Kathryn E. Pearce

TFE3 rearrangements in adult renal cell carcinoma: clinical and pathologic features with outcome in a large series of consecutively treated patients.

Renal cell carcinoma (RCC) with chromosomal rearrangement of transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) at Xp11.2 is a distinct subtype that was initially described in children and has been reported to display an indolent course. Recent reports have identified RCC with TFE3 rearrangements in adults and have suggested a more aggressive course in this population. However, only a few studies have examined these tumors in a large series of consecutively treated adults. We screened 632 RCCs from patients consecutively treated by surgery at a single institution by fluorescence in situ hybridization to detect TFE3 rearrangements. We identified 6 RCCs with TFE3 rearrangement. Patient ages ranged from 25 to 78 years and included 4 women and 2 men. Tumors showed significant histologic variability. Comparison of the clinical and pathologic features between RCCs with TFE3 rearrangements and RCCs without TFE3 rearrangements showed no significant differences. Follow-up period for patients with TFE3-rearranged RCC ranged from 0.8 to 16.5 years, with 4 of 6 dying from the disease. Cancer-specific survival for patients with TFE3-rearranged RCC was significantly worse than for patients with TFE3-rearrangement–negative papillary-type RCC (P<0.001) but not different from that for TFE3-rearrangement–negative clear cell-type RCC. In conclusion, we present an assessment of TFE3 rearrangement status in a large series of adults consecutively treated by surgery for RCC. Our findings confirm that RCCs with TFE3 rearrangement account for only approximately 1% of adult RCCs. The results also suggest that adult RCC with TFE3 rearrangement may be a clinically aggressive tumor.

Molecular cytogenetic analysis for TFE3 rearrangement in Xp11.2 renal cell carcinoma and alveolar soft part sarcoma: validation and clinical experience with 75 cases

Renal cell carcinoma with TFE3 rearrangement at Xp11.2 is a distinct subtype manifesting an indolent clinical course in children, with recent reports suggesting a more aggressive entity in adults. This subtype is morphologically heterogeneous and can be misclassified as clear cell or papillary renal cell carcinoma. TFE3 is also rearranged in alveolar soft part sarcoma. To aid in diagnosis, a break-apart strategy fluorescence in situ hybridization (FISH) probe set specific for TFE3 rearrangement and a reflex dual-color, single-fusion strategy probe set involving the most common TFE3 partner gene, ASPSCR1, were validated on formalin-fixed, paraffin-embedded tissues from nine alveolar soft part sarcoma, two suspected Xp11.2 renal cell carcinoma, and nine tumors in the differential diagnosis. The impact of tissue cut artifact was reduced through inclusion of a chromosome X centromere control probe. Analysis of the UOK-109 renal carcinoma cell line confirmed the break-apart TFE3 probe set can distinguish the subtle TFE3/NONO fusion-associated inversion of chromosome X. Subsequent extensive clinical experience was gained through analysis of 75 cases with an indication of Xp11.2 renal cell carcinoma (n=54), alveolar soft part sarcoma (n=13), perivascular epithelioid cell neoplasms (n=2), chordoma (n=1), or unspecified (n=5). We observed balanced and unbalanced chromosome X;17 translocations in both Xp11.2 renal cell carcinoma and alveolar soft part sarcoma, supporting a preference but not a necessity for the translocation to be balanced in the carcinoma and unbalanced in the sarcoma. We further demonstrate the unbalanced separation is atypical, with TFE3/ASPSCR1 fusion and loss of the derivative X chromosome but also an unanticipated normal X chromosome gain in both males and females. Other diverse sex chromosome copy number combinations were observed. Our TFE3 FISH assay is a useful adjunct to morphologic analysis of such challenging cases and will be applicable to assess the growing spectrum of TFE3-rearranged tumors.

Molecular Cytogenetic Analysis of JAZF1, PHF1, and YWHAE in Endometrial Stromal Tumors: Discovery of Genetic Complexity by Fluorescence in Situ Hybridization

Diagnosis of endometrial stromal tumors (ESTs) can be challenging, particularly endometrial stromal sarcomas (ESSs) because of variable histologic appearance, long latency to recurrence, frequent metastases with unknown primary, and overlap with endometrial stromal nodules and undifferentiated uterine sarcomas. To enhance EST diagnosis, a break-apart strategy fluorescence in situ hybridization panel to detect JAZF1, PHF1, and YWHAE rearrangements was applied to a cohort of primary or metastatic endometrial stromal nodules, ESSs, or undifferentiated uterine sarcomas (36 cases for JAZF1, 24 of which were also assessed for PHF1 and YWHAE), 24 myometrium/endometrium controls, and 37 non-ESTs in the differential diagnosis. JAZF1 was the most frequently altered gene and occurred in all EST types, JAZF1 and/or PHF1 were mutually exclusive from YWHAE involvement, and uterine and extrauterine ESTs have a shared pathogenesis. We further defined frequency of these rearrangements and provided a resource demonstrating the signal complexity that can manifest when evaluating JAZF1. Rearrangement of JAZF1 occurred in 47% of ESTs, most (70%) of which had atypical patterns representing multiple structural alterations and/or more than one clone. YWHAE and PHF1 rearrangements each occurred in 8% of ESTs. An exceptional case was an ESS without JAZF1 or MEAF6 disruption that further disputes correlation of PHF1 involvement with the sex cord-like variant. These results expand our understanding of the genetic heterogeneity that defines ESTs.

