Predicting cytogenetic risk in multiple myeloma using conventional whole-body MRI, spinal dynamic contrast-enhanced MRI, and spinal diffusion-weighted imaging.

OBJECTIVES: Cytogenetic abnormalities are predictors of poor prognosis in multiple myeloma (MM). This paper aims to build and validate a multiparametric conventional and functional whole-body MRI-based prediction model for cytogenetic risk classification in newly diagnosed MM. METHODS: Patients with newly diagnosed MM who underwent multiparametric conventional whole-body MRI, spinal dynamic contrast-enhanced (DCE-)MRI, spinal diffusion-weighted MRI (DWI) and had genetic analysis were retrospectively included (2011-2020/Ghent University Hospital/Belgium). Patients were stratified into standard versus intermediate/high cytogenetic risk groups. After segmentation, 303 MRI features were extracted. Univariate and model-based methods were evaluated for feature and model selection. Testing was performed using receiver operating characteristic (ROC) and precision-recall curves. Models comparing the performance for genetic risk classification of the entire MRI protocol and of all MRI sequences separately were evaluated, including all features. Four final models, including only the top three most predictive features, were evaluated. RESULTS: Thirty-one patients were enrolled (mean age 66 ± 7 years, 15 men, 13 intermediate-/high-risk genetics). None of the univariate models and none of the models with all features included achieved good performance. The best performing model with only the three most predictive features and including all MRI sequences reached a ROC-area-under-the-curve of 0.80 and precision-recall-area-under-the-curve of 0.79. The highest statistical performance was reached when all three MRI sequences were combined (conventional whole-body MRI + DCE-MRI + DWI). Conventional MRI always outperformed the other sequences. DCE-MRI always outperformed DWI, except for specificity. CONCLUSIONS: A multiparametric MRI-based model has a better performance in the noninvasive prediction of high-risk cytogenetics in newly diagnosed MM than conventional MRI alone. CRITICAL RELEVANCE STATEMENT: An elaborate multiparametric MRI-based model performs better than conventional MRI alone for the noninvasive prediction of high-risk cytogenetics in newly diagnosed multiple myeloma; this opens opportunities to assess genetic heterogeneity thus overcoming sampling bias. KEY POINTS: • Standard genetic techniques in multiple myeloma patients suffer from sampling bias due to tumoral heterogeneity. • Multiparametric MRI noninvasively predicts genetic risk in multiple myeloma. • Combined conventional anatomical MRI, DCE-MRI, and DWI had the highest statistical performance to predict genetic risk. • Conventional MRI alone always outperformed DCE-MRI and DWI separately to predict genetic risk. DCE-MRI alone always outperformed DWI separately, except for the parameter specificity to predict genetic risk. • This multiparametric MRI-based genetic risk prediction model opens opportunities to noninvasively assess genetic heterogeneity thereby overcoming sampling bias in predicting genetic risk in multiple myeloma.

Myb overexpression synergizes with the loss of Pten and is a dependency factor and therapeutic target in T-cell lymphoblastic leukemia.

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Although the prognosis of T-ALL has improved over time, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T-ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto-oncogene MYB is highly expressed in diverse hematologic malignancies, including T-ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T-ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event-free survival of pediatric T-ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB-driven T-ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T-cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T-ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T-ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T-ALL.

Fibroepithelial Stromal Polyp of the Vulvovaginal Region as Part of the RB1 Family of Tumors: Friend or Foe?

Fibroepithelial stromal polyps (FSPs) are benign mesenchymal lesions occurring in the vulvovaginal region. Following the identification of loss of Retinoblastoma 1 (RB1) on immunohistochemical staining in routine practice, we stained a series of FSPs and performed additional fluorescence in situ hybridization (FISH) and copy number variation (CNV) sequencing to detect losses/deletions in the Retinoblastoma transcriptional corepressor 1 (RB1) gene. Fifteen FSP cases were stained for RB1, and subsequently, 9 cases were examined by FISH to detect a loss of RB1 (13q). Next, CNV sequencing was performed to assess genomic alterations. The mean age of the patients was 50 years. Loss of RB1 expression on immunohistochemistry was seen in 13 cases, and heterogeneous RB1 staining in the remaining 2 cases. FISH showed deletion of RB1 in all of the cases. CNV sequencing failed in almost all cases due to a low tumor content. Based on our findings, we hypothesize that FSPs are part of a spectrum of genetically related lesions, namely the 13q/RB1 family of tumors (which includes pleomorphic fibromas and spindle cell/pleomorphic lipomas). Due to the clinical, morphologic, and molecular overlap, we suggest that FSPs are pleomorphic fibromas occurring in the specialized stroma of the genital region.

Revealing RB1 loss in an emerging entity: report of two cases of PRRX1-rearranged mesenchymal tumours.

AIMS: PRRX1-rearranged mesenchymal tumours are a recently identified and rare subgroup of soft tissue neoplasms with distinct morphological features and genetic alterations. This study aims to further investigate the immunohistochemical profile and underlying genetic alterations in these tumours in order to get more insight on their underlying biology and the unique profile of these tumours. METHODS: Two new molecular confirmed cases of PRRX1-rearranged mesenchymal tumours were thoroughly studied with immunohistochemical stainings (RB1, CD34, ALK and pan-TRK), fluorescence in situ hybridisation (FISH) RB1/13q12 and RNA-based next-generation sequencing. RESULTS: Both cases exhibited typical morphological and molecular features, confirming the diagnosis of PRRX1-rearranged mesenchymal tumours. Immunohistochemistry revealed RB1 loss in both cases, which was subsequently confirmed through FISH analysis. Additionally, one case showed focal positivity for CD34, ALK and pan-TRK on immunohistochemistry. CONCLUSIONS: We identified loss of RB1 in two cases of PRRX1-rearranged mesenchymal tumours. This could suggest a potential association with RB1-deficient soft tissue tumours, although further research is necessary. Furthermore, the finding of focal positivity for CD34, ALK and pan-TRK on immunohistochemistry enriches the immunohistochemical profile of these tumours.

Targeting hyperactive platelet-derived growth factor receptor-ß signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T cell acute lymphoblastic leukemia (T-ALL) and T cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematological malignancies. Current treatment consists of intensive chemotherapy, leading to 80% overall survival but are associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL and that screening T-ALL/TLBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Dilemmas in the Management of an Infant with Neuroblastoma Metastasized to the Muscles.

The risk stratification of infants with metastatic neuroblastoma (NB) has evolved over time from stage 4/M or IVs/4S/MS/Ms according to various staging systems. Despite these developments for some genetic aberrations, the prognostic value and the impact of soft tissue metastases in infants are not fully understood, nor well described in the different classification systems, hampering the definitions to uniformly treat patients and predict prognosis. A literature review on staging of infants with M/MS disease was performed at the occasion of the diagnosis of NB in an 8-month-old boy who presented with atypical metastatic sites in soft tissue and an aberrant tumor biology. The definitions of stage 4/4S/4s/M/MS/Ms were evaluated and compared to enable tumor risk stratification and inform management. International NB groups use different criteria for defining stage of infants with metastasized NB, resulting in differences in management. Limited literature is available on soft tissue metastases, especially muscular metastases, and is poorly incorporated into management guidelines mainly due to the lack of data. The uncertain prognosis of rare genetic aberrancies may add to the difficulties in treatment decisions. In some rare cases of NB in infants, the international treatment classification is not sufficient for staging and treatment decisions. Based on tumor progression, biology of unknown significance and a lack of evidence to classify a child under 12 months with NB and multiple muscular metastases, the patient was treated as stage 4/M and intermediate-risk protocols with a favorable outcome.

SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry.

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.

High-grade endometrial stromal sarcoma-like' sarcoma in male: Does it exist? A case report and review of the literature.

We report an exceptional case of an undifferentiated round and spindle cell sarcoma, occurring in the periprostatic region of a 54-year-old male, with a 'high-grade endometrial stromal sarcoma-like' (HG-ESS) morphology and harboring a ZC3H7B::BCOR gene fusion identified by RNA-based next-generation sequencing. In this report, we describe the striking overlap of morphologic, immunohistochemical and molecular features of this current case and previously reported similar cases with ZC3H7B::BCOR fusion-positive HG-ESS, and discuss the differential diagnosis and possible pathogenesis of this unusual entity.

Targeted therapy in juvenile myelomonocytic leukemia: Where are we now?

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive clonal neoplasm of early childhood, classified as an overlap myeloproliferative/myelodysplastic neoplasm by the World Health Organization. In 90% of the patients with JMML, typical initiating mutations in the canonical Ras pathway genes NF1, PTPN11, NRAS, KRAS, and CBL can be identified. Hematopoietic stem cell transplantation (HSCT) currently is the established standard of care in most patients, although long-term survival is still only 50-60%. Given the limited therapeutic options and the important morbidity and mortality associated with HSCT, new therapeutic approaches are urgently needed. Hyperactivation of the Ras pathway as disease mechanism in JMML lends itself to the use of targeted therapy. Targeted therapy could play an important role in the future treatment of patients with JMML. This review presents a comprehensive overview of targeted therapies already developed and evaluated in vitro and in vivo in patients with JMML.

Malignant pleural mesothelioma with an EML4-ALK fusion: Expect the unexpected!

Case report of malignant pleural mesothelioma with an ALK gene rearrangement, detected by FISH and confirmed by RNA-based next-generation sequencing. The co-occurrence of ALK gene fusions with the more common genetic alterations in CDKN2A, NF2 and BAP1 has, to our best knowledge, not yet been described in malignant mesothelioma. Furthermore, this unexpected finding could suggest a potential target for therapy in this subset of malignant mesotheliomas.

Shallow whole-genome sequencing: a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue-a glioma-driven study.

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7 +/ 10 - chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 ± 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection.

Extrauterine Mesonephric-like Neoplasms: Expanding the Morphologic Spectrum.

Mesonephric-like adenocarcinomas (MLA) are rare neoplasms arising in the uterine corpus and ovary which have been added to the recent 2020 World Health Organization Classification of Female Genital Tumors. They have similar morphology and immunophenotype and exhibit molecular aberrations similar to cervical mesonephric adenocarcinomas. It is debated as to whether they are of mesonephric or Mullerian origin. We describe the clinical, pathologic, immunohistochemical, and molecular features of 5 cases of extrauterine mesonephric-like proliferations (4 ovary, 1 extraovarian), all with novel and hitherto unreported features. These include an origin of MLA in extraovarian endometriosis, an association of ovarian MLA with high-grade serous carcinoma, mixed germ cell tumor and mature teratoma, and a borderline ovarian endometrioid tumor exhibiting mesonephric differentiation. Four of the cases exhibited a KRAS variant and 3 also a PIK3CA variant. In reporting these cases, we expand on the published tumor types associated with MLA and report for the first time a borderline tumor exhibiting mesonephric differentiation. We show the value of molecular testing in helping to confirm a mesonephric-like lesion and in determining the relationship between the different neoplastic components. We provide further evidence for a Mullerian origin, rather than a true mesonephric origin, in some of these cases. We also speculate that in the 2 cases associated with germ cell neoplasms, the MLA arose out of the germ cell tumor.

The feasibility of using liquid biopsies as a complementary assay for copy number aberration profiling in routinely collected paediatric cancer patient samples.

BACKGROUND: Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. PROCEDURE: The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. RESULTS: Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (<700 bp) and high molecular weight DNA (>700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. CONCLUSION: In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial.

From DNA Copy Number Gains and Tumor Dependencies to Novel Therapeutic Targets for High-Risk Neuroblastoma.

Neuroblastoma is a pediatric tumor arising from the sympatho-adrenal lineage and a worldwide leading cause of childhood cancer-related deaths. About half of high-risk patients die from the disease while survivors suffer from multiple therapy-related side-effects. While neuroblastomas present with a low mutational burden, focal and large segmental DNA copy number aberrations are highly recurrent and associated with poor survival. It can be assumed that the affected chromosomal regions contain critical genes implicated in neuroblastoma biology and behavior. More specifically, evidence has emerged that several of these genes are implicated in tumor dependencies thus potentially providing novel therapeutic entry points. In this review, we briefly review the current status of recurrent DNA copy number aberrations in neuroblastoma and provide an overview of the genes affected by these genomic variants for which a direct role in neuroblastoma has been established. Several of these genes are implicated in networks that positively regulate MYCN expression or stability as well as cell cycle control and apoptosis. Finally, we summarize alternative approaches to identify and prioritize candidate copy-number driven dependency genes for neuroblastoma offering novel therapeutic opportunities.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study.

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

A challenging diagnosis of a nonsecretor plasma cell dyscrasia with pleomorphic plasmablastic morphology.

This report highlights the importance of integrating clinical, radiological, genetic, and pathological laboratory findings to make a correct diagnosis especially with challenging and rare entities.