CCND1 Gain Research Articles

Whole-Exome Sequencing of Vulvar Squamous Cell Carcinomas Reveals an Impaired Prognosis in Patients With TP53 Mutations and Concurrent CCND1 Gains

Very little information is available on the mutational landscape of vulvar squamous cell carcinoma (VSCC), a disease that mainly affects older women. Studies focusing on the mutational patterns of the currently recognized etiopathogenic types of this tumor (human papillomavirus [HPV]-associated [HPV-A], HPV-independent [HPV-I] with TP53 mutation [HPV-I/TP53mut], and HPV-I with wild-type TP53 [HPV-I/TP53wt]) are particularly rare, and there is almost no information on the prognostic implications of these abnormalities.Whole-exome DNA sequencing of 60 VSCC and matched normal tissues from each patient was performed. HPV detection, immunohistochemistry (IHC) for p16, p53, and mismatch repair proteins were also performed. Ten tumors (16.7%) were classified as HPV-A, 37 (61.7%) as HPV-I/TP53mut, and 13 (21.6%) as HPV-I/TP53wt. TP53 was the most frequently mutated gene (66.7%), followed by FAT1 (28.3%), CDKN2A (25.0%), RNF213 (23.3%), NFE2L2 (20%) and PIK3CA (20%). All the 60 tumors (100%) were DNA mismatch repair proficient. Seventeen tumors (28.3%) showed CCND1 gain. Bivariate analysis, adjusted for International Federation of Gynecology and Obstetrics stage, revealed that TP53 mutation, CCND1 gain, and the combination of the 2 alterations were strongly associated with impaired recurrence-free survival (hazard ratio, 4.4; P < .001) and disease-specific survival (hazard ratio, 6.1; P = .002). Similar results were obtained when p53 IHC status was used instead of TP53 status and when considering only HPV-I VSCC. However, in the latter category, p53 IHC maintained its prognostic impact only in combination with CCND1 gains. All tumors carried at least one potentially actionable genomic alteration. In conclusion, VSCCs with CCND1 gain represent a prognostically adverse category among HPV-I/TP53mut tumors. All patients with VSCCs are potential candidates for targeted therapy.

Clonal Bone Marrow Plasma Cells Are Common in Patients with Vexas Syndrome

Introduction Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder. Its prevalence increases with age, reaching 5.3% at 70 years and older. MGUS progression rate to multiple myeloma (MM) or lymphoplasmacytic malignancies is about 1% per year. Patients with autoimmune diseases are at increased risk for developing MGUS and MM. VEXAS ( vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic syndrome), an autoinflammatory disorder caused by somatic mutations in ubiquitin-activating enzyme, UBA1 gene, has been associated with plasma cell disorders, specifically MGUS, but the rate of progression is unknown. Cyclin D1, important in cell cycle progression, is regulated by ubiquitin-proteasome mediated degradation, and deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. We aim to assess marrow characteristics of plasma cell disorders (MGUS and MM) in VEXAS, and the impact of a defective UBA1 gene product on protein expression of cyclin D1. Methods We assessed 28 patients with VEXAS for the presence of plasma cell disorders, and studied the clinical and laboratory characteristics of those with clonal plasma cells (PC) in the marrow. Bone marrow biopsies and aspirates were evaluated by morphology, immunohistochemistry (IHC) dual staining CD138/cyclin D1, dual staining MUM-1/CD20), in situ hybridization (Kappa and Lambda light chains) and flow cytometry (FC) using antibodies for CD45, CD19, CD20, CD38, CD138, CD56, CD27, CD81, and cytoplasmic Kappa and Lambda immunoglobulins. Serum free kappa and lambda light chains, IgG, IgM, and IgA, and level of M paraprotein by serum protein electrophoresis with immunofixation were obtained. Fluorescent in situ hybridization (FISH) analysis for MM was performed in most patients with clonal PCs. Results Patients' characteristics are shown in Table 1. Eight out of 28 (28.6%) patients with VEXAS syndrome, all males, had clonal plasma cells in their bone marrows: 3 MGUS, and 5 MM (3 smoldering, SMM; 2 active MM). Median age at diagnosis of plasma cell disorder was 69.5 years (range 60-75). All patients had pathogenic UBA1 somatic mutations (5 M41T, 1 each with M41V, M41L, and splice). Clinically, monoclonal protein size was small, with no specific selection of the type of M protein. Strikingly, most (7/8) clonal plasma cells showed cyclin D1 expression by IHC (dual immunohistochemical stain cyclinD1/CD138 shows double-positive plasma cells; figure not shown), displaying nuclear immunoreactivity in 6 cases, and cytoplasmic immunoreactivity in 1 case. FC showed positive CD20 expression of clonal plasma cells in 4 cases, dim to negative CD19 expression in 4 cases, variable CD56 (positive in 3 cases, partial positive in 1 case, negative in 2 cases), dim to negative CD27 in 3 cases, dim to negative CD81 in 3 cases. FISH for MM revealed: t(11;14) in 4 cases, gain of 11q (CCND1) in 2 cases (in one case, CCND1 gain was present in addition to t(11;14); in the second case, CCND1 gain was associated with gains of 1q and 17p(TP53). The cyclin D1-negative MM case was negative for t(11;14) but had t(4;14). All cases with t(11;14) were positive for cyclin D1 by IHC (nuclear staining). The one MM case with negative nuclear staining for cyclin D1 but positive cytoplasmic staining, was negative for t(11;14) but had gain of 11q (CCND1).One patient evolved from SMM to active MM after 4 years. No evolution to MM was seen in patients with MGUS with median follow-up of 30 months. Conclusions Patients with VEXAS have a high prevalence of clonal plasma cell disorders. Clonal plasma cells in VEXAS patients are associated with t(11;14), and with nuclear cyclin D1 expression by IHC. Cytoplasmic cyclin D1 staining in the absence or weak nuclear staining was observed with CCND1 gain and absence of t(11;14). Cytoplasmic cyclin D1 expression may be related to lack of cyclin D1 protein degradation by a defective UBA1 gene product. Our results suggest that defective UBA1 and autoinflammation pose an increased risk for developing MGUS or multiple myeloma.

Gain of CCND1 May Occur Too Infrequently in Cutaneous Melanoma, and Too Late in Melanomagenesis, to Be Diagnostically Useful: Genomic Analysis of 88 Cases.

Genomic analysis is an important tool in the diagnosis of histologically ambiguous melanocytic neoplasms. Melanomas, in contrast to nevi, are characterized by the presence of multiple copy number alterations. One such alteration is gain of the proto-oncogene CCND1 at 11q13. In melanoma, gain of CCND1 has been reported in approximately one-fifth of cases. Exact frequencies of CCND1 gain vary by melanoma subtype, ranging from 15.8% for lentigo maligna to 25.1% for acral melanoma. We present a cohort of 72 cutaneous melanomas from 2017-2022 in which only 6 (8.3%) showed evidence of CCND1 gain by chromosomal microarray. This CCND1 upregulation frequency falls well below those previously published and is significantly lower than estimated in the literature ( P < 0.05). In addition, all 6 melanomas with CCND1 gain had copy number alterations at other loci (most commonly CDKN2A loss, followed by RREB1 gain), and 5 were either thick or metastatic lesions. This suggests that CCND1 gene amplification may be a later event in melanomagenesis, long after a lesion would be borderline or equivocal by histology. Data from fluorescence in situ hybridization, performed on 16 additional cutaneous melanomas, further corroborate our findings. CCND1 gain may not be a common alteration in melanoma and likely occurs too late in melanomagenesis to be diagnostically useful. We present the largest chromosomal microarray analysis of CCND1 upregulation frequencies in cutaneous melanoma, conjecture 3 hypotheses to explain our novel observation, and discuss implications for the inclusion or exclusion of CCND1 probes in future melanoma gene panels.

Genomic comparison between an in vitro three-dimensional culture model of melanoma and the original primary tumor.

Three-dimensional (3D) melanoma culture is a personalized in vitro model that can be used for high-fidelity pre-clinical testing and validation of novel therapies. However, whether the genomic landscape of 3D cultures faithfully reflects the original primary tumor which remains unknown. The purpose of our study was to compare the genomic landscapes of 3D culture models with those of the original tumors. Patient-derived xenograft (PDX) tumors were established by engrafting fresh melanoma tissue from each patient. Then, a 3D culture model was generated using cryopreserved PDX tumors embedded in pre-gelled porcine skin decellularized extracellular matrix with normal human dermal fibroblasts. Using whole-exome sequencing, the genomic landscapes of 3D cultures, PDX tumors, and the original tumor were compared. We found that 91.4% of single-nucleotide variants in the original tumor were detected in the 3D culture and PDX samples. Putative melanoma driver mutations (BRAF p.V600E, CDKN2A p.R7*, ADAMTS1 p.Q572*) were consistently identified in both the original tumor and 3D culture samples. Genome-wide copy number alteration profiles were almost identical between the original tumor and 3D culture samples, including the driver events of ARID1B loss, BRAF gain, and CCND1 gain. In conclusion, our study revealed that the genomic profiles of the original tumor and our 3D culture model showed high concordance, indicating the reliability of our 3D culture model in reflecting the original characteristics of the tumor.

