Immunohistochemical and Molecular Features of Melanomas Exhibiting Intratumor and Intertumor Histomorphologic Heterogeneity

Melanoma is a heterogeneous neoplasm at the histomorphologic, immunophenotypic, and molecular levels. Melanoma with extreme histomorphologic heterogeneity can pose a diagnostic challenge in which the diagnosis may predominantly rely on its immunophenotypic profile. However, tumor survival and response to therapy are linked to tumor genetic heterogeneity rather than tumor morphology. Therefore, understating the molecular characteristics of such melanomas become indispensable. In this study, DNA was extracted from 11 morphologically distinct regions in eight formalin-fixed, paraffin-embedded melanomas. In each region, mutations in 50 cancer-related genes were tested using next-generation sequencing (NGS). A tumor was considered genetically heterogeneous if at least one non-overlapping mutation was identified either between the histologically distinct regions of the same tumor (intratumor heterogeneity) or among the histologically distinct regions of the paired primary and metastatic tumors within the same patient (intertumor heterogeneity). Our results revealed that genetic heterogeneity existed in all tumors as non-overlapping mutations were detected in every tested tumor (n = 5, 100%; intratumor: n = 2, 40%; intertumor: n = 3, 60%). Conversely, overlapping mutations were also detected in all the tested regions (n = 11, 100%). Melanomas exhibiting histomorphologic heterogeneity are often associated with genetic heterogeneity, which might contribute to tumor survival and poor response to therapy

Peningkatan Prestasi Belajar CAD Mahasiswa Teknik Otomotif Non-Reguler FT UNY melalui Pembuatan “Pohon Kata” Perintah dalam Program AutoCAD

Penelitian ini bertujuan meningkatkan prestasi belajar mata kuliah Computer Aided Design (CAD) mahasiswa prodi Teknik Otomotif Non-Reguler yang dinyatakan dalam bentuk rerata nilai akhir semester yang berasal dari komponen nilai tugas harian, nilai ujian tengah semester dan nilai ujian akhir semester. Penelitian quasi-eksperimen ini terdiri dari tahapan penelitian diawali dengan penyusunan materi pembelajaran sejumlah pokok bahasan tertentu dalam satu job sheet (lembar kerja), dilanjutkan dengan pembuatan bantuan “Pohon Kata” perintah dalam Auto CAD kepada kelas eksperimen yang ditentukan secara random dari dua kelas peserta kuliah Auto CAD pada Semester Genap 2008/2009. Kedua kelas diamati prestasinya, baik kecepatan penyelesaiannya maupun kualitas kebenaran gambarnya. Prestasi belajar kedua kelas juga diukur melalui pemberian ujian tengah semester dan ujian akhir semester. Setelah data prestasi kedua kelas terkumpul dilanjutkan dengan analisis statistik melalui uji beda (t-test) setelah sebelumnya dilakukan uji persyaratan analisis yang ternyata dapat dipenuhi. Hasil penelitian ini disimpulkan bahwa: prestasi belajar CAD mahasiswa pada kelas yang diberi perlakuan strategi pembelajaran menggunakan “Pohon Kata” perintah dalam Program Auto CAD lebih baik dibanding prestasi belajar CAD mahasiswa pada kelas yang tidak diberi perlakuan (75,41>70,89), dengan demikian pembelajaran CAD menggunakan media “Pohon Kata” perintah dalam Program Auto CAD dapat meningkatkan prestasi belajar mahasiswa Teknik Otomotif Program Non-Reguler

Clinical next generation sequencing to identify actionable aberrations in a phase I program

Purpose We determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer. Methods: We reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA). Results: Of the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified. Conclusion: Multiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials

Clinical Testing for Mismatch Repair in Neoplasms Using Multiple Laboratory Methods

