Publications by authors named "Shuko Harada"

53 Publications

The Interpretation of Sequence Variants in Myeloid Neoplasms.

Am J Clin Pathol 2021 Jun 22. Epub 2021 Jun 22.

Department of Pathology, Division of Genomic Diagnostics and Bioinformatics, University of Alabama at Birmingham, Birmingham, AL, USA.

Objectives: To provide an overview of the challenges encountered during the interpretation of sequence variants detected by next-generation sequencing (NGS) in myeloid neoplasms, as well as the limitations of the technology with the goal of preventing the over- or undercalling of alterations that may have a significant effect on patient management.

Methods: Review of the peer-reviewed literature on the interpretation, reporting, and technical challenges of NGS assays for myeloid neoplasms.

Results: NGS has been integrated widely and rapidly into the standard evaluating of myeloid neoplasms. Review of the literature reveals that myeloid sequence variants are challenging to detect and interpret. Large insertions and guanine-cytosine-heavy areas prove technically challenging while frameshift and truncating alterations may be classified as variants of uncertain significance by tertiary analysis informatics pipelines due to their absence in the literature and databases.

Conclusions: The analysis and interpretation of NGS results in myeloid neoplasia are challenging due to the varied number of detectable gene alterations. Familiarity with the genomic landscape of myeloid malignancies and knowledge of the tools available for the interpretation of sequence variants are essential to facilitate translation into clinical and therapy decisions.
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http://dx.doi.org/10.1093/ajcp/aqab039DOI Listing
June 2021

Diagnostic utility of one-stop fusion gene panel to detect TFE3/TFEB gene rearrangement and amplification in renal cell carcinomas.

Mod Pathol 2021 Jun 19. Epub 2021 Jun 19.

Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL, USA.

MiT family translocation renal cell carcinoma (MiT-RCC) harbors translocations involving the TFE3 or TFEB genes. RCC with TFEB amplification is also identified and is associated with a more aggressive clinical course. Accurate diagnosis of MiT-RCC is crucial for patient management. In this study, we evaluated the performance of the Archer FusionPlex assay for detection of MiT-RCC with TFE3 or TFEB translocations and TFEB amplifications. RNA was extracted from 49 RCC FFPE tissue samples with known TFE3/TFEB status (26 TFE3 FISH positive, 12 TFEB FISH positive, 4 TFEB amplified (1 case both split and amplified), and 8 FISH negative) using the Covaris extraction kit. Target enriched cDNA libraries were prepared using the Archer FusionPlex kit and sequenced on the Illumina NextSeq 550. We demonstrate that the age of the specimen, quality of RNA, and sequencing metrics are important for fusion detection. Fusions were identified in 20 of 21 cases less than 2 years old, and TFE3/TFEB rearrangements were detected in all cases with Fusion QC ≥ 100. The assay identified intrachromosomal inversions in two cases (TFE3-RBM10 and NONO-TFE3), usually difficult to identify by FISH assays. TFEB mRNA expression and the TFEB/TFE3 mRNA expression ratio were significantly higher in RCCs with TFEB fusion and TFEB gene amplification compared to tumors without TFEB fusion or amplification. A cutoff TFEB/TFE3 ratio of 0.5 resulted in 97.3% concordance to FISH results with no false negatives. Our study demonstrates that the FusionPlex assay successfully identifies TFE3 and TFEB fusions including intrachromosomal inversions. Age of the specimen and certain sequencing metrics are important for successful fusion detection. Furthermore, mRNA expression levels may be used for predicting cases harboring TFEB amplification, thereby streamlining testing. This assay enables accurate molecular detection of multiple subtypes of MiT-RCCs in a convenient workflow.
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http://dx.doi.org/10.1038/s41379-021-00858-yDOI Listing
June 2021

Discordance Between Immunohistochemistry and In Situ Hybridization to Detect HER2 Overexpression/Gene Amplification in Breast Cancer in the Modern Age: A Single Institution Experience and Pooled Literature Review Study: Discordance between HER2 overexpression and gene amplification in breast cancer.

Clin Breast Cancer 2021 May 17. Epub 2021 May 17.

Department of Pathology; O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL. Electronic address:

Background: Human epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported.

Methods: We determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis.

Results: Of the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC/ISH and 17 IHC/ISH, representing 1.6% and 11.9% of IHC and IHC cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC/ISH+ and IHC/ISH discordances were seen in all antibody clones and ISH methods used. The IHC/ISH discordance was significantly higher than IHC/ISH (13.8% vs. 3%, P < .0001). The overall discordance constituted 4% of all cases and 5.4% of those with an unequivocal IHC status. Significantly lower incongruities for both IHC/ISH and IHC/ISH were found in those published after 2018. The discordances probably reflect altered biology of HER2 oncogene/oncoprotein. Routinely performing both IHC and ISH may uncover such cases to prevent denial of potentially beneficial targeted therapy.
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http://dx.doi.org/10.1016/j.clbc.2021.05.004DOI Listing
May 2021

NAB2-STAT6 Gene Fusions to Evaluate Primary/Metastasis of Hemangiopericytoma/Solitary Fibrous Tumors.

Am J Clin Pathol 2021 Jun 2. Epub 2021 Jun 2.

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Objectives: Hemangiopericytomas (HPCs) and solitary fibrous tumors (SFTs) were considered two distinct entities, but a common gene fusion, NAB2-STAT6, has been identified in both. Although rare, HPCs and SFTs do metastasize, some many years later after resection. Given the extended disease-free interval, it can be difficult to determine with certainty if an HPC or SFT at a new anatomic location represents a second primary or metastatic disease.

Methods: RNA was extracted from formalin-fixed, paraffin-embedded tissue of two patients with multiple SFT/HPC samples. The fusion gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) and a custom-designed Archer FusionPlex panel (94 target genes) and the Illumina NextSeq 550.

Results: We identified two patients with multiple resections for HPC/SFT during 26 years at our institution. The first patient had a history of HPC and almost 10 years later she was diagnosed with malignant SFT. The HPC and the SFT shared the same fusion breakpoint. The second patient had multiple lesions in the brain and bone/soft tissue over a 27-year span following a diagnosis of meningeal SFT. Three lesions from this patient shared the same fusion breakpoint.

Conclusions: Our study demonstrated the same fusion breakpoints in primary and metastatic SFTs/HPCs at different time points using both RT-PCR and the Archer fusion panel.
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http://dx.doi.org/10.1093/ajcp/aqab045DOI Listing
June 2021

The accuracy of HPV genotyping in isolation and in combination with CD4 and HIV viral load for the identification of HIV-infected women at risk for developing cervical cancer.

Cancer Med 2021 03 19;10(5):1900-1909. Epub 2021 Feb 19.

Department of Medicine/Division of Infectious Diseases, The University of Alabama at Birmingham (UAB), Birmingham, AL, USA.

Background: Human papillomavirus (HPV) genotype testing has limited utility to identify human immunodeficiency virus-infected (HIV+) women's risk for developing cervical cancer (CC) due to high positivity rate of high-risk (HR) HPVs. We investigated the accuracy of HPV testing in isolation/in combination with CD4 and HIV viral load (VL) to identify HIV+ women at risk for developing CC.

Methods: Study consisted of 344 HIV+ women on combination antiretroviral therapy (cART), tested for cervical cytology/HPV using the Cobas test and had data on absolute CD4 count and VL measurements. We calculated the positive predictive value (PPV) and negative predictive value (NPV) of HPV testing, pre-, post-cART, and current CD4 and VL in isolation and in combinations to identify those with or free of higher than atypical squamous cells of unknown significance (ASCUS+) or low-grade intraepithelial lesions (LSIL+).

Results: HPV test in combination with pre-/post-cART or current CD4 counts and VL had higher PPVs compared to HPV test alone for identifying ASCUS+ or LSIL+. PPV of HPV-CD4 combinations yielded higher PPVs compared to HPV-VL combinations. The NPVs with pre-, post-cART, or current CD4 count and VL in isolation or in combinations were comparable to that of HPV test alone.

Conclusions: Our results provide a more accurate tool for managing HIV+ women by combining Cobas HPV with CD4 and VL, especially those who had an undesirable pre-cART CD4 and VL status. Our results also indicate the usefulness of CD4 and VL measurements to identify those at lower risk in the absence of HPV testing.
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http://dx.doi.org/10.1002/cam4.3785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940247PMC
March 2021

miR-31 Displays Subtype Specificity in Lung Cancer.

Cancer Res 2021 Apr 8;81(8):1942-1953. Epub 2021 Feb 8.

Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama.

miRNA rarely possess pan-oncogenic or tumor-suppressive properties. Most miRNAs function under tissue-specific contexts, acting as either tumor suppressors in one tissue, promoting oncogenesis in another, or having no apparent role in the regulation of processes associated with the hallmarks of cancer. What has been less clear is the role of miRNAs within cell types of the same tissue and the ability within each cell type to contribute to oncogenesis. In this study, we characterize the role of one such tissue-specific miRNA, miR-31, recently identified as the most oncogenic miRNA in lung adenocarcinoma, across the histologic spectrum of human lung cancer. Compared with normal lung tissue, miR-31 was overexpressed in patient lung adenocarcinoma, squamous cell carcinoma, and large-cell neuroendocrine carcinoma, but not small-cell carcinoma or carcinoids. miR-31 promoted tumor growth in mice of xenografted human adenocarcinoma and squamous cell carcinoma cell lines, but not in large- or small-cell carcinoma lines. While miR-31 did not promote primary tumor growth of large- and small-cell carcinoma, it did promote spontaneous metastasis. Mechanistically, miR-31 altered distinct cellular signaling programs within each histologic subtype, resulting in distinct phenotypic differences. This is the first report distinguishing diverse functional roles for this miRNA across the spectrum of lung cancers and suggests that miR-31 has broad clinical value in human lung malignancy. SIGNIFICANCE: These findings demonstrate the oncogenic properties of miR-31 in specific subtypes of lung cancer and highlight it as a potential therapeutic target in these subtypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/1942/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137562PMC
April 2021

Acute Myeloid Leukemia Case Harboring Unusual FLT3 Variant: Somatic vs Germline?

Lab Med 2021 May;52(3):e53-e56

Department of Pathology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama.

FLT3 mutations are considered a prognostic and predictive marker. Here we report on a patient with a rare FLT3 germline variant in the context of relapsed acute myeloid leukemia (AML). A female patient aged 57 years presented with AML with mutations in the IDH2, ASXL1, and DNMT3A genes. She underwent allogenic hematopoietic stem cell transplant but relapsed 2 years posttransplant. Targeted next generation sequencing identified a new missense variant in the FLT3 tyrosine kinase domain c.2440G > T (p.A814S). The treating team considered the possibility of patient eligibility for an FLT3 inhibitor. Because both somatic and germline mutations can be identified in tumor tissue with high-throughput sequencing, it becomes important to distinguish the origin of these alterations when possible-especially, in this challenging case, to define the treatment modality. Simultaneous tumor/germline sequencing allows for the identification of rare germline mutations and may help in determining their significance in the pathogenesis of disease.
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http://dx.doi.org/10.1093/labmed/lmaa080DOI Listing
May 2021

TFEB rearranged renal cell carcinoma. A clinicopathologic and molecular study of 13 cases. Tumors harboring MALAT1-TFEB, ACTB-TFEB, and the novel NEAT1-TFEB translocations constantly express PDL1.

Mod Pathol 2021 04 18;34(4):842-850. Epub 2020 Nov 18.

Department of Diagnostic and Public Health, Section of Pathology, University of Verona, Verona, Italy.

Renal cell carcinomas with t(6;11) chromosome translocation has been classically characterized by the rearrangement of the TFEB gene, located on chromosome 6, and MALAT1 gene, located on chromosome 11. Recently, a few other genes have been described as fusion partners in TFEB rearranged renal cell carcinomas. Although most of TFEB rearranged renal cell carcinomas have an indolent behavior, in the rare cases of advanced metastatic disease targeted therapy and predictive markers remain lacking. In the present study, we collected 13 TFEB rearranged renal cell carcinomas, confirmed by FISH, analyzing their morphology and exploring the novel gene partners. Looking for predictive markers, we have also performed PDL1 immunohistochemical analysis by using four different assays (E1L3N, 22C3, SP142, and SP263). MALAT1 gene rearrangement has been found in ten tumors, five cases showing classical biphasic morphology with "rosettes", five cases without "rosettes" mimicking other renal cell carcinomas or epithelioid angiomyolipoma/pure epithelioid PEComa. We identified two different partner genes, ACTB and NEAT1, the latter previously unreported and occurring in a tumor with an unusual solid and cystic appearance. In both cases, the "rosettes" were absent. In one case no gene partner was identified. Overall, in 12 of 13 TFEB-rearranged renal cell carcinomas staining for PDL1 SP263 was observed, whereas the other antibodies were less reliable or more difficult to interpret. In conclusion, we described the third case of ACTB-TFEB rearranged renal cell carcinoma and a novel NEAT1-TFEB rearranged renal cell carcinoma, both without the distinctive biphasic morphology typical of t(6;11) renal cell carcinoma. Finally, PDL1 SP263 was constantly expressed in TFEB rearranged renal cell carcinoma with possible clinical benefit which requires further investigations.
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http://dx.doi.org/10.1038/s41379-020-00713-6DOI Listing
April 2021

Gastric Sclerosing Epithelioid Fibrosarcoma Harboring a Rare - Fusion.

Int J Surg Pathol 2021 Aug 23;29(5):565-570. Epub 2020 Sep 23.

Depatment of Pathology, University of Alabama at Birmingham, Birmingham AL, USA.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare, aggressive soft-tissue tumor, commonly occurring in upper and lower extremities, the limb girdle, and the head and neck, which shows morphologic and molecular overlap with low-grade fibromyxoid sarcoma. For SEF in soft tissues, 100 case reports have been published. To our knowledge, the present case is the first to be reported in English literature for a primary SEF of the stomach with a rare fusion. We report a case of gastric SEF in a 35-year-old female who presented with nonspecific symptoms, including night sweat, cough, and iron deficiency anemia for the past few months. Further workup showed, on computed tomography, a large, heterogeneously enhancing and centrally necrotic left upper quadrant mass, which measured approximately 8.4 cm. A laparoscopic partial gastrectomy with distal pancreatectomy and splenectomy was performed. Histological examination and immunohistochemical staining suggested the diagnosis of primary gastric SEF, which was later confirmed by sarcoma fusion panel showing fusion. In this article, we report this first case of SEF in the stomach with a rare fusion, which has been previously reported only once in SEFs of soft tissue.
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http://dx.doi.org/10.1177/1066896920961174DOI Listing
August 2021

Association of Event-Free and Distant Recurrence-Free Survival With Individual-Level Pathologic Complete Response in Neoadjuvant Treatment of Stages 2 and 3 Breast Cancer: Three-Year Follow-up Analysis for the I-SPY2 Adaptively Randomized Clinical Trial.

JAMA Oncol 2020 09;6(9):1355-1362

Division of Hematology and Oncology, University of California, San Francisco.

Importance: Pathologic complete response (pCR) is a known prognostic biomarker for long-term outcomes. The I-SPY2 trial evaluated if the strength of this clinical association persists in the context of a phase 2 neoadjuvant platform trial.

Objective: To evaluate the association of pCR with event-free survival (EFS) and pCR with distant recurrence-free survival (DRFS) in subpopulations of women with high-risk operable breast cancer treated with standard therapy or one of several novel agents.

Design, Setting, And Participants: Multicenter platform trial of women with operable clinical stage 2 or 3 breast cancer with no prior surgery or systemic therapy for breast cancer; primary tumors were 2.5 cm or larger. Women with tumors that were ERBB2 negative/hormone receptor (HR) positive with low 70-gene assay score were excluded. Participants were adaptively randomized to one of several different investigational regimens or control therapy within molecular subtypes from March 2010 through 2016. The analysis included participants with follow-up data available as of February 26, 2019.

Interventions: Standard-of-care neoadjuvant therapy consisting of taxane treatment with or without (as control) one of several investigational agents or combinations followed by doxorubicin and cyclophosphamide.

Main Outcomes And Measures: Pathologic complete response and 3-year EFS and DRFS.

Results: Of the 950 participants (median [range] age, 49 [23-77] years), 330 (34.7%) achieved pCR. Three-year EFS and DRFS for patients who achieved pCR were both 95%. Hazard ratios for pCR vs non-pCR were 0.19 for EFS (95% CI, 0.12-0.31) and 0.21 for DRFS (95% CI, 0.13-0.34) and were similar across molecular subtypes, varying from 0.14 to 0.18 for EFS and 0.10 to 0.20 for DRFS.

Conclusions And Relevance: The 3-year outcomes from the I-SPY2 trial show that, regardless of subtype and/or treatment regimen, including 9 novel therapeutic combinations, achieving pCR after neoadjuvant therapy implies approximately an 80% reduction in recurrence rate. The goal of the I-SPY2 trial is to rapidly identify investigational therapies that may improve pCR when validated in a phase 3 confirmatory trial. Whether pCR is a validated surrogate in the sense that a therapy that improves pCR rate can be assumed to also improve long-term outcome requires further study.

Trial Registration: ClinicalTrials.gov Identifier: NCT01042379.
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http://dx.doi.org/10.1001/jamaoncol.2020.2535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378873PMC
September 2020

Intracranial anaplastic solitary fibrous tumor/hemangiopericytoma: immunohistochemical markers for definitive diagnosis.

Neurosurg Rev 2021 Jun 15;44(3):1591-1600. Epub 2020 Jul 15.

Department of Neurosurgery, Ehime University Graduate School of Medicine, 454 Shitsukawa, Toon, Ehime, 791-0295, Japan.

Intracranial anaplastic hemangiopericytoma (AHPC) is a rare and malignant subset of solitary fibrous tumor/hemangiopericytoma (SFT/HPC) as per the WHO 2016 Classification of Tumors of the Central Nervous System. AHPC portends a poor prognosis and is associated with higher rates of recurrence/metastasis in comparison with SFT/HPC. Accordingly, it is critical to continue to define the clinical course of patients with AHPC and in so doing further refine clinicopathologic/immunohistochemical (IHC) criteria needed for definitive diagnosis. Herein, we describe clinical/histological characteristics of six patients with AHPC. In addition, we reviewed and analyzed the expression of various IHC markers reported within the literature (i.e., a total of 354 intracranial SFT/HPCs and 460 meningiomas). Histologically, tumors from our six patients were characterized by a staghorn-like vascular pattern, mitotic cells, and strong nuclear atypia. Immunohistochemically, all tumors displayed positive nuclear staining for STAT6; other markers, including CD34 and Bcl-2, were expressed only in three patients. Analysis of IHC expression patterns for SFT/HPC and meningioma within the literature revealed that nuclear expression of STAT6 had the highest specificity (100%) for SFT/HPC, followed by ALDH1 (97.2%) and CD34 (93.6%). Of note, SSTR2A (95.2%) and EMA (85%) displayed a high specificity for meningioma. Anaplastic SFT/HPC is a tumor with poor prognosis that is associated with higher rates of recurrence and metastasis in comparison with SFT/HPC. Given that anaplastic SFT/HPC requires more aggressive treatment than meningioma despite of a similar presentation on imaging, it is crucial to be able to distinguish between these tumors.
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http://dx.doi.org/10.1007/s10143-020-01348-6DOI Listing
June 2021

Comparison of Tissue Molecular Biomarker Testing Turnaround Times and Concordance Between Standard of Care and the Biocartis Idylla Platform in Patients With Colorectal Cancer.

Am J Clin Pathol 2020 07;154(2):266-276

Department of Pathology and Laboratory Medicine, Comprehensive Care and Research Center, Cancer Treatment Centers of America Philadelphia, Philadelphia, PA.

Objectives: Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti-epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories.

Methods: Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods.

Results: There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days.

Conclusions: The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.
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http://dx.doi.org/10.1093/ajcp/aqaa044DOI Listing
July 2020

Rare MDM2 amplification in a fat-predominant angiomyolipoma.

Virchows Arch 2020 Nov 15;477(5):661-666. Epub 2020 May 15.

Pathology Department, University of Alabama at Birmingham, Birmingham, AL, USA.

Angiomyolipomas (AMLs) are triphasic tumors (smooth muscle, vascular and adipocytic components) with myomelanocytic differentiation, arising most commonly in the kidneys, which can show predominant epithelioid morphology and fat-predominant or fat-poor variants. Fat-predominant AMLs can show areas of hypercellularity and lipoblast-like cells, and these features can mimic well-differentiated liposarcoma (WDLS). To date, only one documented metastatic epithelioid AML showed unequivocal MDM2 amplification by fluorescence in situ hybridization. We describe our findings in a series of 35 AMLs including epithelioid, fat-poor, and fat-predominant variants, following interrogation of the MDM2 locus by FISH and CISH assays. MDM2 amplification was detected in 1 fat-predominant AML. Our findings demonstrate that rare MDM2 amplifications can occur in AMLs. We favor that this finding likely represents a "molecular bystander" event since these tumors are mainly driven by aberrations in the TSC1/TSC2 genes. Nevertheless, the presence of MDM2 amplification in a fat-predominant AML could present a potential diagnostic pitfall, particularly when confronted with the differential diagnosis of fat-predominant AML and WDLS in limited material from the retroperitoneum.
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http://dx.doi.org/10.1007/s00428-020-02813-9DOI Listing
November 2020

Identification of ALDH1A3 as a Viable Therapeutic Target in Breast Cancer Metastasis-Initiating Cells.

Mol Cancer Ther 2020 05 3;19(5):1134-1147. Epub 2020 Mar 3.

Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama.

The development of efficacious therapies targeting metastatic spread of breast cancer to the brain represents an unmet clinical need. Accordingly, an improved understanding of the molecular underpinnings of central nervous system spread and progression of breast cancer brain metastases (BCBM) is required. In this study, the clinical burden of disease in BCBM was investigated, as well as the role of aldehyde dehydrogenase 1A3 (ALDH1A3) in the metastatic cascade leading to BCBM development. Initial analysis of clinical survival trends for breast cancer and BCBM determined improvement of breast cancer survival rates; however, this has failed to positively affect the prognostic milestones of triple-negative breast cancer (TNBC) brain metastases (BM). ALDH1A3 and a representative epithelial-mesenchymal transition (EMT) gene signature (mesenchymal markers, CD44 or Vimentin) were compared in tumors derived from BM, lung metastases (LM), or bone metastases (BoM) of patients as well as mice after injection of TNBC cells. Selective elevation of the EMT signature and ALDH1A3 were observed in BM, unlike LM and BoM, especially in the tumor edge. Furthermore, ALDH1A3 was determined to play a role in BCBM establishment via regulation of circulating tumor cell adhesion and migration phases in the BCBM cascade. Validation through genetic and pharmacologic inhibition of ALDH1A3 via lentiviral shRNA knockdown and a novel small-molecule inhibitor demonstrated selective inhibition of BCBM formation with prolonged survival of tumor-bearing mice. Given the survival benefits via targeting ALDH1A3, it may prove an effective therapeutic strategy for BCBM prevention and/or treatment.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716183PMC
May 2020

An Overview of Molecular Genetic Diagnosis Techniques.

Curr Protoc Hum Genet 2020 03;105(1):e97

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama.

Our understanding of genetic disease(s) has increased exponentially since the completion of human genome sequencing and the development of numerous techniques to detect genetic variants. These techniques have not only allowed us to diagnose genetic disease, but in so doing, also provide increased understanding of the pathogenesis of these diseases to aid in developing appropriate therapeutic options. Additionally, the advent of next-generation or massively parallel sequencing (NGS/MPS) is increasingly being used in the clinical setting, as it can detect a number of abnormalities from point mutations to chromosomal rearrangements as well as aberrations within the transcriptome. In this article, we will discuss the use of multiple techniques that are used in genetic diagnosis. © 2020 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cphg.97DOI Listing
March 2020

Validation of the Spartan RXCYP2C19 Genotyping Assay Utilizing Blood Samples.

Clin Transl Sci 2020 03 29;13(2):260-264. Epub 2019 Nov 29.

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

The antiplatelet agent clopidogrel, a prodrug that requires bioactivation through the cytochrome P450 2C19 (CYP2C19) enzyme, is commonly prescribed post-percutaneous coronary intervention (PCI). Genetic variation in CYP2C19 contributes to individual variability in clopidogrel response, and can lead to adverse cardiovascular events. Incorporating CYP2C19 testing during routine clinical care helps identify high-risk patients, and provides the opportunity for pharmacotherapeutic interventions in the early post-PCI period. The Spartan RX CYP2C19 System has emerged as an optimal genotyping assay for use in clinical care due to ease of use, utilization of buccal swabs, and rapid turnaround time. However, workflow constraints related to sample collection and processing, storage, time, and personnel were encountered when integrating testing into clinical care. To improve clinical workflow and successfully implement CYP2C19 genotyping at our institution, we validated the Spartan RX System to return genotype utilizing blood samples. Our Molecular Diagnostic Laboratory tested 26 known reference materials and both blood and buccal swab samples from 23 patients and volunteers using the Spartan RX Assay. Genotype results were 100% concordant between DNA from blood and buccal swabs for all patients or volunteers, and consistent with expected results for the 26 reference materials. For reproducibility, three samples were tested in at least four separate runs, with all resulting genotypes in agreement between runs. Post-validation, the laboratory began offering CYP2C19 testing during clinical care. DNA extracted from blood can serve as a genomic DNA source for the Spartan RX Assay. Alteration of the methodology allowed for clinical implementation to support genotype-guided therapy.
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http://dx.doi.org/10.1111/cts.12714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070822PMC
March 2020

Molecular Pathology of Colorectal Cancer.

Adv Anat Pathol 2020 Jan;27(1):20-26

The University of Alabama at Birmingham, Birmingham, AL.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer. This review gives an overview of the current knowledge of molecular mechanisms of colorectal carcinogenesis and the role of molecular testing in the management of CRC. The majority of CRCs arise from precursor lesions such as adenoma, transforming to adenocarcinoma. Three molecular carcinogenesis pathways have been identified; (1) chromosomal instability, (2) microsatellite instability (MSI), and (3) CpG island methylator phenotype, each account for ~85%, 15%, and 17%, respectively. Evaluation of MSI status, extended RAS mutation analysis, and BRAF mutation analysis are recommended by the guideline published by joint effort from professional societies. MSI testing is important for identification of Lynch syndrome patients and prognostic and predictive markers. Extended RAS testing is an important predictive marker for antiepidermal growth factor receptor therapy. BRAF p.V600 mutation status can be used as prognostic marker, but not predictive marker for antiepidermal growth factor receptor therapies. Emerging technologies utilizing high throughput sequencing have introduced novel biomarkers and testing strategies. Tumor mutation burden predicts immunotherapy response in addition to MSI status. Liquid biopsy can be utilized when adequate tissue sample is not available or for monitoring therapy response. However, assay standardization and guidelines and recommendations for utilization of these assay will be needed. The advancement in CRC research and technologies will allow better prognostication and therapy stratification for the management of patients with CRCs.
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http://dx.doi.org/10.1097/PAP.0000000000000247DOI Listing
January 2020

Randomized trial of weight loss in primary breast cancer: Impact on body composition, circulating biomarkers and tumor characteristics.

Int J Cancer 2020 05 5;146(10):2784-2796. Epub 2019 Sep 5.

O'Neal Comprehensive Cancer Center at UAB, Birmingham, AL.

Obesity adversely impacts overall and cancer-specific survival among breast cancer patients. Preclinical studies demonstrate negative energy balance inhibits cancer progression; however, feasibility and effects in patients are unknown. A two-arm, single-blinded, randomized controlled weight-loss trial was undertaken presurgery among 32 overweight/obese, Stage 0-II breast cancer patients. The attention control arm (AC) received basic nutritional counseling and upper-body progressive resistance training whereas the weight loss intervention (WLI) arm received identical guidance, plus counseling on caloric restriction and aerobic exercise to promote 0.68-0.92 kg/week weight loss. Anthropometrics, body composition, blood and survey data were collected at baseline and presurgery ∼30 days later. Tumor markers (e.g., Ki67) and gene expression were assessed on biopsy and surgical specimens; sera were analyzed for cytokines, growth and metabolic factors. Significant WLI vs. AC differences were seen in baseline-to-follow-up changes in weight (-3.62 vs. -0.52 kg), %body fat (-1.3 vs. 0%), moderate-to-vigorous physical activity (+224 vs. +115 min/week), caloric density (-0.3 vs. 0 kcal/g), serum leptin (-12.3 vs. -4.0 ng/dl) and upregulation of tumor PI3Kinase signaling and cell cycle-apoptosis related genes (CC-ARG; all p-values <0.05). Cytolytic CD56 NK cell expression was positively associated with weight loss; CC-ARG increased with physical activity. Increased tumor (nuclear) TNFα and IL-1β, CX3CL1 and CXCL1 gene expression was observed in the WLI. Tumor Ki67 did not differ between arms. Feasibility benchmarks included 80% accrual, 100% retention, no adverse effects and excellent adherence. Short-term weight loss interventions are feasible; however, mixed effects on tumor biology suggest unclear benefit to presurgical caloric restriction, but possible benefits of physical activity.
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http://dx.doi.org/10.1002/ijc.32637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155016PMC
May 2020

Clear cell papillary renal cell carcinoma: molecular profile and virtual karyotype.

Hum Pathol 2019 09 6;91:52-60. Epub 2019 Jun 6.

Department of Urology, University of Alabama at Birmingham, Birmingham, AL 35233, USA; Department of Radiology, University of Alabama at Birmingham, Birmingham, AL 35233, USA.

Clear cell papillary renal cell carcinoma (CCP-RCC) is a recently recognized tumor that shares morphologic features of both clear cell renal cell carcinoma and papillary renal cell carcinoma but behaves in a more indolent fashion. To date, there is little molecular information available on CCP-RCC. DNA was extracted from formalin-fixed, paraffin-embedded tissue blocks of 22 cases of CCP-RCC at the University of Alabama at Birmingham. Targeted next-generation sequencing and single-nucleotide polymorphism array were performed on all cases. Next-generation sequencing analysis found 30 somatic variants across 63.3% of cases. Seventeen variants (56.7%) were predicted to be deleterious or possibly/probably damaging. Single-nucleotide polymorphism array analysis found copy number abnormalities and/or loss of heterozygosity in 22.7% of cases. We analyzed the genetic characteristics of a group of CCP-RCCs cases and found them to be genetically different from one another. Some cases were genetically similar to clear cell renal cell carcinoma.
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http://dx.doi.org/10.1016/j.humpath.2019.05.011DOI Listing
September 2019

Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics.

Am J Clin Pathol 2019 06;152(1):27-35

University of Alabama at Birmingham.

Objectives: To compare the effects of two methods of formalin-fixed paraffin-embedded (FFPE) tissue harvesting on DNA quality and next-generation sequencing (NGS) quality metrics.

Methods: DNA integrity number (DIN) and NGS quality metrics resulting from DNA extraction and sequencing of 199 sequential samples harvested via the Pinpoint Slide DNA Isolation System and the punch method were compared.

Results: DNA extracted from FFPE tissue punches had higher DIN than that extracted from Pinpoint samples (mean ± SD, 6.18 ± 0.83 vs 5.09 ± 0.91; P < .0001), indicating less degradation. Lower DIN correlated with lower-quality metrics of NGS, that is lower percentage of unique on-target reads, average depth of coverage, and percentage of positions with coverage depth greater than or equal to 100×, 400×, and 1,000×.

Conclusions: Our study demonstrated methods to harvest tissue from FFPE blocks may affect quality of DNA, which in turn has an effect on other NGS quality metrics.
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http://dx.doi.org/10.1093/ajcp/aqz014DOI Listing
June 2019

Adamantinoma-Like Ewing Sarcoma of the Thyroid: A Case Report and Review of the Literature.

Head Neck Pathol 2019 Dec 8;13(4):618-623. Epub 2019 Feb 8.

Department of Pathology, University of Alabama at Birmingham, 3548 North Pavilion, 1802 6th Ave. S, Birmingham, AL, 35249, USA.

Currently considered a variant of Ewing sarcoma, adamantinoma-like Ewing sarcoma is a rare malignancy that shows classic Ewing sarcoma-associated gene fusions but also epithelial differentiation. Here we present the 6th reported case of adamantinoma-like Ewing sarcoma involving the thyroid gland. Sections of the thyroid tumor from a 20-year old woman showed sheets, lobules and trabeculae of primitive, uniform, small round blue cells that diffusely expressed pankeratin, p40 and CD99. Fluorescent in situ hybridization revealed an EWSR1 gene rearrangement and an EWSR1-FLI1 fusion was detected by RT-PCR. Neck lymph nodes were not involved, and the patient was treated with a Ewing sarcoma chemotherapy protocol and radiation and is disease free 7 months after surgery. The unusual histology and immunohistochemical profile of adamantinoma-like Ewing sarcoma makes diagnosis and classification very challenging. We also present a literature review of adamantinoma-like Ewing sarcoma involving the thyroid.
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http://dx.doi.org/10.1007/s12105-019-01021-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854136PMC
December 2019

First-Line Treatment of Widely Metastatic -Mutated Salivary Duct Carcinoma With Combined BRAF and MEK Inhibition.

J Natl Compr Canc Netw 2018 10;16(10):1166-1170

Salivary duct carcinoma (SDC) is a rare and aggressive malignancy for which limited data exist to guide treatment decisions. With the advent of advanced molecular testing and tumor genomic profiling, clinicians now have the ability to identify potential therapeutic targets in difficult-to-treat cancers such as SDC. This report presents a male patient with widely metastatic SDC found on targeted next-generation sequencing to have a p.V600E mutation. He experienced a prolonged and robust response to first-line systemic chemotherapy with dabrafenib and trametinib. During his response interval, new data emerged to justify subsequent treatment with both an immune checkpoint inhibitor and androgen blockade after his disease progressed. To our knowledge, this is the first report of frontline -directed therapy eliciting a response in metastatic SDC.
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http://dx.doi.org/10.6004/jnccn.2018.7056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292446PMC
October 2018

Spindle Epithelial Tumor with Thymus-Like Differentiation (SETTLE): A Next-Generation Sequencing Study.

Head Neck Pathol 2019 Jun 7;13(2):162-168. Epub 2018 May 7.

Caris Life Sciences, Phoenix, AZ, USA.

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a malignant biphasic neoplasm of the thyroid or neck with propensity for late metastasis. Unlike synovial sarcoma, its main morphologic mimic, SETTLE lacks synovial sarcoma-associated translocations. A single case of SETTLE has shown a KRAS mutation but to date no comprehensive next generation sequencing studies of this rare neoplasm have been undertaken. Herein, we subjected 5 well defined cases of SETTLE to direct sequence analysis of 592 genes and fusion gene analysis of 52 genes frequently rearranged in human cancers. We identified one case with two pathogenic variants in the KMT2D gene, one being in an intron splice site (c.674-1A>G) and the other being a frameshift variant (p.M2829fs). This same case also had a pathogenic nonsense variant in the KMT2C gene (p.R1237*). A second case of SETTLE carried a pathogenic NRAS missense variant, Q61R. No other molecular alterations, microsatellite instability, gene fusions or amplifications were identified.
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http://dx.doi.org/10.1007/s12105-018-0927-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514029PMC
June 2019

Comparison of Two Point-of-Care Genotyping Assays for Genotype-Guided Antiplatelet Therapy.

Ann Clin Lab Sci 2017 Nov;47(6):738-743

Department of Pathology, University of Alabama at Birmingham, Birmingham AL, USA

Background: polymorphisms contribute about 12% of the variability in the antiplatelet effect of clopidogrel, which is commonly prescribed for patients undergoing percutaneous coronary intervention. For these patients, rapid turnaround time of genotyping may be critical. We validated and compared the performance of two point-of-care genotype tests, Nanosphere Verigene CYP2C19 Nucleic Acid Test and Spartan RX CYP2C19 System.

Materials And Methods: Our CLIA certified Molecular Diagnostic Laboratory performed 99 Verigene tests and 108 Spartan RX CYP2C19 assays. We compared performance and genotype results between the two platforms, across runs, and among technologists. Based on our validation results, we started offering genotyping using the Spartan RX CYP2C19 assay for post-percutaneous coronary intervention patients.

Results: Laboratory validation genotype results were consistent between both assays when the assays produced results (100% accuracy); however, the Verigene CYP2C19 had a 33% no call rate. In contrast, Spartan consistently showed accurate results. Using a newly established clinical workflow, we assayed 342 post-percutaneous coronary intervention patients with the Spartan test. Within one hour of submitting patient samples, ordering physicians were notified of any clinically significant results and provided clinical decision support.

Conclusions: Every approach has its limitations, but our practice of using the Spartan RX CYP2C19 test in our acute cardiac workflow provides accurate and rapid results to guide clinical decision-making at the point-of-care. Prospective follow-up is ongoing to evaluate outcomes and effectiveness of testing.
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November 2017

Hepatitis C Virus Genie: A Web 2.0 Interpretation and Analytics Platform for the Versant Hepatitis C Virus Genotype Line Probe Assay Version 2.0.

J Pathol Inform 2017 3;8:41. Epub 2017 Oct 3.

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Context: Hepatitis C virus (HCV) genotyping at our institution is performed using the Versant HCV genotype 2.0 Line Probe Assay (LiPA). The last steps of this procedure are manual, laborious, and error-prone process that involves the comparison of the banding pattern on a test strip to a physical reference table.

Aim: We developed a web-based HCV genotype interpretation platform that utilizes a scanned image to generate the genotypes, thus minimizing interpretation time and reducing error.

Subjects And Methods: HCV Genie 2 utilizes a database of banding patterns in conjuncture with image analysis algorithms to determine the genotype for any number of scanned LiPA strips. HCV Genie 2 is built with client-side JavaScript; allowing the program to run in the user' browser rather than on an unknown server, essentially eliminating data and patient privacy concerns.

Results: HCV Genie 2 was tested over 2 months and proved identical to human expert interpretation for 148 samples (>1000 bands identified). Manual intervention was required only for two faint bands and one false-positive band; this was done utilizing the built-in-user interface. Utilizing the original method, the trained laboratory technician interpretation time for 16 samples was 13.8 (±0.96) min as compared to 5.0 (±1.09) min with HCV Genie 2, a 63.8% decrease. In addition to the time savings, the new method provides an additional validation step, which decreases the potential for errors.

Conclusions: Our institution has moved exclusively to utilize the new techniques and tools described here. Both experienced technicians and the molecular pathologists at our institution prefer the workflow using HCV Genie. It is easier for the technicians to prepare and document, and the pathologists are more rapidly able to review and confirm results. The use of this tool will lead to increase the quality of patient care delivered through this test methodology by decreasing the potential for error. The algorithms developed here can be ported to similar band identification platforms, most directly to other LiPAs.
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http://dx.doi.org/10.4103/jpi.jpi_44_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653960PMC
October 2017

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention.

JACC Cardiovasc Interv 2018 01 1;11(2):181-191. Epub 2017 Nov 1.

Department of Medicine, University of Maryland, Baltimore, Maryland.

Objectives: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI).

Background: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI.

Methods: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele. Major adverse cardiovascular events (defined as myocardial infarction, stroke, or death) within 12 months of PCI were compared between patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy. Risk was also compared between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy. Cox regression was performed, adjusting for group differences with inverse probability of treatment weights.

Results: Among 1,815 patients, 572 (31.5%) had a loss-of-function allele. The risk for major adverse cardiovascular events was significantly higher in patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy (23.4 vs. 8.7 per 100 patient-years; adjusted hazard ratio: 2.26; 95% confidence interval: 1.18 to 4.32; p = 0.013). Similar results were observed among 1,210 patients with acute coronary syndromes at the time of PCI (adjusted hazard ratio: 2.87; 95% confidence interval: 1.35 to 6.09; p = 0.013). There was no difference in major adverse cardiovascular events between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy (adjusted hazard ratio: 1.14; 95% confidence interval: 0.69 to 1.88; p = 0.60).

Conclusions: These data from real-world observations demonstrate a higher risk for cardiovascular events in patients with a CYP2C19 loss-of-function allele if clopidogrel versus alternative therapy is prescribed. A future randomized study of genotype-guided antiplatelet therapy may be of value.
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http://dx.doi.org/10.1016/j.jcin.2017.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775044PMC
January 2018

Implementation and utilization of the molecular tumor board to guide precision medicine.

Oncotarget 2017 Aug 14;8(34):57845-57854. Epub 2017 Jun 14.

Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA.

Background: With rapid advances in genomic medicine, the complexity of delivering precision medicine to oncology patients across a university health system demanded the creation of a Molecular Tumor Board (MTB) for patient selection and assessment of treatment options. The objective of this report is to analyze our progress to date and discuss the importance of the MTB in the implementation of personalized medicine.

Materials And Methods: Patients were reviewed in the MTB for appropriateness for comprehensive next generation sequencing (NGS) cancer gene set testing based on set criteria that were in place. Because profiling of stage IV lung cancer, colon cancer, and melanoma cancers were standard of care, these cancer types were excluded from this process. We subsequently analyzed the types of cases referred for testing and approved with regards to their results.

Results: 191 cases were discussed at the MTB and 132 cases were approved for testing. Forty-six cases (34.8%) had driver mutations that were associated with an active targeted therapeutic agent, including , and . An additional 56 cases (42.4%) had driver mutations previously reported in some type of cancer. Twenty-two cases (16.7%) did not have any clinically significant mutations. Eight cases did not yield adequate DNA. 15 cases were considered for targeted therapy, 13 of which received targeted therapy. One patient experienced a near complete response. Seven of 13 had stable disease or a partial response.

Conclusions: MTB at University of Alabama-Birmingham is unique because it reviews the appropriateness of NGS testing for patients with recurrent cancer and serves as a forum to educate our physicians about the pathways of precision medicine. Our results suggest that our detection of actionable mutations may be higher due to our careful selection. The application of precision medicine and molecular genetic testing for cancer patients remains a continuous educational process for physicians.
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http://dx.doi.org/10.18632/oncotarget.18471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593688PMC
August 2017

Immunogenomics: using genomics to personalize cancer immunotherapy.

Virchows Arch 2017 Aug 20;471(2):209-219. Epub 2017 May 20.

Department of Pathology, University of Alabama at Birmingham, 1802 6th Avenue South, NP3540, Birmingham, AL, 35249, USA.

While the use of genomic data has the potential to revolutionize patient care, there is still much work to be done with regard to the transformation of host-tumor interactions into favorable clinical outcomes for our patients. High-throughput technologies, such as next-generation sequencing (NGS), have rapidly advanced our understanding of oncology, and we are learning that most tumors do not simply possess consistently mutated genes that are responsible for tumorigenesis, facilitating the need for personalized cancer therapy. A T cell-dependent mechanism of cancer progression was discovered in 2012, providing a potential link to cancer immunotherapy. Since then, an antibody against cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), ipilimumab, and three programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) inhibitors, pembrolizumab (Keytruda), nivolumab (Opdivo), and atezolizumab (Tecentriq), were approved by the Food and Drug Administration (FDA) in the USA. In this review article, based on evidence that has been emerging in the literature over the last decade, we will discuss the basis for including genomic data in immunotherapy regimens, the current progress in identifying biomarkers targetable by immune checkpoint blockade, and the application of these therapies in modern oncology programs. Going forward, the clinical application of NGS in personalized oncology programs could include dose monitoring and adjustment or the development of individualized vaccines or other personalized therapies based on the mutational landscape. The continued identification of new neoantigens and the efficient mobilization of tumor-reactive lymphocytes in patients with cancer will promote the advancement of immunotherapy using personalized NGS-guided technologies.
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http://dx.doi.org/10.1007/s00428-017-2140-0DOI Listing
August 2017

Detection of Chromosomal Translocation in Hematologic Malignancies by a Novel DNA-Based Looped Ligation Assay (LOLA).

Clin Chem 2017 Jul 17;63(7):1278-1287. Epub 2017 May 17.

Departments of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD;

Background: Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots.

Methods: Oligonucleotide sets that probe breakpoints of (immunoglobulin heavy-apoptosis regulator) in follicular lymphoma (FL), (MYC proto-oncogene, bHLH transcription factor-immunoglobulin heavy) in Burkitt lymphoma (BL) and (RhoGEF and GTPase activating protein-ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis. We also tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)-PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL).

Results: LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1%. Signal intensity was log linear to the DNA concentration ( = 0.94). Furthermore, we were able to detect a LOLA peak in DNA from 53.3% of FL patients and 87.5% of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent.

Conclusions: Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.
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http://dx.doi.org/10.1373/clinchem.2016.270140DOI Listing
July 2017
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