Single Time Frame Overview of the Genetic Changes in Conjunctival Melanoma from Intraepithelial Disease to Invasive Melanoma: A Study of 4 Exenteration Specimens Illustrating the Potential Role of Cyclin D1

Introduction: Despite advances in the understanding of the molecular pathogenesis of cutaneous melanoma, relatively little is known about the genetic changes that occur in the progression of conjunctival melanocytic intraepithelial lesions to invasive conjunctival melanoma. Methods: We exposed 4 exenteration specimens that each contained varying grades of intraepithelial conjunctival melanocytic neoplasia and invasive neoplasia to a combination of various techniques, including array comparative genomic hybridization (aCGH), ribonucleic acid sequencing (RNA-seq), fluorescence in situ hybridization (FISH), and immunohistochemistry. Results: Three out of 4 of the invasive melanomas showed gains in 11q13 (CCND1 locus) by aCGH. FISH demonstrated CCND1 gain in invasive melanoma and in conjunctival melanocytic intraepithelial lesions (CMILs) of all grades (low-grade CMILs and in situ melanoma), and this was paralleled by increased expression of Cyclin D1 protein within the atypical melanocytes by immunohistochemistry, using a double-staining method with a red end point for Melan A cytoplasmic staining and a brown end point for nuclear Cyclin D1 expression. Higher grades of melanocytic intraepithelial lesions showed more cells expressing Cyclin D1 than lower grade melanocytic intraepithelial lesions. The Cyclin D1 protein expression was in the same location as the amplified CCND1 signal by FISH. One out of 3 of these cases also showed the amplification of the 12q13-15 locus corresponding to MDM2 and FISH confirmed gains in the conjunctival melanocytic intraepithelial neoplasia and invasive melanoma. The remaining fourth case showed a homozygous deletion of 9p21 (CDKN2A) by aCGH only, with immunohistochemistry showing clonal loss of p16 protein expression in the invasive and conjunctival melanocytic intraepithelial lesion. Two out of 4 of the invasive melanomas harboured classical driver mutations in NRAS and NF-1, respectively. None of the cases showed mutations in BRAF, KIT, and TERT mutations. RNA-seq data showed secondary mutations in ARAF, PLCB4, MET, EZH2, MAP2K2, CTNNB1, CIITA, NF2, TP53, and MEN1, some of which are implicated in the MAPK pathway. Conclusion: CMILs harbour amplifications of CCND1 (3 cases), MDM2 (1 case), and loss of CDKN2A (1 case), which are also present when the lesion progresses to invasive melanoma, implicating these amplifications in the early pathogenesis of CMILs. This study represents the first attempt to capture the mutational landscape of all stages of conjunctival melanoma in a single tissue excision.

Palbociclib in advanced acral melanoma with genetic aberrations in the cyclin-dependent kinase 4 pathway

BackgroundGenetic aberrations in the cyclin-dependent kinase (CDK)4 pathway occur in 82% of patients with acral melanoma (AM), which is the predominant subtype of melanoma in China. We aimed to evaluate the anti-tumour activity of palbociclib, a selective CDK4/6 inhibitor, in patients with advanced AM with CDK4 pathway gene aberrations. MethodsIn this phase II trial, patients with advanced AM with CDK4 or/and CCND1 gain or/and CDKN2A loss were treated with oral palbociclib (125 mg) on days 1–21 of a 28-day cycle. The primary end-point was overall response rate (ORR). Secondary end-points were progression-free survival (PFS), overall survival (OS), and treatment-related adverse events (TRAEs). Whole-exome sequencing and multiplex immunohistochemistry of the available formalin-fixed, paraffin-embedded samples of nine patients were analysed to explore the predictive biomarkers of palbociclib response. ResultsFifteen patients were enrolled. Three (20.0%) patients achieved tumour shrinkage at 8 weeks, including one with confirmed partial response. At data cut-off date, treatment was ongoing for one patient. The median PFS was 2.2 mo (range: 1.5–13.3 mo; 95% confidence interval [CI]: 1.9–2.5), and the median OS was 9.5 mo (range: 2.6–14.1 mo, 95% CI: 5.7–13.4). Eight patients died due to disease progression. The most common TRAEs were leukopenia (87%; Grade III/IV, 27%), neutropenia (80%; grade III/IV, 27%), and fatigue (53%; grade III/IV, 7%). Significant JAK2 deletions and SH2B3 amplifications were observed in patients who did not achieve any clinical benefit (CB) with palbociclib treatment. MCM7 amplification or protein expression level was found to be associated with CB. ConclusionsPalbociclib monotherapy demonstrated preliminary efficacy and an acceptable safety profile in advanced AM patients with CDK4 pathway aberrations. Patients with amplification or high protein levels of MCM7 were more prone to benefit from palbociclib. The JAK-STAT pathway might play a role in the mechanism of action of palbociclib in AM. Trial registration numberNCT03454919. The date of registrationMarch 6, 2018.

A case-control study to identify molecular risk factors for local recurrence in young breast cancer patients

PurposeTo investigate risk factors for local recurrence (LR) after breast conserving therapy in young breast cancer patients randomized in the “Young Boost Trial”. Material & methodsIn the “Young Boost Trial” 2421 patients ≤50 years old were randomized between a 16 Gy and 26 Gy boost after breast conserving surgery and whole breast radiation (50 Gy). We performed a case-control study comparing patients who developed a LR (cases) and for each of them three control patients free of recurrence (controls). Clinicopathological factors, copy number- and gene expression profiles of primary tumors were compared between cases and controls, and between primary tumors and local recurrences. ResultsThe cumulative 5-year LR rate was 1.07% (95% CI 0.72-1.59%) and 10-year LR rate 2.56% (1.81–3.62%). Analysis of a subset of primary tumors and local recurrences showed similar histopathological characteristics (n = 15), copy number (n = 13) and gene expression profiles (n = 14). Basal subtype was strongly associated with LR in univariable and multivariable analysis. Gains of CCND1 were identified more frequently among controls, while more frequent gains of FGFR1 and IGF1R were observed among cases. Upregulation of genes involved in the p53-pathway was observed in recurring tumors compared to non-recurring tumors. We could not identify a genomic classifier for LR. ConclusionsAll investigated local recurrences were true genomic recurrences. Although differences in copy number variation and gene expression pathways were observed in recurring tumors compared to non-recurring tumors, no genomic classifier for LR could be identified.

Abstract 4741: Exploring possible drug resistance and sensitivity mechanisms in treatment-refracotry betel-nuts related HNSCC cell line(TW2.6) by whole exon sequencing and molecular signaling for future drug combinations

Abstract Background: Betel nut chewing might contribute to (1)strong inflammation, angiogenesis, &amp; invasion; (2)resistance to traditional therapies. Betel-nuts related HNSCC in Taiwan might belong to mesenchymal differentiation and have the worst prognosis. Our group found betel-nuts exposed HNSCC cell line, TW2.6, was resistant to traditional therapies. PDL1 western blotting was strong over TW2.6 with defective p53 mutation, p16 loss, and BCL2 overexpression. In our previous studies, Astragalus polysaccharides, PI3Kalpha inhibitor, AKT inhibitor, FGFR inhibitor, ALK/IGF1R inhibitor, CDK4/6 inhibitor, BCl2 inhibitor, WEE1 inhibitor, ATR inhibitor, DNA-PK inhibitor, &amp; VEGFR2/ PDGFR/FGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, maybe through the inhibition of mesenchymal transformation, pRB, &amp; PI3K/AKT /mTOR signaling and the modulation of stemness &amp; PD1/PDL1 pathway. Afatinib &amp; CDK4/6 inhibitors also have immuno-modulatory effects in our studies. Purpose &amp; Methods: NGS studies will be introduced to representative HNSCC cell lines to study molecular phenotypes of these HNSCC cell lines (including SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, &amp; TW2.6) for future drug combinations &amp; basic/translational research. Results: TW2.6 had PIK3CA H1047R mutation, high TMB(8.42 muts/Mb)/MSS, and VEGF-A amplification. We found afatinib response on several cell lines not so correlated to EGFR expression &amp; CNV status. Palbociclib response seemed to correlate to CCND1 gain and p16 loss; but FaDu had not so good palbociclib response even with these two changes and TW2.6 has good response even without these two. TW2.6 also had other genomic changes, such as HRAS &amp; DDR2 mutations, FAT1 loss, amplification of FGF10/CCND3/SOX9, and deletions of STK11/ARID1B/TNFAIP3. Conclusions: PI3K inhibitor, anti-angiogenesis therapies, and immunotherapy-containing regimens will be future combination options for betel-nuts related HNSCC by TW2.6 molecular signatures. Afatinib, CDK4/6 inhibitors, or DDR interventions could also enhance ICIs efficacy. The genomic landscapes of TW2.6 also deserve further studies about CDK4/6 inhibitor resistance, immunotherapy resistance, epigenetic modifications, novel targeted therapy, &amp; biomarkers exploration. Mutations in TW2.6TW2.6GenesN=1%TP531100%SPEN1100%FAT11100%MUC61100%BUB1B1100%EPHB11100%HRAS1100%PTPRD1100%ATM1100%CDK121100%CDK81100%DDR21100%GNAS1100%MYC1100%PDGFRB1100%PIK3CA1100%TNFSF111100%TSC21100% Citation Format: Jo-Pai Chen, Jui-Ying Chang, Ruey-Long Hong. Exploring possible drug resistance and sensitivity mechanisms in treatment-refracotry betel-nuts related HNSCC cell line(TW2.6) by whole exon sequencing and molecular signaling for future drug combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4741.

Ancillary values of fluorescence in situ hybridization with different gene combination in diagnosis of malignant melanoma

Objective: To investigate the clinical value of the first multicolor fluorescence in situ hybridization (FISH) assay on multiple genes, and combined with 9p21 and 8q24 evaluation in the differential diagnosis of melanoma. Methods: Fifty-six melanomas and 36 benign melanocytic nevi diagnosed in Fudan University Shanghai Cancer Center from 2017 to 2019 were included. Each specimen was examined by first multicolor FISH assay targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and CEP6, as well as 9p21 (CDKN2A) and 8q24 (MYC). The results of FISH assay in all cases were recorded according to Gerami's criteria. Basing on the sensitivity and specificity of the first FISH assay, the refinement of diagnosis by adding combined 9p21 and 8q24 probes was further evaluated, as well as their association with different clinicopathological features. Results: In 86 cases, the FISH signals were adequate for analysis. Of the 56 melanoma cases, 52 cases were adequate for analysis; 36 cases (69.2%) were positive in the first FISH assay. The most frequent chromosomal anomaly was gain of RREB1 (30/52, 57.7%), followed by gain of CCND1 (20/52, 38.5%), loss of MYB relative to CEP6 (18/52, 34.6%) and gain of RREB1 relative to CEP6 (17/52, 32.7%). The frequency of homozygous deletions in 9p21 was 15.4% (8/52) and gain of 8q24 was 36.5% (19/52). Among the 36 melanocytic nevi cases, FISH results could be accurately evaluated in 34 cases, and none showed a positive result in the first FISH assay or 9p21 and 8q24 FISH analysis. Compared with the first FISH assay, the sensitivity of combination with 9p21 and 8q24 FISH analysis increased from 69.2% to 76.9% (40/52) and the specificity remained 100.0%. Statistical data showed that the rates of FISH positivity in patients with acral-lentiginious melanoma and nodual melanoma subtypes were higher than that in patients with superficial spreading melanoma and lentigo maligna melanoma subtypes, and patients with Breslow thickness>2.0 mm had higher positive FISH frequency than patients with Breslow thickness ≤2.0 mm. Conclusion: Multisite FISH analysis is a highly effective ancillary tool for the differentiation of unequivocal malignant from benign melanocytic lesions. By combining the first FISH assay with CDKN2A and MYC assay, the clinical utility of FISH analysis is further optimized in differential diagnosis of melanoma. Patients with Breslow thickness>2.0 mm, or acral-lentiginious melanoma and nodual melanoma subtypes tend to have higher FISH positivity. There remains a need to further explore the ancillary value of FISH analysis in diagnosis of ambiguous lesions.

Four-color fluorescence in-situ hybridization is useful to assist to distinguish early stage acral and cutaneous melanomas from dysplastic junctional or compound nevus

Background/objectiveAcral and cutaneous melanomas are usually difficult to accurately diagnose in the early stage, owing to the similarity in clinical manifestations and morphology with those of dysplastic nevus (DN). In this study, we aimed to evaluate the diagnostic value of four-color fluorescence in-situ hybridization (FISH) probes specific to the RREB1,CCND1,and MYB genes, and centromere of chromosome 6, in distinguishing DN and melanoma.MethodsFifty one DN and 58 melanoma cases were collected and tested with four-color FISH. Histological features were reviewed and concordant morphologic diagnosis by three pathologists was considered the golden criterion.ResultsFifty DN and 59 melanoma cases, with 37 melanomas in situ and 22 melanomas in Clark level 2, were confirmed finally; among them, 42 (71.2%) cases were acral. A comparison of clinicopathological features between the two entities showed that several features were considerably more frequently observed in the melanoma group, including more mitotic figures, stratum corneum pigmentation, lymphocyte infiltration, cell atypia, successive or pagetoid melanocyte growth pattern in the epidermis, larger tumor size, and older age at diagnosis. FISH was positive in 3 (6.0%) DN and 56 (94.9%) melanoma cases according to Gerami’s criteria. In distinguishing the two groups, the sensitivity of the four-color FISH was 94.9% and specificity was 94.0%.We found that CCND1 gain was the most sensitive, either in Gerami’s or Gaiser’s criteria. Further analysis showed that CCND1gain was more obvious in the acral group of melanoma.ConclusionsWe conclude that the four-color FISH test was highly sensitive and specific in distinguishing early-stage acral and cutaneous melanomas from dysplastic nevus in Chinese population, and the most sensitive criterion was the gain of CCND1.

Targeted Genomic Profiling of Female Adnexal Tumors of Probable Wolffian Origin (FATWO)

Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic neoplasm of low-malignant potential presumed to be derived from mesonephric remnants in the upper female genital tract. Similarly, mesonephric remnants in the lower female genital tract are thought to be the origin for mesonephric carcinoma. Although the molecular alterations in mesonephric carcinoma have been recently reported, the pathogenesis of and molecular alterations in FATWO are not well understood. The aims of this study were to examine the molecular alterations in FATWO and to establish whether these neoplasms are molecularly similar to mesonephric carcinoma. Eight FATWOs underwent massively parallel sequencing to detect single nucleotide variations, copy number variations, and structural variants by surveying exonic DNA sequences of 300 cancer genes and 113 introns across 35 genes. Good quality DNA was isolated from 7 of 8 cases. Novel KMT2D variants (1 frameshift, 3 missense) were identified in 4 of 7 cases (57%), but were variants of uncertain biologic significance. STK11 mutations (both frameshift) were identified in 2 of 7 cases (29%); one of these was in a patient with a known history of Peutz-Jeghers syndrome. A mutation in the chromatin remodeling gene ARID1B was identified in 1 of 7 cases (14%). No cases harbored KRAS, NRAS, TP53, PIK3CA, PTEN, or DICER1 mutations. There were relatively low numbers of copy number variations, and no recurrent copy number variations were identified. One case demonstrated moderate copy gain of CCND1. No structural variants were identified. In summary, FATWO is characterized molecularly by the absence of KRAS/NRAS mutations (characteristic of mesonephric carcinoma), absence of DICER1 mutations (characteristic of Sertoli-Leydig cell tumor) and frequent KMT2D mutations of unknown biologic significance. FATWOs exhibit a limited number of molecular aberrations that are significantly different from those reported in tumors in the differential diagnosis, and our results question the relationship of mesonephric carcinoma with FATWO. Disease-defining molecular alterations for FATWO have yet to be discovered.

PS1367 COMPREHENSIVE GENETIC ANALYSIS OF MULTIPLE MYELOMA IN JAPAN

Background:Next generation sequencing (NGS) technology has enabled comprehensive analyses of genomic abnormalities occurring in cancer. Previous genetic studies using large cohorts of patients with multiple myeloma (MM) have revealed the genomic landscape of MM. Although some of these genomic abnormalities are believed to be associated with the molecular pathogenesis of MM and/or clinical outcome, these relationships are not fully understood. In addition, most previous studies have been performed in European and American cohorts. Thus, the genomic profiles of Asian or Japanese MM patients remains unclear.Aims:We aimed to clarify the genomic profiles of Japanese patients with MM and to explore genomic alterations correlated to the clinical or pathological features of MM.Methods:The cohort in this study included 138 Japanese patients, newly diagnosed with MM between Dec. 2013 and Jan. 2018. The median age at diagnosis was 69 years (36–87), and the male to female ratio was 1:1. Tumor DNA was isolated from CD138‐positive bone marrow plasma cells, and germline control DNA was derived from peripheral blood mononuclear cells. A total of 340 genes related to MM and immunoglobulin (Ig) gene rearrangement regions were analyzed for somatic mutations and clonal Ig rearrangements by targeted capture sequencing. Single nucleotide polymorphism‐baits were also included to detect copy number alterations (CNA) by sequencing. IgH‐CCND1 translocations were detected, and expression levels of CCND1 mRNA were measured using FISH and RQ/RT‐PCR, respectively.Results:Clonal Ig gene rearrangements in myeloma‐derived cells were identified in all patients. In total, 136/138 patients (98.6%) harbored at least one single‐nucleotide variant or indel, with a median of 4 (0–12). KRAS was the most commonly mutated gene (48/138, 34.8%), and abnormalities in RAS/MAPK pathway genes were present in 85/138 patients (61.6%). In copy number analysis, 130 /138 patients (94.2%) had some kind of CNA and 52/138 (37.7%) were considered to be hyperdiploid subtypes according to WHO classification. Analysis of relationships between somatic mutations and CNA revealed MAX gene mutations in 9 patients (6.5%). All of these were accompanied by loss of heterozygosity (LOH) at chr14q23. An expression analysis was performed in 92 (66.7%) patients. Overexpression of CCND1 was observed in all 24 patients with t(11;14) as previously reported. Moreover, patients with a gain of CCND1 and uniparental disomy (UPD) of chr11 showed CCND1 overexpression. Del(17p) was observed in 13/138 patients (9.4%) and was predictive of shorter overall survival (OS) (p &lt; 0.001).In cases with t(11;14), the presence of a KRAS mutation also tended to predict shorter OS (p = 0.0379).Figure:Frequency of mutated genes in this cohort.Summary/Conclusion:To the best of our knowledge, this is the first report of a comprehensive genetic analysis of Japanese MM patients. Although most of our results were compatible with those of the previous reports, hyperdiploid cases were less frequent, and KRAS mutations were more frequent than in other cohorts. These characteristics, although based on small numbers, may represent racial differences. In addition, all of the patients who had MAX mutations displayed LOH in this cohort. Because the function of MAX is not fully understood in MM, this correlation may add to our understanding of the molecular pathogenesis of myeloma.image

Poor Prognosis Associated with Gains of CCND1 in Mantle Cell Lymphoma Treated By First Line Immuno-Chemotherapy — a Study By the Lysa Group

BackgroundMantle cell lymphoma (MCL) is a lymphoproliferative disorder characterized by the t(11;14) balanced translocation, involving on chromosome 11 the gene encoding cyclin D1, CCND1. The enhancer of IGH, on chromosome 11, induces aberrant Cyclin D1 overexpression. A few reports have shown that the CCND1 oncogene locus is a recurrently amplified region in MCL. However, the prognostic value of this copy number abnormality (CNA) is not known. The incidence and clinical impact of CCND1 CNA were investigated on diagnostic samples from patients enrolled in the first-line randomized controlled trial LyMa (S. Le Gouill et al. NEJM 2017).Patients and methodsA series of 100 lymph node biopsies performed at diagnosis for patients enrolled in the LyMa trial (n=299) was selected. After DNA extraction, CNAs were investigated in these samples using the innovative Oncoscan® SNP-array technique adapted to analyze highly degraded DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Ninety-four samples were informative for CNAs. CCND1 gains were controlled by fluorescence in situ hybridization on interphase nuclei, following standard procedures using LSI IGH/CCND1 XT Dual color, dual fusion translocation probes (Abbott Molecular, Des Plaines, IL). Progression-free and overall survivals were calculated from the date of inclusion until relapse/death or last news and death or last news respectively. Cumulative incidence of relapse (CIR) was estimated from the date of inclusion until the date of relapse or last newsResultsAmplifications of a large portion of 11q, beginning at the t(11;14) breakpoint, were observed with Oncoscan® in 7 patients. All these cases with a gain of 11q13-14 (CCND1) also had a gain of the 14q32 locus (IGH) suggesting amplification of the rearranged IGH-CCND1 region of t(11;14). This was confirmed by FISH analysis which disclosed three different configurations. The first was the classical CCND1-IGH rearrangement (CCND1 on normal chromosome 11, IGH on normal chromosome 14 and two fusion signals from the t(11;14)(CCND1-IGH)). The second configuration associated CCND1-IGH fusion signals and a gain of both CCND1 and IGH signals. The third configuration was a duplication of a CCND1-IGH fusion signals (3 signals). Both in SNP and in FISH, the amplification was always identified as sub-clonal, concerning only part of the cells. In some patients, different configurations coexisted.Compared to other patients of the series, those with amplification of IGH and CCND1 regions had a higher-risk bio-MIPI (11q13-14, p=0.015; 14q32, p=0.004). Patients with large gains at 11q13-14 (CCND1 N=7) had poorer median PFS (18 months vs not reached (NR); p=0.004), OS (35 months vs NR; p=0.01) and CIR (33 months vs not NR; p=0.004). The same was observed for patients with gains at 14q32 (IGH; N=8), with significantly different median PFS (23 months vs NR; p<0.001), OS (38 months vs NR; p=0.001) and CIR (28 months vs NR; p<0.001).ConclusionGains of the IGH-CCND1 rearrangement or involved genes appear to be a potential new biomarker predictive of poor response to first line immunochemotherapy in young MCL patients. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding; Erythec: Research Funding; Celgene Corporation: Research Funding.

Frequent Genetic Aberrations in the CDK4 Pathway in Acral Melanoma Indicate the Potential for CDK4/6 Inhibitors in Targeted Therapy.

Purpose: Effective therapies for the majority of metastatic acral melanoma patients have not been established. Thus, we investigated genetic aberrations of CDK4 pathway in acral melanoma and evaluated the efficacy of CDK4/6 inhibitors in targeted therapy of acral melanoma.Experimental Design: A total of 514 primary acral melanoma samples were examined for the copy number variations (CNV) of CDK4 pathway-related genes, including Cdk4, Ccnd1, and P16INK4a , by QuantiGenePlex DNA Assay. The sensitivity of established acral melanoma cell lines and patient-derived xenograft (PDX) containing typical CDK4 aberrations to CDK4/6 inhibitors was evaluated.Results: Among the 514 samples, 203 cases, 137 cases, and 310 cases, respectively, showed Cdk4 gain (39.5%), Ccnd1 gain (26.7%), and P16INK4a loss (60.3%). The overall frequency of acral melanomas that contain at least one aberration in Cdk4, Ccnd1, and P16INK4a was 82.7%. The median overall survival time for acral melanoma patients with concurrent Cdk4 gain with P16INK4a loss was significantly shorter than that for patients without such aberrations (P = 0.005). The pan-CDK inhibitor AT7519 and selective CDK4/6 inhibitor PD0332991 could inhibit the cell viability of acral melanoma cells and the tumor growth of PDX with Cdk4 gain plus Ccnd1 gain, Cdk4 gain plus P16INK4a loss, and Ccnd1 gain plus P16INK4a loss.Conclusions: Genetic aberration of CDK4 pathway is a frequent event in acral melanoma. Acral melanoma cell lines and PDX containing CDK4 pathway aberrations are sensitive to CDK4/6 inhibitors. Our study provides evidence for the testing of CDK4/6 inhibitors in acral melanoma patients. Clin Cancer Res; 23(22); 6946-57. ©2017 AACR.

Mechanism of resistance to endocrine therapy in breast cancer: the important role of PI3K/Akt/mTOR in estrogen receptor-positive, HER2-negative breast cancer.

Endocrine therapy is a crucial treatment for estrogen receptor-positive (ER+) breast cancer, with proven clinical benefits. However, adaptive mechanisms emerge in the tumor, causing resistance to endocrine therapy. A better understanding of resistance mechanisms is needed to overcome this problem and to develop new, precise treatment strategies. Accumulating genetic and cancer biological studies demonstrate the importance of understanding the PI3K/Akt/mTOR and CDK4/6/RB pathways in ER+ HER2- breast cancer. PIK3CA (which encodes phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit α) is frequently mutated in breast cancer, and 30% of advanced ER+ HER2- breast cancers have an activating PIK3CA mutation. AKT1 mutations (E17K) have been found in 1.4-8% of breast cancer patients. ER+ breast cancer patients preferentially demonstrate gain of CCND1 (cyclin D1; 58% in luminal B vs. 29% in luminal A) and CDK4 (25% in luminal B vs. 14% in luminal A) and loss of CDKN2A (p16) and CDKN2C (p18), which are negatively regulated with the cell cycle and are correlated with the CDK4/6/RB pathway. Abnormalities in PI3K/Akt/mTOR and CDK4/6/RB pathways due to genetic alterations result in deregulated kinase activity and malignant transformation. This review focuses on the recent reports of the essential role of PI3K/Akt/mTOR and CDK4/6/RB pathways in ER+ HER2- breast cancer.

Fluorescence in situ hybridisation as an ancillary tool in the diagnosis of acral melanoma: a review of 44 cases

Acral melanoma is associated with outcomes which are more unfavourable than those of other melanoma subtypes, and acral melanoma has higher mortality. However, histological distinction of acral melanoma from acral naevi may be difficult. Fluorescence in situ hybridisation (FISH) targeting specific genes has been used as an ancillary method for differential diagnosis of melanocytic tumours, but most previous studies have focused on non-acral lesions which may have genetic alterations different from acral lesions. We evaluated use of multi-site FISH in the diagnosis of acral melanoma in a series of 82 acral melanocytic tumours. Two probe groups were applied. Probe set 1 involved a 4-probe FISH targeting 6p25 (RREB1), CEP6 (centromere 6), 6q23 (MYB) and 11q13 (CCND1). Probe set 2 involved a 3-probe FISH targeting 8q24 (MYC), 9p21 (CDKN2A) and CEP9 (centromere 9). In 44 primary acral melanomas, sensitivity was 70.5% (31/44) using probe set 1 alone, and 59.1% (26/44) using probe set 2 alone. When both probe sets were combined, sensitivity increased to 88.6% (39/44). The frequency of each gene alteration was as follows: MYC gain in 54.5% cases (24/44), RREB1 gain in 52.3% cases (23/44), CCND1 gain in 45.4% cases (20/44), MYB loss relative to CEP6 in 25.0% cases (11/44), and CDKN2A homozygous deletion in 20.5% cases (9/44). For lesions with both in situ and invasive disease, FISH findings in these two components were similar. No gene alterations were detected in any of 36 benign acral naevi. In this study FISH exhibited sensitivity and specificity in diagnosis of acral melanoma which allows its application as an auxiliary diagnostic test in acral melanocytic tumours.

Malignant Melanoma of the Nail Apparatus

Background Malignant melanoma of the nail apparatus is exceedingly rare. Increasingly, genetic studies have been employed to aid in distinguishing between malignant melanoma and benign melanocytic nevi. Methods Archived nail apparatus melanomas were analyzed by fluorescence in situ hybridization (FISH) using probes targeting the genes at 6p25 (RREB1), 11q13 (CCND1), 8q24.1 (MYC), 6q23 (MYB), 9p21 (CDKN2A) and the centromeres of chromosomes 8 (D8Z2) and 6 (D6Z1). The results were correlated with clinical and demographic information. Results Mean patient age was 57.8 years (range 23-92 years). In all, 5 of 7 (71%) cases involved the upper extremity digits. RREB1 gain was seen in all cases. CCND1 gain was seen in 6 of 7 (86%) cases, 3 of which were amplified. MYB loss and MYC gain were both seen in 5 of 7 (71%) cases. Homozygous loss of CDKN2A was not observed in any case. Two of 7 (28.6%) patients had lymph node metastasis and died of widely metastatic disease. These 2 patients harbored the most genetic aberrations: gains of RREB1, CCND1, and MYC, and MYB loss. Both benign melanocytic nevi controls showed normal FISH results. Conclusions RREB1 and CCND1 gains are common in nail apparatus melanoma as in most melanomas, and an increased number of genetic aberrations may be associated with a poorer prognosis, though the limited number of cases precludes definitive correlation. FISH appears to be a useful adjunct in the diagnosis of nail apparatus melanomas and improves diagnostic confidence even in the setting of unambiguous histomorphology.

Clinical relevance of copy number profiling in oral and oropharyngeal squamous cell carcinoma.

Current conventional treatment modalities in head and neck squamous cell carcinoma (HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas (OPSCC) and 164 oral cavity squamous cell carcinomas (OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus (HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I–II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature