Implementation of the 2018 American Society of Clinical Oncology/College of American Pathologists Guidelines on HER2/neu Assessment by FISH in breast cancers: predicted impact in a single institutional cohort.

The 2018 American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) update modified the interpretation guidelines for human epidermal growth factor receptor 2 (HER2) testing by incorporating immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results in a subset of cases. Importantly, the new guidelines eliminate "equivocal" results, as well as the use of alternative chromosome 17 probes as the primary strategy for resolving the indeterminate FISH results. Herein, we investigate the predicted impact of implementing the 2018 ASCO/CAP guidelines on HER2 assessment by FISH in breast cancers, using data from a single institution. We compared the HER2 status of 1542 consecutive cases of breast carcinoma, interpreted by 2013 and 2018 ASCO/CAP guidelines. In total, 10.7% (165/1542) of the cases had a different final interpretation by 2018 guidelines compared with 2013 guidelines, including 70 previously HER2-positive cases reclassified as negative, four previously negative cases reclassified as positive, and 91 previously equivocal cases reclassified as negative. Overall, the number of HER2-positive cancers was reduced by 66 cases (4.3% reduction in the HER2 positivity rate). The newly HER2-negative cases were mostly estrogen receptor positive (90%), progesterone receptor positive (80%), stage 1 (60.9%), and grade 1-2 (59.4%) cancers; 70% of them had been designated as HER2 positive only after the use of an alternative chromosome 17 FISH probe after an intially equivocal result from the standard CEP17 probe. Overall, implementing the revised 2018 HER2 guidelines is predicted to change the HER2 results of 10.7% of breast cancers, mainly by reclassifying previously equivocal to negative results.

Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

The Dohner fluorescence in situ hybridization prognostic classification of chronic lymphocytic leukaemia (CLL): the CLL Research Consortium experience.

This study revisited the Dohner prognostic hierarchy in a cohort of 1585 well-documented patients with chronic lymphocytic leukaemia. The duration of both time to first treatment (TTFT) and overall survival (OS) were significantly longer than observed previously, and this is at least partly due to improved therapeutic options. Deletion 13q remains the most favourable prognostic group with median TTFT and OS from fluorescence in situ hybridization (FISH) testing of 72 months and >12 years, respectively. Deletion 11q had the poorest median TTFT (22 months) and 17p deletion the poorest median OS (5 years). The percentages of abnormal nuclei were significantly associated with differential TTFT for the trisomy 12, 13q and 17p deletion cohorts but not for the 11q deletion cohort. From the date of the first FISH study, patients with >85% 13q deletion nuclei had a notably shorter TTFT (24 months). Patients with ≤20% 17p deletion nuclei had longer median TTFT and OS from the date of the first FISH study (44 months and 11 years), and were more likely to be IGHV mutated.

Translocation t(2;11) in CLL cells results in CXCR4/MAML2 fusion oncogene.

Recent investigations of chromosomal aberrations in chronic lymphocytic leukemia (CLL) led to a better understanding of the molecular causes of CLL. Here we report a rearrangement between MAML2 (mastermind-like protein 2) and CXCR4 (specific receptor for CXC chemokine stromal cell-derived factor-1) in CLL cells of a patient with a t(2;11)(q22.1;q21) chromosomal translocation. The rearrangement between MAML2 and CXCR4, created by a t(2;11)(q22.1;q21) translocation, results in a new fusion gene in which a portion of CXCR4 is linked to the MAML2 gene. This fusion gene encodes for CXCR4/MAML2 protein chimera in which the N-terminal basic domain of MAML2 is replaced by the N-terminal domain of CXCR4.

Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of B-cell chronic lymphocytic leukemia.

CONTEXT: Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. OBJECTIVE: To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. DESIGN, SETTING, AND PATIENTS: CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. MAIN OUTCOME MEASURES: Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. RESULTS: In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). CONCLUSIONS: A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.

Breast cancers with a HER2/CEP17 ratio of 2.0 or greater and an average HER2 copy number of less than 4.0 per cell: frequency, immunohistochemical correlation, and clinicopathological features.