Background: A deficiency in DNA mismatch repair function in neoplasms can be assessed by an immunohistochemical (IHC) analysis of the deficiency/loss of the mismatch repair proteins (dMMR) or by PCR-based methods to assess high microsatellite instability (MSI-H). In some cases, however, there is a discrepancy between the IHC and MSI analyses. Several studies have addressed the issue of discrepancy between IHC and MSI deficiency assessment, but there are limited studies that also incorporate genetic/epigenetic alterations. Methods: In this single-institution retrospective chart-review study, we reviewed 706 neoplasms assessed between 2015 and 2021. All eligible neoplasms were assessed by IHC testing, MSI analysis by PCR-based assay, and tumor-normal paired next-generation sequencing (NGS) analysis. Eighty percent of neoplasms with MLH1 protein loss had a concurrent MLH1 promoter methylation analysis. Mutation data for MMR genes, IHC, MSI analysis, and tumor histology were correlated with each other. Results: Fifty-eight (8.2%) of 706 neoplasms had MSI-H by PCR and/or dMMR by IHC. Of the 706 analyzed neoplasms, 688 neoplasms (98%) had concordant results: MSI-H/dMMR (n = 44), microsatellite-stable (MSS)/proficient MMR (pMMR) (n = 625), and MSI-Low (L)/pMMR (n = 19). Of the remaining 18 neoplasms, 9 had a major discordance: MSS/loss of MSH2 and MSH6 (n = 3), MSS/loss of MSH6 (n = 2), MSS/Loss of MLH1 and PMS2 (n = 1), and MSI-High/pMMR (n = 3). In total, 57% of cases with dMMR and 61% of cases with MSI-H had a null mutation of an MMR gene mutation (or methylation of the MLH1 promoter), whereas this figure was 1% for neoplasms with a normal IHC or MSI pattern (p < 0.001). Among 9 cases with major discordance between MSI and IHC, only 3 cases (33%) had an underlying genetic/epigenetic etiology, whereas 37 (76%) of 49 cases with MSI-H and/or dMMR and without major discordance had an underlying genetic abnormality (p = 0.02). Discussion: For most neoplasms, IHC and PCR-based MSI testing results are concordant. In addition, an underlying genetic abnormality (a null mutation of an MMR gene or MLH1 promoter methylation) was attributable to dMMR and/or MSI-H findings. For neoplasms with major discordance in IHC and MSI testing, the addition and integration of NGS results and MLH1 promoter methylation analyses can be beneficial for resolving borderline cases, thereby facilitating patient management

HCVAD plus imatinib or dasatinib in lymphoid blastic phase chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) may progress to blast phase (BP) at the rate of 1% to 1.5% per year. With the use of single-agent tyrosine kinase inhibitors, median overall survival ranges between 7 and 11 months.MethodsThe outcome was analyzed for 42 patients with lymphoid BP-CML who were treated with hyperfractionated cyclophosphamide, vincristine, Adriamycin, dexamethasone (HCVAD) plus imatinib or dasatinib.ResultsComplete hematological response was achieved in 90% of patients, complete cytogenetic remission in 58%, and complete molecular remission in 25%. Flow cytometry minimal residual disease negativity was achieved by 42% of evaluable patients after induction. Eighteen patients received allogeneic stem cell transplant (SCT) while in first complete hematological response. Median remission duration was 14 months and was longer among SCT recipients (P = .01) on multivariate analysis. Median overall survival was 17 months (range, 7-27 months) and was longer among SCT recipients (P < .001) and patients treated with dasatinib (P = .07) on multivariate analysis. Although a high rate of hematologic toxicity (100%) and infectious complications (59%) were observed, the related rate of treatment discontinuation was low (7% and 9%, respectively).ConclusionsHCVAD combined with tyrosine kinase inhibitors is an effective regimen for the management of BP-CML, particularly when followed by allogeneic SCT

HCVAD plus imatinib or dasatinib in lymphoid blastic phase chronic myeloid leukemia

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Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era.

ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease