageal Squamous Cell Carcinoma

Squamous cell carcinoma of the esophagus (SCC) is the most common form of esophageal cancer in the world and is typically diagnosed at an advanced stage when successful treatment is challenging. Understanding the mutational profile of this cancer may identify new treatment strategies. Because somatic retrotransposition has been shown in tumors of the gastrointestinal system, we focused on LINE‐1 (L1) mobilization as a source of genetic instability in this cancer. We hypothesized that retrotransposition is ongoing in SCC patients. The expression of L1 encoded proteins is necessary for retrotransposition to occur; therefore, we evaluated the expression of L1 open reading frame 1 protein (ORF1p). Using immunohistochemistry, we detected ORF1p expression in all four SCC cases evaluated. Using L1‐seq, we identified and validated 74 somatic insertions in eight tumors of the nine evaluated. Of these, 12 insertions appeared to be somatic, not genetically inherited, and sub‐clonal (i.e., present in less than one copy per genome equivalent) in the adjacent normal esophagus (NE), while clonal in the tumor. Our results indicate that L1 retrotransposition is active in SCC of the esophagus and that insertion events are present in histologically NE that expands clonally in the subsequent tumor.

Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer

Significance Much of our genome is repetitive sequence. This property poses challenges for investigators because differences in repetitive sequences are difficult to detect. With hundreds of thousands of similar repeats, it has been difficult to discern how one person’s genome differs from another person’s genome or how tumor DNA differs from normal DNA. To solve this issue, we developed methods to target next-generation sequencing to the insertion sites of the most variable repeats. Computational pipelines to make these studies scalable and more widely accessible were needed, however. Here, we report a pipeline that accomplishes this goal. We use it to demonstrate insertions of the long interspersed element-1 (LINE-1) acquired in ovarian cancer that may contribute to the development of these tumors. Mammalian genomes are replete with interspersed repeats reflecting the activity of transposable elements. These mobile DNAs are self-propagating, and their continued transposition is a source of both heritable structural variation as well as somatic mutation in human genomes. Tailored approaches to map these sequences are useful to identify insertion alleles. Here, we describe in detail a strategy to amplify and sequence long interspersed element-1 (LINE-1, L1) retrotransposon insertions selectively in the human genome, transposon insertion profiling by next-generation sequencing (TIPseq). We also report the development of a machine-learning–based computational pipeline, TIPseqHunter, to identify insertion sites with high precision and reliability. We demonstrate the utility of this approach to detect somatic retrotransposition events in high-grade ovarian serous carcinoma.

Diagnostic utility of 5-hydroxymethylcytosine immunohistochemistry in melanocytic proliferations

Decreased hydroxymethylated cytosine (5‐hydroxymethycytosine, 5‐hmC) is reported to correlate with melanocyte dysplasia. The purpose of this study was to assess the diagnostic utility of this observation. 5‐hmC immunohistochemistry was performed on tissue microarrays containing 171‐melanocytic lesions from two different institutions. An immunohistochemical staining score representing the percentage and intensity of nuclear staining was assigned. The performance characteristics of 5‐hmC immunohistochemistry for discriminating between a nevus and melanoma were determined. Additional cases of melanoma arising in a nevus (n = 8), nodal nevi (n = 5) and melanoma micrometastases to a lymph node (n = 6) were also assessed. Pronounced 5‐hmC loss was observed in melanomas when compared with nevi (mean ± standard deviation = 6.71 ± 11.78 and 55.19 ± 23.66, respectively, p < 0.0001). While the mean immunohistochemical staining score values for melanocytic nevi and melanoma were distinct, there was considerable variability in immunohistochemical staining score within a single diagnostic category. The sensitivity and specificity of this assay for nevus vs. melanoma is 92.74 and 97.78%, respectively. Distinct biphasic staining patterns were observed in cases of melanoma arising in association with a nevus. Relative changes of 5‐hmC expression within a single lesion may be more informative than absolute values when using 5‐hmC as a diagnostic adjunct.

Translocation junctions in TCF3-PBX1 acute lymphoblastic leukemia/lymphoma cluster near transposable elements

BackgroundHematolymphoid neoplasms frequently harbor recurrent genetic abnormalities. Some of the most well recognized lesions are chromosomal translocations, and many of these are known to play pivotal roles in pathogenesis. In lymphoid malignancies, some translocations result from erroneous V(D)J-type events. However, other translocation junctions appear randomly positioned and their underlying mechanisms are not understood.ResultsWe tested the hypothesis that genomic repeats, including both simple tandem and interspersed repeats, are involved in chromosomal translocations arising in hematopoietic malignancies. Using a database of translocation junctions and RepeatMasker annotations of the reference genome assembly, we measured the proximity of translocation sites to their nearest repeat. We examined 1,174 translocation breakpoints from 10 classifications of hematolymphoid neoplasms. We measured significance using Student’s t-test, and we determined a false discovery rate using a random permutation statistics technique.ConclusionsMost translocations showed no propensity to involve genomic repeats. However, translocation junctions at the transcription factor 3 (TCF3)/E2A immunoglobulin enhancer binding factors E12/E47 (E2A) locus clustered within, or in proximity to, transposable element sequences. Nearly half of reported TCF3 translocations involve a MER20 DNA transposon. Based on this observation, we propose this sequence is important for the oncogenesis of TCF3-PBX1 acute lymphoblastic leukemia.