The 2013 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines classified breast cancers with a fluorescence in situ hybridization dual-probe HER2/CEP17 ratio of 2 or greater as "amplified," inclusive of cases with a HER2 copy number less than 4. The 2018 ASCO/CAP update assigns HER2/neu status for the latter group in a fashion that is highly dependent on the associated immunohistochemical findings. Herein, the authors define the frequency, immunohistochemical correlates, and other clinicopathological features of breast cancers with HER2/CEP17 ratio of 2 or greater and HER2/neu copy number less than 4 (group A), based on an analysis of an institutional cohort assessed for HER2/neu status by both florescence in situ hybridization and immunohistochemistry and scored using 2013 ASCO/CAP criteria. Group A cases were compared with a group B of HER2/neu-amplified breast cancers with a HER2/neu copy number of 4 or greater regarding a variety of clinicopathological features. One hundred sixty-nine (14%) of 1201 cases were HER2/neu amplified, 18 (10.7%) in group A and 151 (89.3%) in group B. By immunohistochemistry, 61.1% of group A cases were HER2/neu negative, 7 (38.9%) were equivocal, and none were positive. In contrast, 66.9% of group B cases were HER2 positive (3+). We could not demonstrate statistically significant differences between the 2 groups regarding standard clinicopathological variables. In summary, our group A cases account for 1.5% of breast cancers, and 10.7% of all HER2/neu-amplified cancers classified as such based on 2013 ASCO/CAP criteria. They are predominantly HER2/neu negative by immunohistochemistry, which suggests that they are biologically different from classically HER2/neu-amplified cases and which validates the 2018 ASCO/CAP guideline against automatically classifying such cases as HER2/neu amplified.

Comparative Pathologic Analysis of Breast Cancers Classified as HER2/neu-Amplified by FISH Using a Standard HER2/CEP17 Dual Probe and an Alternative Chromosome 17 Control Probe.

At our institution, breast cancer cases that generate an equivocal HER2/neu (HER2) result by fluorescence in situ hybridization (FISH) using the dual HER2/chromosome enumeration probe (CEP17) are reflexed to an assay that utilizes an alternative control probe (lissencephaly gene1 [LIS1] [17p13.3]/retinoic acid receptor α [RARA] [17q21.2]). This study examines whether cancers that are classified as HER2-amplified with an alternate probe are clinicopathologically similar to those that are classified as such using the HER2/CEP17 probe. Reports for 1201 breast cancers were reviewed, and clinicopathologic findings were compared between HER2/CEP17-equivocal cases that became HER2-amplified using the alternate probe (group A: n=48), HER2-amplified cases using the HER2/CEP17 probe (group B: n=169), and HER2-nonamplified cases using the HER2/CEP17 probe (group C: n=910). Of 1201 cases tested using the HER2/CEP17 probe, 169 (14%) were HER2-amplified, 122 (10%) were equivocal, and 910 (76%) were nonamplified. Additional testing with the alternative probe on the 122 equivocal cases reclassified 48 (39%) of them to HER2-amplified, and such cases comprised 22% of all HER2-amplified tumors. A higher proportion of tumors with HER2 copy number between 5.0 and 5.9 became positive upon additional testing when compared with those with a priori HER2 copy numbers between 4.0 and 4.9 (P=0.0362). Group A cases, compared with group B cases, were more frequently positive for estrogen receptor (97.91% vs. 72.18%, P<0.0001) and progesterone receptor (85.41% vs. 59.17%, P=0.0009). Most group A cases (71%) were HER2 equivocal (score 2+) by immunohistochemistry, whereas most group B cases (60%) were positive (score 3+). Groups A and B showed no significant differences regarding patient age, lymph node status, tumor grade, histotype, and stage distribution. In summary, among our HER2-amplified cohort of breast cancers, alternative probe-detected cases were more frequently estrogen receptor and progesterone receptor positive than HER2/CEP17-detected cases, and were more frequently discordant with HER2 immunohistochemistry results. These findings raise the possibility of underlying biologic differences between these 2 groups, which warrants further study. However, the tumors were largely comparable regarding all other clinicopathologic variables. As it is unknown whether HER2-targeted therapy is truly beneficial in this subgroup of patients, future clinical trials should specifically evaluate this subset.

Her2/neu Status Determination in Breast Cancer: A Single Institutional Experience Using a Dual-Testing Approach With Immunohistochemistry and Fluorescence In Situ Hybridization.

OBJECTIVES: According to current guidelines, either immunohistochemistry (IHC) or in situ hybridization (ISH) can be used to determine human epidermal growth factor receptor 2 (Her2/neu) status in breast carcinoma. While the guidelines explicitly delineate result interpretation, there is no consensus on the most appropriate testing algorithm. METHODS: The Her2/neu statuses of 369 consecutive cases of invasive breast cancer (from 351 patients) were assessed in a dual-testing algorithm that uses both IHC and fluorescence ISH (FISH). FISH was performed using dual-color HER2/ chromosome enumeration probe 17 ( CEP17 ) probes, and if equivocal results were obtained, reflex testing using HER2/lissencephaly gene 1 ( LIS1 ) probes was used. Results from both modalities were scored and reported using American Society of Clinical Oncology/College of American Pathologists 2013 criteria. RESULTS: Sixty-one (16.5%) of the 369 tumors were found to be Her2/neu positive by at least one modality. The overall concordance between IHC and FISH results was 97.6%. Six of the 369 tumors were reclassified as Her2/neu positive after a negative IHC result. FISH was also able to identify significantly more Her2/neu-positive cases than IHC. CONCLUSIONS: The commonly used reflex strategy based on IHC results may deny potentially beneficial targeted therapy for a small cohort of patients, which should be considered as testing guidelines are formulated and the cost-benefit analyses of various testing algorithms are assessed.

Successful treatment of both double minute of C-MYC and BCL-2 rearrangement containing large B-cell lymphoma with subsequent unfortunate development of therapy-related acute myeloid leukemia with t(3;3)(q26.2;q21).

Double minute chromosomes (DMs), although relatively frequently encountered in solid tumors, are rare in hematologic neoplasms such as acute myeloid leukemia (AML), and even rarer in lymphoid neoplasms. t(3;3)(q26.2;q21) is a very rare genetic alteration observed in myeloid neoplasm. Herein we report an interesting and unique case of concomitant C-MYC DMs and t(14;18)-containing large B-cell lymphoma, which was successfully treated with R-hyper-CVAD; unfortunately, the patient has developed a therapy-related AML (t-AML) 2 years since the start of his lymphoma treatment. His t-AML contains both t(3;3)(q26.2;q21) and monosomy 7, and the patient died of AML 10 months after the initial diagnosis of t-AML despite clinical remission. To the best of our knowledge, this is the first reported case of C-MYC DM-containing de novo large B-cell lymphoma, which was successfully treated with complete remission, but unfortunately died of t-AML harboring t(3;3)(q21;q26).

Nodal involvement by marginal zone B-cell lymphoma harboring t(14;22)(q32;q11) involving immunoglobulin heavy chain and light chain lambda as the sole karyotypically recognizable abnormality in a patient with systemic lupus erythematosus.

Recurrent non-random balanced chromosomal translocation, usually involving the immunoglobulin heavy chain (IgH) gene or an immunoglobulin light chain gene and a proto-oncogene, which results in the overexpression of the latter under the control of an enhancer or promoter of the former, is a hallmark of many types of non-Hodgkin lymphoma (NHL) of B-cell origin. However, translocations between IgH and the immunoglobulin (Ig) light chain lambda gene (IgL), namely, a t(14;22)(q32;q11), have rarely been described in B-cell NHL. Herein we report the first case of marginal zone B-cell lymphoma harboring a t(14;22)(q32;q11) as its sole genetic abnormality in a patient with a 12-year history of systemic lupus erythematosus (SLE). Other interesting findings of this case include: 1) the neoplastic B-cells lack expression of both surface and cytoplasmic Ig light chain as revealed by flow cytometry and 2) monoclonal rearrangement of Ig light chain kappa (IgK) only due to k-deleting element (kde) recombination event. This case illustrates the necessity of utilizing a multi-modality approach in the diagnosis of B-cell NHL.

Myeloid neoplasm with t(3;8)(q26;q24): report of six cases and review of the literature.

Balanced translocation between chromosomes 3q26 and 8q24 is a very rare event. Here we report six patients with t(3;8)(q26;q24) either as a sole or as a part of genetic abnormalities. Five of the six patients were men with ages ranging from 41 to 84 years old. One patient had a long history of granulocyte colony stimulating factor (G-CSF) treatment. Three of the patients were initially diagnosed with acute myeloid leukemia, two with myelodysplastic syndrome and one with chronic myelogenous leukemia with blast crisis. The peripheral blood in all patients showed severe to moderate anemia; one had absolute neutropenia, one with neutrophilia; four had thrombocytopenia, two with thrombocytosis. The bone marrows from all patients showed dysmegakaryopoiesis with additional erythroid (three patients) and granulocytic (two patients) dysplasia. Cytogenetics revealed t(3;8)(q26;q24) as the sole abnormality in three patients. The majority of patients (4/6) had a poor clinical course, with an average survival of 10 months.

t(4;22)(q12;q11.2) involving presumptive platelet-derived growth factor receptor A and break cluster region in a patient with mixed phenotype acute leukemia.

The patient is a 45-year-old woman with a history of breast cancer who had been treated 1 year ago with radiation and chemotherapy. Flow cytometric analysis of bone marrow aspirate revealed 81% blasts positive for CD4, CD11c (partial), CD13, CD19 (partial), cytoplasmic CD22, CD34, CD36, CD45, cytoplasmic CD79a, CD117 (partial), HLA-DR, and terminal deoxynucleotide transferase, consistent with a mixed phenotype acute leukemia (B/myeloid lineage). Conventional karyotypic analysis revealed a t(4;22)(q12;q11.2) in 12 of 13 cells analyzed. Fluorescence in situ hybridization analysis using a dual-color, dual-fusion break cluster region/ABL probe set showed no break cluster region/ABL translocation but an extra break cluster region signal in 85% (170/200) of cells, consistent with a translocation involving the break cluster region gene at 22q11.2. A FIP1L1/CHIC2/platelet-derived growth factor receptor α deletion/fusion probe showed signal separation in 96.5% (193/200) of interphase nuclei. Reverse transcriptase-polymerase chain reaction using sense break cluster region primers and an antisense platelet-derived growth factor receptor α primer resulted in a product of approximately 590 base pairs, consistent with the presence of a break cluster region/platelet-derived growth factor receptor α fusion gene. Because of the presumptive platelet-derived growth factor receptor α translocation and its sensitivity to tyrosine-kinase inhibitor, the patient was treated with imatinib mesylate, cytarabine, and idarubicin as induction and maintenance therapy; and she has remained free of disease for 5 months since the initial diagnosis.

CD20dim-positive T-cell large granular lymphocytic leukemia in a patient with concurrent hairy cell leukemia and plasma cell myeloma.

We report a CD20dim- positive T-cell large granular lymphocytic (T-LGL) leukemia in a patient with concurrent hairy cell leukemia and plasma cell myeloma. This patient was first diagnosed with T-LGL leukemia with dim CD20 expression, which by itself was a rare entity. He received no treatment for T-LGL leukemia. The patient later developed a hairy cell leukemia, which went into complete clinical remission after one cycle of 2-CdA. Five years later, he was diagnosed with a third malignancy, plasma cell myeloma. Complex cytogenetic aberrancies were present at the time when plasma cell myeloma was diagnosed. This is the first report, to the best of our knowledge, in the English literature with the aforementioned three distinct hematopoietic malignancies in one patient.

Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) Study.

Cytogenetic abnormalities are important prognostic indicators in CLL. Historically, only interphase cytogenetics was clinically useful in CLL, because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL cells. The CLL Research Consortium tested the effectiveness and reproducibility of CpG-ODN stimulation for detecting chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with the traditional pokeweed mitogen plus 12-O-tetradecanoylphorbol-13-acetate (PWM+TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control sample and 12 CLL samples showed that, excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Abnormal clones in CLL were more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. With CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture, but did enhance detection of abnormalities and complexity in CLL. Because karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful for identifying this additional prognostic factor in CLL.

Standardization of fluorescence in situ hybridization studies on chronic lymphocytic leukemia (CLL) blood and marrow cells by the CLL Research Consortium.

Five laboratories in the Chronic Lymphocytic Leukemia (CLL) Research Consortium (CRC) investigated standardizing and pooling of fluorescence in situ hybridization (FISH) results as a collaborative research project. This investigation used fixed bone marrow and blood cells available from previous conventional cytogenetic or FISH studies in two pilot studies, a one-day workshop, and proficiency test. Multiple FISH probe strategies were used to detect 6q-, 11q-, +12, 13q-, 17p-, and IGH rearrangements. Ten specimens were studied by participants who used their own probes (pilot study 1). Of 312 FISH interpretations, 224 (72%) were true-negative, 74 (24%) true-positive, 6 (2%) false-negative, and 8 (3%) false-positive. In pilot study no. 2, each participant studied two specimens using identical FISH probe sets to control for variation due to probe sets and probe strategies. Of 80 FISH interpretations, no false interpretations were identified. At a subsequent workshop, discussions produced agreement on scoring criteria. The proficiency test that followed produced no false-negative results and 4% (3/68) false-positive interpretations. Interpretation disagreements among laboratories were primarily attributable to inadequate normal cutoffs, inconsistent scoring criteria, and the use of different FISH probe strategies. Collaborative organizations that use pooled FISH results may wish to impose more conservative empiric normal cutoff values or use an equivocal range between the normal cutoff and the abnormal reference range to eliminate false-positive interpretations. False-negative results will still occur, and would be expected in low-percentage positive cases; these would likely have less clinical significance than false positive results. Individual laboratories can help by closely following rigorous quality assurance guidelines to ensure accurate and consistent FISH studies in their clinical practice and research.

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias.

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673-676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253-258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia.