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.

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).

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.

Myelodysplastic Syndrome with concomitant t(5;21)(q15;q22) and del(5)(q13q33): case report and review of literature.

Chromosomal abnormalities lead to the development of hematologic malignancies such as Myelodysplastic Syndrome (MDS). Known chromosomal changes causing MDS include deletion of the long arm of chromosome 5, runt-related transcription factor 1 (RUNX1) also known as acute myeloid leukemia 1 protein (AML1), and very rarely fusion genes involving RUNX1 at t(5;21)(q15;q22). We present a case of a 71-year-old female with MDS, refractory anemia with excess blasts, type 1, with a combination of two cytogenetic abnormalities, specifically a concomitant translocation between chromosomes 5q15 and 21q22 and deletion of chromosome 5q13q33. Fluorescence in-situ hybridization (FISH) using a probe for RUNX1 (AML1), localized to 21q22, showed three FISH signals for RUNX1, consistent with rearrangement of RUNX1. Therapy was started with Lenalidomide leading to normal blood counts. Most significantly, repeat cytogenetics revealed normal karyotype and resolution of deletion on the long arm of chromosome 5 and a t(5;21). FISH negative for deletion 5q. The results altogether meet criteria for a complete cytogenetic remission (CR). We report a new case of t(5;21)(q15;q22) involving the RUNX1 gene and del(5)(q13q33) in a MDS patient, a combination of chromosomal abnormalities heretofore not reported in the literature. RUNX1 rearrangement is usually associated with an adverse prognosis in AML and MDS. Deletions of 5q are typically associated with poor prognosis in AML, however it is usually associated with a favorable prognosis in MDS. Our patient responded very well to Lenalidomide therapy with achievement of CR. Lenalidomide is approved for treatment of anemia in low and intermediate risk MDS with del (5q), however based on a search of literature it seems that RUNX1 mutations are also more prominent in patients who have responded to Lenalidomide therapy. MDS is a genomically unstable disease. Hence, it is conceivable that our patient started with a 5q minus syndrome and then acquired the second hit RUNX1 translocation leading to an accelerated phase of myeloid neoplasm or refractory anemia with excess blasts, type 1. Hence, the temporal relationship between acquisition of del 5q and RUNX1 rearrangement may have influenced the clinical outcome and possibly response to therapy.

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.

High-Risk Microgranular Acute Promyelocytic Leukemia with a Five-Way Complex Translocation Involving PML-RARA.

Acute promyelocytic leukemia (APL) is classically characterized by chromosomal translocation (15;17), resulting in the PML-RARA fusion protein leading to disease. Here, we present a case of a 50-year-old man who presented with signs and symptoms of acute leukemia with concern for APL. Therapy was immediately initiated with all-trans retinoic acid. The morphology of his leukemic blasts was consistent with the hypogranular variant of APL. Subsequent FISH and cytogenetic analysis revealed a unique translocation involving five chromosomal regions: 9q34, 17q21, 15q24, 12q13, and 15q26.1. Molecular testing demonstrated PML/RARA fusion transcripts. Treatment with conventional chemotherapy was added and he went into a complete remission. Given his elevated white blood cell count at presentation, intrathecal chemotherapy for central nervous system prophylaxis was also given. The patient remains on maintenance therapy and remains in remission. This is the first such report of a 5-way chromosomal translocation leading to APL. Similar to APL with chromosomal translocations other than classical t(15;17) which result in the typical PML-RARA fusion, our patient responded promptly to an ATRA-containing regimen and remains in complete remission.

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.

Use of a structured template to facilitate practice-based learning and improvement projects.

BACKGROUND: The Accreditation Council for Graduate Medical Education (ACGME) requires residency programs to meet and demonstrate outcomes across 6 competencies. Measuring residents' competency in practice-based learning and improvement (PBLI) is particularly challenging. PURPOSE: We developed an educational tool to meet ACGME requirements for PBLI. The PBLI template helped programs document quality improvement (QI) projects and supported increased scholarly activity surrounding PBLI learning. METHODS: We reviewed program requirements for 43 residency and fellowship programs and identified specific PBLI requirements for QI activities. We also examined ACGME Program Information Form responses on PBLI core competency questions surrounding QI projects for program sites visited in 2008-2009. Data were integrated by a multidisciplinary committee to develop a peer-protected PBLI template guiding programs through process, documentation, and evaluation of QI projects. All steps were reviewed and approved through our GME Committee structure. RESULTS: An electronic template, companion checklist, and evaluation form were developed using identified project characteristics to guide programs through the PBLI process and facilitate documentation and evaluation of the process. During a 24 month period, 27 programs have completed PBLI projects, and 15 have reviewed the template with their education committees, but have not initiated projects using the template. DISCUSSION: The development of the tool generated program leaders' support because the tool enhanced the ability to meet program-specific objectives. The peer-protected status of this document for confidentiality and from discovery has been beneficial for program usage. The document aggregates data on PBLI and QI initiatives, offers opportunities to increase scholarship in QI, and meets the ACGME goal of linking measures to outcomes important to meeting accreditation requirements at the program and institutional level.

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.

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.

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.

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.