Actionable mutational profiling in solid tumors using hybrid-capture-based next-generation sequencing in a real-world setting in Spain.

OBJECTIVE: This study aimed to describe the performance of a next-generation sequencing (NGS) panel for the detection of precise genomic alterations in cancer in Spanish clinical practice. The impact of tumor characteristics was evaluated on informative NGS and actionable mutation rates. MATERIALS AND METHODS: A cross-sectional study was conducted at the Fundación Jiménez Díaz University Hospital (May 2021-March 2022) where molecular diagnostic of 537 Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of diverse solid tumors (lung, colorectal, melanoma, gastrointestinal stromal, among others) was performed using AVENIO Tumor Tissue Targeted Kit. A descriptive analysis of the features of all samples was carried out. Multivariable logistic analysis was conducted to assess the impact of sample characteristics on NGS performance defined by informative results rate (for all tumors and for lung tumors), and on actionable mutations rate (for lung tumors only). RESULTS: AVENIO performance rate was 75.2% in all tumor samples and 75.3% in lung cancer samples, and the multivariable analysis showed that surgical specimens are most likely to provide informative results than diagnostic biopsies. Regarding the mutational findings, 727 pathogenic, likely pathogenic, or variant of unknown significance mutations were found in all tumor samples. Single nucleotide variant was the most common genomic alteration, both for all tumor samples (85.3% and 81.9% for all solid tumors and lung samples, respectively). In lung tumors, multivariable analysis showed that it is more likely to find actionable mutations from non-smokers and patients with adenocarcinoma, large cell, or undifferentiated histologies. CONCLUSION: This is the largest cohort-level study in Spain to profile the analyses of biopsy samples of different tumors using NGS in routine clinical practice. Our findings showed that the use of NGS routinely provides good rates of informative results and can improve tumor characterization and identify a greater number of actionable mutations.

Leukaemic Presentation of Small-Cell Alk-Positive Anaplastic Large Cell Lymphoma in a Young Woman-Report of a Case with 9-Year Survival.

Anaplastic large cell lymphoma (ALCL) with leukaemic presentation (either ab initio or along the course of the disease) has been rarely reported. Irrespective of ALK expression in the neoplastic cells, it features a dismal prognosis. We report a rare case of leukaemic, small cell variant ALK-positive ALCL with 9-year survival in a young woman who was treated upfront with corticosteroids and standard chemotherapy, and review thoroughly the previously published cases. Such an unexpected, good outcome hints at the existence of different clinical subgroups in the leukaemic variant of ALK-positive ALCL.

Anaplastic large cell lymphomas with the 6p25.3 rearrangement are a heterogeneous group of tumours with a diverse molecular background.

ALK-negative anaplastic large cell lymphoma (ALCL) cases with 6p25.3 rearrangement are characterized by peculiar morphological and immunohistochemical features compare to 6p25.3-negative ALK-negative ALCL cases. A subgroup of 6p25.3-positive ALK-negative ALCL cases show the t(6,7) (p25.3;q32.3) rearrangement. Aims: To analyse the differences between 6p25.3-rearranged cases with and without t(6,7) (p25.3;q32.3). Using RNA-sequencing we studied a series of 17 samples showing 6p25.3-rearrangement, identified by FISH, consisting of seven systemic and eight primary cutaneous cases including two examples of secondary skin involvement by systemic ALCL. RNA-sequencing exclusively detected a translocation involving a gene in the 6p25.3 region (either IRF4 or DUSP22) in 7/14 cases (50%). In six of these seven cases the partner proved to be the LINC-PINT region in chromosome 7, while an EXOC2::DUSP22 rearrangement was found in one case. All cases but one were primary cutaneous ALCLs. They all were CD3 positive and BCL2 negative, while most of them expressed p-STAT3. On the contrary, cases without the t(6,7) (p25.3;q32.3) were mainly systemic (71%, 5/7) against just two pcALCL. In general, they lose CD3 (50% positive) and p-STAT3 (25% positive) expression, being all of them BCL2 positive. Moreover, in 60% of them other gene fusions were found. At the transcriptional level, they were characterized by the overexpression of TCF3 (TCF7L1/E2A), DLL3, CD58 and BCL2 genes 75%(6/8) of pcALCL with 6p25.3 rearrangement featured the so-called "biphasic morphologic pattern, which was not found in cutaneous involvement from systemic ALCL. 83% (5/6) of the pcALCL cases with the "biphasic morphologic pattern" showed the t(6,7) (p25.3;q32.3) rearrangement. ALK-negative ALCL cases with 6p25.3 rearrangement are a subgroup of tumours that are heterogeneous with respect to the presence or absence of the t(6,7) (p25.3;q32.3) translocation.

Novel Germline TET2 Mutations in Two Unrelated Patients with Autoimmune Lymphoproliferative Syndrome-Like Phenotype and Hematologic Malignancy.

Somatic mutations in the ten-eleven translocation methylcytosine dioxygenase 2 gene (TET2) have been associated to hematologic malignancies. More recently, biallelic, and monoallelic germline mutations conferring susceptibility to lymphoid and myeloid cancer have been described. We report two unrelated autoimmune lymphoproliferative syndrome-like patients who presented with T-cell lymphoma associated with novel germline biallelic or monoallelic mutations in the TET2 gene. Both patients presented a history of chronic lymphoproliferation with lymphadenopathies and splenomegaly, cytopenias, and immune dysregulation. We identified the first compound heterozygous patient for TET2 mutations (P1) and the first ALPS-like patient with a monoallelic TET2 mutation (P2). P1 had the most severe form of autosomal recessive disease due to TET2 loss of function resulting in absent TET2 expression and profound increase in DNA methylation. Additionally, the immunophenotype showed some alterations in innate and adaptive immune system as inverted myeloid/plasmacytoid dendritic cells ratio, elevated terminally differentiated effector memory CD8 + T-cells re-expressing CD45RA, regulatory T-cells, and Th2 circulating follicular T-cells. Double-negative T-cells, vitamin B12, and IL-10 were elevated according to the ALPS-like suspicion. Interestingly, the healthy P1's brother carried a TET2 mutation and presented some markers of immune dysregulation. P2 showed elevated vitamin B12, hypergammaglobulinemia, and decreased HDL levels. Therefore, novel molecular defects in TET2 confirm and expand both clinical and immunological phenotype, contributing to a better knowledge of the bridge between cancer and immunity.

Comprehensive Approach to Genomic and Immune Profiling: Insights of a Real-World Experience in Gynecological Tumors.

Gynecological cancer accounts for an elevated incidence worldwide requiring responsiveness regarding its care. The comprehensive genomic approach agrees with the classification of certain tumor types. We evaluated 49 patients with gynecological tumors undergoing high-throughput sequencing to explore whether identifying alterations in cancer-associated genes could characterize concrete histological subtypes. We performed immune examination and analyzed subsequent clinical impact. We found 220 genomic aberrations mostly distributed as single nucleotide variants (SNV, 77%). Only 3% were classified as variants of strong clinical significance in BRCA1 and BRCA2 of ovarian high-grade serous (HGSC) and uterine endometrioid carcinoma. TP53 and BRCA1 occurred in 72% and 28% of HGSC. Cervical squamous cell carcinoma was entirely HPV-associated and mutations occurred in PIK3CA (60%), as well as in uterine serous carcinoma (80%). Alterations were seen in PTEN (71%) and PIK3CA (60%) of uterine endometrioid carcinoma. Elevated programmed death-ligand 1 (PD-L1) was associated with high TILs. Either PD-L1 augmented in deficient mis-matched repair (MMR) proteins or POLE mutated cases when compared to a proficient MMR state. An 18% received genotype-guided therapy and a 4% immunotherapy. The description of tumor subtypes is plausible through high-throughput sequencing by recognizing clinically relevant alterations. Additional concomitant assessment of immune biomarkers identifies candidates for immunotherapy.

Double CD4/CD8-Positive, Nonpoikilodermic Mycosis Fungoides Expressing CD56 in a Young Man.

ABSTRACT: We report a case of mycosis fungoides (MF) in an 18-year-old man whose neoplastic T cells expressed CD4, CD8, and CD56, with no evidence of TCR-delta or Epstein-Barr virus (EBER) expression. Clinically, neither hypopigmentation nor hyperpigmentation nor poikilodermatous skin lesions were present, and the lesions subsided with oral corticoids and retinoids and environmental solar ultraviolet exposure. Our case represents the oldest patient reported so far with nonpoikilodermatous, CD8/CD56 MF and adds to the phenotypic diversity of MF in the pediatric population. This distinct phenotype does not seem to be linked to a more aggressive course than the classic CD-4 positive one.

Plasmacytoid Dendritic Cell Dermatosis Associated to Myeloproliferative/Myelodysplastic Neoplasms.

Cutaneous lesions in the setting of myeloproliferative neoplasms and myelodysplastic syndromes are poorly understood. We report 6 patients with pruritic papular eruptions composed of mature T-lymphocytes with large clusters of CD123-positive cells. Double immunohistochemical studies demonstrated a lack of myeloid cell nuclear differentiation antigen in the CD123-positive cells, which expressed SPIB, confirming that they were mature plasmacytoid dendritic cells. Four patients were diagnosed with chronic myelomonocytic leukemia and 2 with myelodysplastic syndromes (AREB-I and myelodysplastic syndromes with 5q deletion, respectively). All patients had a long history of hematological alterations, mainly thrombocytopenia, preceding the cutaneous disorder. Nevertheless, the skin lesions developed in all cases coincidentally with either progression or full-establishment of their hematological disease. Most cutaneous lesions disappeared spontaneously or after corticosteroid treatment. Molecular studies performed in both bone marrow and cutaneous lesions in 2 patients demonstrated the same mutational profile, confirming the specific, neoplastic nature of these mature plasmacytoid dendritic cells-composed cutaneous lesions.

Redefining the high-grade B cell lymphoma with double/triple rearrangements of MYC and BCL2/BCL6 genes. Learning from a case report.

We report a patient initially diagnosed with a triple hit high-grade B cell lymphoma (HGBL-TH), in which further morphologic, immunohistochemical, and next-generation sequencing studies of subsequent specimens disclosed it to be a germinal center diffuse large B cell lymphoma (GC-DLBCL) with BCL2/BCL6 gene translocations, PVT1-deletion, and gain of MYC genes evolving from a previous follicular lymphoma. However, fluorescence in situ hybridization (FISH) studies with the break-apart probe for MYC gene showed a fusion and two separated signals (red and green, respectively) leading to the interpretation of MYC gene translocation and a false diagnosis of a TH-lymphoma, according to the recent WHO classification. Nevertheless, PVT1 deletion plus MYC gain/amplification has been described as a cause of the double-hi transcription profile. These data highlight the need for new criteria to identify these highly aggressive lymphomas.

Subcutaneous panniculitis-like T-cell lymphoma, lupus erythematosus profundus, and overlapping cases: molecular characterization through the study of 208 genes.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic cutaneous lymphoma. Differential diagnosis with lupus erythematosus panniculitis (LEP) can be challenging and overlapping cases have been described. In this study, we investigate whether gene expression profiling may or not identify markers that can be used to improve our understanding of the disease and to make a precise differential diagnosis. SPTCL, LEP, and overlapping cases were analyzed using a customized NanoString platform including 208 genes related to T-cell differentiation, stromal signatures, oncogenes, and tumor suppressor genes. Gene expression unsupervised analysis of the samples differentiated SPTCL from LEP samples. Most overlapping cases were clustered with LEP cases. Differentially expressed genes were observed when comparing SPTCL with LEP cases; and overlapping with LEP cases. Gene set enrichment analysis recognized gene sets defining each group. In conclusion, SPTCL and LEP have distinctive molecular profiles and the molecular background of overlapping cases more closely resembles LEP.

PD-L1 expression in peripheral T-cell lymphomas is not related to either PD-L1 gene amplification or rearrangements.

Nodal peripheral T-cell lymphomas (n-PTCL) are aggressive lymphomas with no specific treatment. Programmed death 1 (PD-1) inhibits T-cell activation and proliferation, and the expression of its ligand PD-L1 has been associated with worse prognosis in some tumors. We performed immunohistochemistry for PD-1, p-STAT3, and PD-L1 (Clones SP142/263/22C3/28.8) and FISH studies for PD-L1/2 genes in chromosome 9p in a series of 168 formalin-fixed, paraffin-embedded n-PTCL samples. PD-L1 (clone 263) was the most frequently detected in both tumor cells (especially in the ALCL subgroup) and the microenvironment (especially in the AITL subgroup). In five ALCL cases, 3-4 copies of the two loci of chromosome 9 were found, suggestive of polyploidy. PD-L1 correlated with p-STAT3 on tumor cells. PD-1 expression in tumor cells was related to expression of PD-L1 in microenvironment. The expression of PD-L1 on tumor cells or microenvironment suggests that some n-PTCL cases might benefit from immune check-point modulation therapy.

Lymphoplasmacytic lymphoma associated with diffuse large B-cell lymphoma: Progression or divergent evolution?

AIM: Lymphoplasmacytic lymphoma (LPL) is an indolent mature B-cell-neoplasm with involvement of the bone marrow. At least 90% of LPLs carry MYD88-L265P mutation and some of them (~10%) transform into diffuse large B-cell-lymphoma (DLBCL). MATERIAL AND METHODS: Over the past 15 years we have collected 7 cases where the both LPL and DLBCL were diagnosed in the same patient. Clinical records, analytical data and histopathological specimens were reviewed. FISH studies on paraffin-embedded tissue for MYC, BCL2 and BCL6 genes were performed, as well as MYD88-L265P mutation and IGH rearrangement analysis by PCR. A mutational study was done by massive next generation sequencing (NGS). RESULTS: There were 4 women and 3 men between 36-91 years of age. Diagnoses were made simultaneously in 4 patients. In two cases the LPL appeared before the DLBCL and in the remaining case the high-grade component was discovered 5 years before the LPL. In 6 cases both samples shared the MYD88-L265P mutation. IGH rearrangement analysis showed overlapping features in two of 6 cases tested. Mutational study was evaluable in three cases for both samples showing shared and divergent mutations. CONCLUSIONS: These data suggest different mechanisms of DLBCL development in LPL patients.

Validation and clinical application of a targeted next-generation sequencing gene panel for solid and hematologic malignancies.

BACKGROUND: Next-generation sequencing (NGS) is a high-throughput technology that has become widely integrated in molecular diagnostics laboratories. Among the large diversity of NGS-based panels, the Trusight Tumor 26 (TsT26) enables the detection of low-frequency variants across 26 genes using the MiSeq platform. METHODS: We describe the inter-laboratory validation and subsequent clinical application of the panel in 399 patients presenting a range of tumor types, including gastrointestinal (GI, 29%), hematologic (18%), lung (13%), gynecological and breast (8% each), among others. RESULTS: The panel is highly accurate with a test sensitivity of 92%, and demonstrated high specificity and positive predictive values (95% and 96%, respectively). Sequencing testing was successful in two-thirds of patients, while the remaining third failed due to unsuccessful quality-control filtering. Most detected variants were observed in the TP53 (28%), KRAS (16%), APC (10%) and PIK3CA (8%) genes. Overall, 372 variants were identified, primarily distributed as missense (81%), stop gain (9%) and frameshift (7%) altered sequences and mostly reported as pathogenic (78%) and variants of uncertain significance (19%). Only 14% of patients received targeted treatment based on the variant determined by the panel. The variants most frequently observed in GI and lung tumors were: KRAS c.35G > A (p.G12D), c.35G > T (p.G12V) and c.34G > T (p.G12C). CONCLUSIONS: Prior panel validation allowed its use in the laboratory daily practice by providing several relevant and potentially targetable variants across multiple tumors. However, this study is limited by high sample inadequacy rate, raising doubts as to continuity in the clinical setting.

Epstein-Barr virus-associated large B-cell lymphoma transformation in marginal zone B-cell lymphoma: a series of four cases.

AIMS: To present four examples of clonally related Epstein-Barr virus (EBV)-associated large-cell transformation of marginal zone lymphoma (MZL) (of nodal, extranodal and splenic types), occurring 120, 11 and 5 months after the initial diagnosis in three instances, and concurrently in one case; and to discuss several interesting features of EBV infection. METHODS AND RESULTS: Somatic mutations were detected by use of a customised panel for next-generation sequencing and polymerase chain reaction studies of IgH in both low-grade and high-grade components of each case. In case 1, the initial biopsy of nodal MZL showed scattered EBV-positive cells, which might constitute an indication of EBV-induced progression. Case 2 showed heterogeneous EBV expression, a phenomenon attributable to loss of the EBV episomes during cell division, or to a secondary superinfection or reactivation of the virus. In case 3, p53 overexpression related to gene mutation and EBV-encoded small RNAs were identified in the same neoplastic component. In case 4, the mucosa-associated lymphoid tissue-type MZL and the high-grade component were identified concurrently in a patient previously treated with methotrexate for an autoimmune disorder. CONCLUSION: These data suggest that the presence of EBV should be added to the list of potential markers to be analysed for MZL prognosis.

Large Cells With CD30 Expression and Hodgkin-like Features in Primary Cutaneous Marginal Zone B-Cell Lymphoma: A Study of 13 Cases.

The presence of CD30 cells in cutaneous lymphomas has come to prominence in recent years as a potential diagnostic and therapeutic marker. In primary cutaneous marginal zone B-cell lymphomas, the presence of large CD30 cells with Hodgkin-like features and their significance have not yet been studied. Here we describe the main clinical, histologic, immunophenotypic, and molecular characteristics of 13 cases of primary cutaneous marginal zone lymphomas featuring >10% of CD30 large cells, and analyze their relationship with histologic and clinical progression of the disease and with other morphologic and immunophenotypic features. We report 10 male and 3 female patients, 4 with early-local disease and 8 with locoregional advanced disease without extracutaneous involvement but with a high relapse rate of 69%. We describe an association between a high level of CD30 expression and disease progression, with increased clinical recurrence in cases with >15% of CD30 cells. We also discuss the differential diagnosis with other cutaneous and systemic lymphomas, especially Hodgkin lymphoma.

The Spectrum of EBV-Positive Mucocutaneous Ulcer: A Study of 9 Cases.

We describe a series of 9 patients with Epstein-Barr virus (EBV)-positive mucocutaneous lymphoproliferative lesions that broadens the concept of EBV-positive mucocutaneous ulcer. We report 5 female and 4 male patients, with an average age of 74 years (range, 55 to 87 y), 2 of whom were HIV-positive. The lesions were located in the oropharynx, skin, and rectal and/or genital mucosa. Histopathologically, 6 cases showed a polymorphic pattern and 3 had a monomorphic and diffuse one, with angiotropism in 4 cases (2 each with the polymorphic and monomorphic patterns). Three of the cases expressed PDL1. In addition to its presence in the neoplastic lymphoid cells, EBV was also detected in adjacent epithelial cells in an oropharyngeal lesion. All cases responded to local therapy or adapted systemic chemotherapy in selected cases. This series extends the spectrum of this disorder to include some HIV-positive cases, patients with multiple lesions confined to a single anatomic area, lesions with an angiocentric pattern, and some cases with monomorphous large-cell cytology. We discuss the differential clinicopathologic diagnosis of this disorder and that of classic EBV large B-cell lymphoma.

Mycosis fungoides progression could be regulated by microRNAs.

Differentiating early mycosis fungoides (MF) from inflammatory dermatitis is a challenge. We compare the differential expression profile of early-stage MF samples and benign inflammatory dermatoses using microRNA (miRNA) arrays. 114 miRNAs were found to be dysregulated between these entities. The seven most differentially expressed miRNAs between these two conditions were further analyzed using RT-PCR in two series comprising 38 samples of early MFs and 18 samples of inflammatory dermatitis. A series of 51 paraffin-embedded samples belonging to paired stages of 16 MF patients was also analyzed. MiRNAs 26a, 222, 181a and 146a were differentially expressed between tumoral and inflammatory conditions. Two of these miRNAs (miRNA-181a and miRNA-146a) were significantly deregulated between early and advanced MF stages. Bioinformatic analysis showed FOXP3 expression to be regulated by these miRNAs. Immunohistochemistry revealed the level of FOXP3 expression to be lower in tumoral MFs than in plaque lesions in paraffin-embedded tissue. A functional study confirmed that both miRNAs diminished FOXP3 expression when overexpressed in CTCL cells. The data presented here suggest that the analysis of a restricted number of miRNAs (26a, 222, 181a and 146a) could be sufficient to differentiate tumoral from reactive conditions. Moreover, these miRNAs seem to be involved in MF progression.

Overlap at the molecular and immunohistochemical levels between angioimmunoblastic T-cell lymphoma and a subgroup of peripheral T-cell lymphomas without specific morphological features.

The overlap of morphology and immunophenotype between angioimmunoblastic T-cell lymphoma (AITL) and other nodal peripheral T-cell lymphomas (n-PTCLs) is a matter of current interest whose clinical relevance and pathogenic background have not been fully established. We studied a series of 98 n-PTCL samples (comprising 57 AITL and 41 PTCL-NOS) with five TFH antibodies (CD10, BCL-6, PD-1, CXCL13, ICOS), looked for mutations in five of the genes most frequently mutated in AITL (TET2, DNMT3A, IDH2, RHOA and PLCG1) using the Next-Generation-Sequencing Ion Torrent platform, and measured the correlations of these characteristics with morphology and clinical features. The percentage of mutations in the RHOA and TET2 genes was similar (23.5% of cases). PLCG1 was mutated in 14.3%, IDH2 in 11.2% and DNMT3A in 7.1% of cases, respectively. In the complete series, mutations in RHOA gene were associated with the presence of mutations in IDH2, TET2 and DNMT3A (p < 0.001, p = 0.043, and p = 0.029, respectively). Fourteen cases featured RHOA mutations without TET2 mutations. A close relationship was found between the presence of these mutations and a TFH-phenotype in AITL and PTCL-NOS patients. Interestingly, BCL-6 expression was the only TFH marker differentially expressed between AITL and PTCL-NOS cases. There were many fewer mutated cases than there were cases with a TFH phenotype. Overall, these data suggest alternative ways by which neoplastic T-cells overexpress these proteins. On the other hand, no clinical or survival differences were found between any of the recognized subgroups of patients with respect to their immunohistochemistry or mutational profile.