BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Comparative Performance of Breast Cancer Human Epidermal Growth Factor Receptor 2 Fluorescence In Situ Hybridization and Brightfield In Situ Hybridization on College of American Pathologists Proficiency Tests.

CONTEXT.­: Fluorescence in situ hybridization (FISH) and brightfield in situ hybridization (ISH) are 2 clinically approved laboratory methods for detecting ERBB2 (HER2) amplification in breast cancer. OBJECTIVE.­: To compare the performance of FISH and brightfield ISH on proficiency testing administered by the College of American Pathologists Laboratory Accreditation Program. DESIGN.­: Retrospective review was performed on 70 tissue core samples in 7 separate proficiency testing surveys conducted between 2009 and 2013. RESULTS.­: The samples included 13 consensus-amplified tissue cores, 53 consensus-nonamplified cores, and 4 cores that did not reach consensus for FISH and/or brightfield ISH. There were 2552 individual responses for FISH and 1871 individual responses for brightfield ISH. Consensus response rates were comparable for FISH (2474 of 2524; 98.0%) and brightfield ISH (2135 of 2189; 97.5%). The FISH analysis yielded an average HER2 copy number per cell that was significantly higher (by 2.86; P = .02) compared with brightfield ISH for amplified cores. For nonamplified cores, FISH yielded slightly, but not significantly, higher (by 0.17; P = .10) HER2 copy numbers per cell. There was no significant difference in the average HER2 to control ratio for either consensus-amplified or consensus-nonamplified cores. Participants reported "unable to analyze" more frequently for brightfield ISH (244 of 2453; 9.9%) than they did for FISH (160 of 2684; 6.0%). CONCLUSIONS.­: Our study indicates a high concordance rate in proficiency testing surveys, with some significant differences noted in the technical performance of these assays. In borderline cases, updated American Society of Clinical Oncology/College of American Pathologists cutoff thresholds that place greater emphasis on HER2 copy number per cell could accentuate those differences between FISH and brightfield ISH.

A 26-Year-Old Female with Systemic Mastocytosis with Associated Myeloid Neoplasm with Eosinophilia and Abnormalities of PDGFRB, t(4;5)(q21;q33).

Various translocations involving the PDGFRB gene are identified in myeloid neoplasms. However, the PRKG2/PDGFRB fusion gene associated with t(4;5)(q21;q33) has previously been reported in only 3 patients. We present the case of a 26-year-old woman with microcytic anemia, basophilia, thrombocytosis, and massive splenomegaly, who was found to have systemic mastocytosis and associated clonal hematological non-mast cell lineage disease (SM-AHNMD), with myeloid neoplasm with PRKG2/PDGFRB rearrangement. Initial findings included basophilia (37%, 4.1 k/µL), hypercellular marrow with eosinophilia, and increased and atypical megakaryocytes, suggestive of myeloproliferative neoplasm. Additional studies revealed large clusters of CD25 positive mast cells, fulfilling the criteria for the diagnosis of systemic mastocytosis. Consistent with prior reports of this translocation, our patient has responded well to imatinib. This case, in conjunction with others in the literature, suggests a possible connection between t(4;5)(q21;q33) PRKG2/PDGFRB and systemic mastocytosis and highlights their favorable response to imatinib.

Clinical and Pathologic Correlation of Increased MYC Gene Copy Number in Diffuse Large B-Cell Lymphoma.

BACKGROUND: Only a few studies have investigated the presence of increased MYC gene copy number (ICN) as a prognostic indicator in patients with diffuse large B-cell lymphoma (DLBCL), and the results have been variable. We compared overall survival in patients with ICN to MYC-negative patients and investigated the prognostic significance of increased MYC gene copy number. PATIENTS AND METHODS: Two groups, those with MYC ICN (n = 33) and those with no MYC aberrations (n = 43), identified by fluorescence in-situ hybridization DNA probes for the MYC region at 8q24, were compared for survival (1-9 years), MYC immunohistochemical (IHC) protein expression, and treatment protocol. Comparison of cases of DLBCL with MYC ICN to those with no MYC aberration demonstrated no significant difference in survival (P = .58). Additionally, no difference in survival was found between patients with increased MYC protein expression (IHC MYC ≥ 40%) compared to those with IHC MYC < 40% (P = .5). RESULTS: Comparison of Ki-67 proliferation rates, stratified into low and high groups, did not achieve statistical significance (P = .67). Patients with MYC ICN showed a slightly increased MYC protein expression (P > .05). Importantly, the majority of patients in both groups (79% of patients with ICN and 81% of patients with no MYC aberrations) were treated with rituximab-based therapies. CONCLUSION: No significant difference in survival was found between patients with DLBCL with MYC ICN and patients with no MYC aberrations (P = .58).