Distinct ALK-rearranged and VCL-negative papillary renal cell carcinoma variant in two adults without sickle cell trait.

Distinct ALK-rearranged and VCL-negative papillary renal cell carcinoma variant in two adults without sickle cell trait

Development of an NPM1/MLF1 D-FISH Probe Set for the Detection of t(3;5)(q25;q35) Identified in Patients with Acute Myeloid Leukemia

The t(3;5)(q25;q35) NPM1/MLF1 fusion has an incidence of approximately 0.5% in acute myeloid leukemia (AML) and has an intermediate prognosis at diagnosis. We have developed a dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the MLF1 and NPM1 genes. A blinded investigation was performed using 25 normal bone marrow specimens and 26 bone marrow samples from patients with one or more metaphases with a t(3;5)(q21-q25;q31-q35) or a der(5)t(3;5)(q21-q25;q31-q35) previously identified by chromosome analysis. Once unblinded, the results indicate our D-FISH method identified NPM1/MLF1 fusion in 15 of the 26 fully evaluated patient samples. Excluding three samples with a single abnormal t(3;5) metaphase, 15 of 17 (88%) patient samples with a balanced t(3;5) demonstrated NPM1/MLF1 fusion, and 0 of 6 patient samples with a der(5)t(3;5) demonstrated NPM1/MLF1 fusion, suggesting only the balanced form of this 3;5 translocation as observed by karyotype is associated with NPM1/MLF1 fusion. Overall, the FISH results demonstrated five different outcomes (NPM1/MLF1 fusion, MLF1 disruption, MLF1 duplication, NPM1 deletion, and normal), indicating significant molecular heterogeneity when the 3;5 translocation is identified. The development of this sensitive D-FISH strategy for the detection of NPM1/MLF1 fusion adds to the AML FISH testing repertoire and is effective in the detection of this translocation at diagnosis as well as monitoring residual disease in AML patients.

KMT2A (MLL) rearrangements observed in pediatric/young adult T-lymphoblastic leukemia/lymphoma: A 10-year review from a single cytogenetic laboratory

T‐lymphoblastic leukemia/lymphoma (T‐ALL/LBL) accounts for approximately 15% of pediatric and 25% of adult ALL. While the underlying frequency of KMT2A (MLL) gene rearrangements has been identified in approximately 4‐8% of T‐ALL/LBL cases, a paucity of literature is available to characterize further the KMT2A rearrangements in pediatric/young adult T‐ALL/LBL. A 10‐year retrospective review was performed to identify KMT2A rearrangements in specimens sent for T‐ALL/LBL fluorescence in situ hybridization studies in patients under the age of 30 years. Of 806 T‐ALL/LBL FISH studies performed on unique individuals, 27 (3.3%) harbored KMT2A rearrangements. Nineteen patients were male and eight were female (M:F ratio, 2.4:1) with ages ranging from 1 to 20 years (mean 12, median 12). Of the 27 cases, nine (33%) had KMT2A/MLLT1 fusions, eight (30%) had KMT2A/AFDN fusions, two (7%) had KMT2A/ELL fusions, and one (4%) had a KMT2A/MLLT10 fusion. In addition, five (19%) had KMT2A rearrangements with unidentified gene fusion partners and two (7%) had 3’KMT2A deletions. Our results indicate that MLLT1 and AFDN account for the majority (63%) of KMT2A gene partners in pediatric/young adult T‐ALL/LBL, while no KMT2A/AFF1 or KMT2A/MLLT3 fusions were observed despite their common identification in B‐ALL and acute myeloid leukemia, respectively. In addition to diagnostic and prognostic value, detecting specific KMT2A fusions may also be of clinical importance in the era of targeted therapies.

Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia

Acute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints.

Differences in genomic abnormalities among African individuals with monoclonal gammopathies using calculated ancestry

Multiple myeloma (MM) is two- to three-fold more common in African Americans (AAs) compared to European Americans (EAs). This striking disparity, one of the highest of any cancer, may be due to underlying genetic predisposition between these groups. There are multiple unique cytogenetic subtypes of MM, and it is likely that the disparity is associated with only certain subtypes. Previous efforts to understand this disparity have relied on self-reported race rather than genetic ancestry, which may result in bias. To mitigate these difficulties, we studied 881 patients with monoclonal gammopathies who had undergone uniform testing to identify primary cytogenetic abnormalities. DNA from bone marrow samples was genotyped on the Precision Medicine Research Array and biogeographical ancestry was quantitatively assessed using the Geographic Population Structure Origins tool. The probability of having one of three specific subtypes, namely t(11;14), t(14;16), or t(14;20) was significantly higher in the 120 individuals with highest African ancestry (≥80%) compared with the 235 individuals with lowest African ancestry (<0.1%) (51% vs. 33%, respectively, p value = 0.008). Using quantitatively measured African ancestry, we demonstrate a major proportion of the racial disparity in MM is driven by disparity in the occurrence of the t(11;14), t(14;16), and t(14;20) types of MM.