RSV-Related Thrombocytopenia Associated with Transient Cytogenetic Abnormalities in a Recipient of Umbilical Cord Blood Transplantation.

Respiratory syncytial virus (RSV) infections are associated with thrombocytopenia. The underlying mechanism of thrombocytopenia in this setting is unknown. Herein, we report a case of RSV-related thrombocytopenia associated with transient cytogenetic abnormalities that occurred following umbilical cord blood transplantation.

EGFR and MYC gene copy number aberrations are more common in squamous cell carcinoma than keratoacanthoma: a FISH study.

Epidermal growth factor receptor (EGFR) and MYC genomic aberrations have been described in cutaneous squamous cell carcinoma (SCC) but have not been widely investigated in keratoacanthoma (KA). EGFR and MYC were evaluated by fluorescence in situ hybridization and immunohistochemistry in 8 verrucae, 19 involuting KA (IKA), 23 classic KA (CKA), 6 atypical KA (AKA) and 19 SCC. Increased EGFR gene copy number was seen in 9 of 23 CKA and 14 of 19 SCC (p = 0.03). Increased MYC gene copy number was observed in 7 of 23 CKA and 17 of 19 SCC (p = 0.0001). MYC gene amplification was more common in SCC than CKA (p = 0.005), while EGFR gene amplification was rare and not significant. MYC protein overexpression was identified in 6 of 23 CKA and 14 of 19 SCC (p = 0.005). There was no statistical difference in EGFR protein overexpression in SCC and CKA (p = 0.06). EGFR and MYC aberrations were rare in IKA. AKA showed EGFR and MYC anomalies at an incidence intermediate between CKA and SCC. EGFR and MYC gene copy number aberrations are more common in SCC than KA. The incidence of aberrations parallels the degree of cytologic atypia in KA.

Test verification and validation for molecular diagnostic assays.

With our ever-increasing understanding of the molecular basis of disease, clinical laboratories are implementing a variety of molecular diagnostic tests to aid in the diagnosis of hereditary disorders, detection and monitoring of cancer, determination of prognosis and guidance for cancer therapy, and detection and monitoring of infectious diseases. Before introducing any new test into the clinical laboratory, the performance characteristics of the assay must be "verified," if it is a US Food and Drug Administration (FDA)-approved or FDA-cleared test, or "validated," if it is a laboratory-developed test. Although guidelines exist for how validation and verification studies may be addressed for molecular assays, the specific details of the approach used by individual laboratories is rarely published. Many laboratories, especially those introducing new types of molecular assays, would welcome additional guidance, especially in the form of specific examples, on the process of preparing a new molecular assay for clinical use.

Validation of KRAS testing for anti-EGFR therapeutic decisions for patients with metastatic colorectal carcinoma.

CONTEXT: KRAS mutation status is a molecular marker for predicting patient response to treatment with anti-EGFR antibodies (cetuximab and panitumumab) in metastatic colorectal carcinoma. Different approaches may be taken to detect KRAS mutations. There currently are no US Food and Drug Administration-approved assays for the detection of KRAS mutations. For assays that are not approved by the US Food and Drug Administration, the performance characteristics of the assay must be determined and validated by the clinical laboratory before implementation. OBJECTIVE: To provide an example of how a KRAS mutation-analysis assay may be validated in a clinical laboratory. DESIGN: Describing the approach used by an individual laboratory to compare different assays for validation of KRAS mutation analysis in metastatic colon carcinoma. RESULTS: Specific validation data are provided, illustrating how a laboratory established assay performance characteristics for KRAS mutation analysis. CONCLUSIONS: All clinical laboratories must establish several performance specifications mandated by the Clinical Laboratory Improvement Amendments of 1988 before implementation of any laboratory-developed test. Approaches to the validation of such assays may vary among laboratories. We describe an approach used for validation of a KRAS mutation-analysis assay by one laboratory.

Design and analytic validation of BCR-ABL1 quantitative reverse transcription polymerase chain reaction assay for monitoring minimal residual disease.

CONTEXT: Monitoring minimal residual disease by quantitative reverse transcription polymerase chain reaction has proven clinically useful, but as yet there are no Food and Drug Administration-approved tests. Guidelines have been published that provide important information on validation of such tests; however, no practical examples have previously been published. OBJECTIVE: To provide an example of the design and validation of a quantitative reverse transcription polymerase chain reaction test. DESIGN: To describe the approach used by an individual laboratory for development and validation of a laboratory-developed quantitative reverse transcription polymerase chain reaction test for BCR-ABL1 fusion transcripts. RESULTS: Elements of design and analytic validation of a laboratory-developed quantitative molecular test are discussed using quantitative detection of BCR-ABL1 fusion transcripts as an example. CONCLUSIONS: Validation of laboratory-developed quantitative molecular tests requires careful planning and execution to adequately address all required analytic performance parameters. How these are addressed depends on the potential for technical errors and confidence required for a given test result. We demonstrate how one laboratory validated and clinically implemented a quantitative BCR-ABL1 assay that can be used for the management of patients with chronic myelogenous leukemia.

Validation of fluorescence in situ hybridization using an analyte-specific reagent for detection of abnormalities involving the mixed lineage leukemia gene.

CONTEXT: Fluorescence in situ hybridization (FISH) is a molecular cytogenetic assay that is commonly used in laboratory medicine. Most FISH assays are not approved by the US Food and Drug Administration but instead are laboratory-developed tests that use analyte-specific reagents. Although several guidelines exist for validation of FISH assays, few specific examples of FISH test validations are available in the literature. OBJECTIVE: To provide an example of how a FISH assay, using an analyte-specific reagent probe, may be validated in a clinical laboratory. DESIGN: We describe the approach used by an individual laboratory for validation of a FISH assay for mixed lineage leukemia (MLL) gene. RESULTS: Specific validation data are provided illustrating how initial assay performance characteristics in a FISH assay for MLL may be established. CONCLUSIONS: Protocols for initial validation of FISH assays may vary between laboratories. However, all laboratories must establish several defined performance specifications prior to implementation of FISH assays for clinical use. We describe an approach used for assessing performance specifications and validation of an analyte-specific reagent FISH assay using probes for MLL rearrangement in interphase nuclei.

College of American Pathologists/American College of Medical Genetics proficiency testing for constitutional cytogenomic microarray analysis.

PURPOSE: To evaluate the feasibility of administering a newly established proficiency test offered through the College of American Pathologists and the American College of Medical Genetics for genomic copy number assessment by microarray analysis, and to determine the reproducibility and concordance among laboratory results from this test. METHODS: Surveys were designed through the Cytogenetic Resource Committee of the two colleges to assess the ability of testing laboratories to process DNA samples provided and interpret results. Supplemental questions were asked with each Survey to determine laboratory practice trends. RESULTS: Twelve DNA specimens, representing 2 pilot and 10 Survey challenges, were distributed to as many as 74 different laboratories, yielding 493 individual responses. The mean consensus for matching result interpretations was 95.7%. Responses to supplemental questions indicate that the number of laboratories offering this testing is increasing, methods for analysis and evaluation are becoming standardized, and array platforms used are increasing in probe density. CONCLUSION: The College of American Pathologists/American College of Medical Genetics proficiency testing program for copy number assessment by cytogenomic microarray is a successful and efficient mechanism for assessing interlaboratory reproducibility. This will provide laboratories the opportunity to evaluate their performance and assure overall accuracy of patient results. The high level of concordance in laboratory responses across all testing platforms by multiple facilities highlights the robustness of this technology.

Histiocytic/dendritic cell sarcoma arising from follicular lymphoma involving the bone: a case report and review of literature.

Histiocytic/dendritic cell sarcomas arising from follicular lymphoma are very rare and poorly understood lesions. We describe a case, which is unique in that it presented with a hipbone lesion simultaneously with axillary lymphadenopathy. Biopsy of the axillary lymph node showed a low-grade follicular lymphoma. The hipbone lesion was comprised two cell populations, one representing diffuse large B cell lymphoma and the other a histiocytic/dendritic sarcoma. The cells of all three lesions contained an IGH/BCL2 rearrangement, suggesting that both diffuse large B cell lymphoma and histiocytic/dendritic sarcoma differentiation developed from the same low grade precursor (follicular lymphoma). This case illustrates that sarcomatous transdifferentiation of follicular lymphoma can be an unpredictable local phenomenon and that it can occur extra nodally in the bone marrow. It may also occur concurrently with the progression of follicular lymphoma to a diffuse large B cell lymphoma.

Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines.

CONTEXT: Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES: To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN: We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS: Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION: Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

Detection of copy number alterations in metastatic melanoma by a DNA fluorescence in situ hybridization probe panel and array comparative genomic hybridization: a southwest oncology group study (S9431).

PURPOSE: Gene copy number alteration (CNA) is common in malignant melanoma and is associated with tumor development and progression. The concordance between molecular cytogenetic techniques used to determine CNA has not been evaluated on a large set of loci in malignant melanoma. EXPERIMENTAL DESIGN: A panel of 16 locus-specific fluorescence in situ hybridization (FISH) probes located on eight chromosomes was used to identify CNA in touch preparations of frozen tissue samples from 19 patients with metastatic melanoma (SWOG-9431). A subset (n = 11) was analyzed using bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) of DNA isolated directly from touch-preparation slides. RESULTS: By FISH, most samples showed loss near or at WISP3/6p21, CCND3/6q22, and CDKN2A/9p21 (>75% of samples tested). More than one third of CDKN2A/9p21 losses were biallelic. Gains of NEDD9/6p24, MET/7q31, and MYC/8q24 were common (57%, 47%, and 41%, respectively) and CNA events involving 9p21/7p12.3 and MET were frequently coincident, suggesting gain of the whole chromosome 7. Changes were confirmed by aCGH, which also uncovered many discreet regions of change, larger than a single BAC. Overlapping segments observed in >45% of samples included many of the loci analyzed in the FISH study, in addition to other WNT pathway members, and genes associated with TP53 pathways and DNA damage response, repair, and stability. CONCLUSIONS: This study outlines a set of CNAs at the gene and regional level, using FISH and aCGH, which may provide a benchmark for future studies and may be important in selection of individual therapy for patients with metastatic malignant melanoma.

Aneusomy for detection of bladder cancer recurrence: a Southwest Oncology Group study.

The high risk of recurrence in superficial transitional cell cancer (TCC) of the bladder prompted evaluation of whether chromosome changes detected at first recurrence were correlated with relapse or with response to fluoroquinolone treatment. Fluorescence in situ hybridization analysis was applied to desquamated cells from bladder washings obtained immediately before surgical resection. Cells were screened for numeric changes in chromosomes 7 and 9. Aberrations were identified in 38/54 patients eligible for evaluation. Although no clear associations were established owing to sample size, the results suggested that risk of progression/relapse was positively associated with loss of chromosome 9 and with polysomy, and negatively associated with gain of chromosome 7, the latter in contrast to published data. A short-term survival advantage was noted anecdotally with gain of chromosome 9.

Biclonal post-transplant B-cell lymphoma: report of a case with two distinct cell populations, XX,t(14;18) and XY,t(11;14).

Lymphoproliferative disorders are more likely to occur in transplant patients compared to the general population. Typically in these patients, lymphomas occur within 6-10 months following transplant and are Epstein-Barr virus (EBV) positive. We report a biclonal apparently EBV negative lymphoma occurring in a patient ten years after renal transplant, with karyotypes XX,t(14;18) and XY,t(11;14). Though the biclonal populations also had different sex chromosome compositions, complete evaluation showed that both clones most likely evolved from the patient's native lymphocytes.

Infant acute lymphoblastic leukemia with t(11;16)(q23;p13.3) and lineage switch into acute monoblastic leukemia.

Rearrangements of the mixed-lineage leukemia (MLL) gene have been associated with a poor prognosis in infant acute lymphoblastic leukemia (ALL). Previously, MLL translocations involving the CREP-binding protein (CREBBP) gene at chromosome band 16p13.3 have primarily been reported in treatment-related acute myeloid leukemia, after chemotherapy for other primary malignancies using topoisomerase II inhibitors. We report a case of de novo infant ALL with t(11;16)(q23;p13.3). After chemotherapy, this patient developed an acute monoblastic leukemia (M5b) with retention of the t(11;16)(q23;p13.3), indicating that this is a lineage switch of the original leukemic clone. To our knowledge, these findings have not been previously reported.

Cytogenetic heteromorphisms: survey results and reporting practices of giemsa-band regions that we have pondered for years.

CONTEXT: Cytogenetic heteromorphisms (normal variants) pose diagnostic dilemmas. Common Giemsa-band heteromorphisms are not described in the literature, although Giemsa-banding is the method most frequently used in cytogenetic laboratories. OBJECTIVE: To summarize the responses from more than 200 cytogeneticists concerning the definition and reporting of cytogenetic heteromorphisms, to offer these responses as a reference for use in clinical interpretations, and to provide guidance for interpretation of newly defined molecular cytogenetic heteromorphisms. DESIGN: The Cytogenetics Resource Committee of the College of American Pathologists and the American College of Medical Genetics administered a proficiency testing survey in 1997 to 226 participant cytogenetic laboratories. Supplemental questions asked whether participants considered particular Giemsa-banded chromosomal features to be heteromorphisms and if these would be described in a cytogenetic clinical report. RESULTS: Responses were obtained from 99% of participants; 61% stated they would include selected heteromorphism data in a clinical report. More than 90% considered prominent short arms, large or double satellites, or increased stalk length on acrocentric chromosomes to be heteromorphisms; 24% to 36% stated that they would include these in a clinical report. Heterochromatic regions on chromosomes 1, 9, 16, and Y were considered heteromorphisms by 97% of participants, and 24% indicated they would report these findings. Pericentric inversions of chromosomes 1, 2, 3, 5, 9, 10, 16, and Y were considered heteromorphisms with more than 75% of respondents indicating they would report these findings. CONCLUSIONS: Responses were not unanimous, but a clear consensus is presented describing which Giemsa-band regions were considered heteromorphisms and which would be reported.

Use of a multitarget fluorescence in situ hybridization assay to diagnose bladder cancer in patients with hematuria.

PURPOSE: We evaluated the multitarget UroVysion fluorescence in situ hybridization assay for the diagnosis of bladder cancer in patients with hematuria and no history of bladder cancer. MATERIALS AND METHODS: A multicenter, blinded trial was performed to compare the sensitivity of the fluorescence in situ hybridization assay to that of voided cytology in patients with gross or microscopic hematuria. Confirmation of hematuria was required. Voided urine was sent to a central laboratory for each study before cystoscopy. Suspicious lesions on cystoscopy were biopsied or resected. A centrally reviewed histopathological interpretation was used to confirm cancer and assign grade and stage. RESULTS: A total of 497 patients were enrolled at 23 centers and in 473 (95.2%) fluorescence in situ hybridization and cytology results were interpretable. Bladder cancer was diagnosed histologically in 50 patients (10.1%) and ureteral cancer was diagnosed in 1. Fluorescence in situ hybridization assay detected 69% of cases and cytology detected 38% (95% CI 25 to 52). When low grade, low stage (TaG1) tumors were excluded, fluorescence in situ hybridization detected 25 of 30 cancers (84%), while cytology detected only 15 (50%). Of 265 current or past smokers with hematuria and positive fluorescence in situ hybridization assay findings bladder cancer was detected in 65% with a history of greater than 40 pack-years compared to 13.6% to 24.2% in those with no, less than a 20 or a 20 to 40-pack-year smoking history. CONCLUSIONS: The UroVysion fluorescence in situ hybridization assay is significantly more sensitive than voided cytology for detecting bladder cancer in patients evaluated for gross or microscopic hematuria for all grades and stages. Based on these data UroVysion was approved by the Food and Drug Administration for use in patients with hematuria.