Impact of NOTCH1 expression in primary breast adenoid cystic carcinoma.

AIMS: Adenoid cystic carcinoma (AdCC) originates from salivary-type like glands in the head and neck, lung, and breast. AdCC shows chromosomal translocation, resulting in MYB::NFIB fusion and overexpression of MYB. Recently, NOTCH1 pathway alteration has been recognised in a subset of patients with salivary gland AdCC and has been shown to be associated with poor survival. In this study, we investigated the correlation of NOTCH1 pathway alteration with the clinical outcome of patients with primary breast AdCC by examining NOTCH1 immunoreactivity in attempts to better predict clinical outcomes. METHODS: We identified 25 cases of breast AdCC, reviewed the clinical outcome and performed immunohistochemical (IHC) staining for NOTCH1 on FFPE sections. RESULTS: IHC evaluation of NOTCH1 expression in 25 cases of primary breast AdCCs revealed a positive correlation between NOTCH1 expression and primary tumour size. All cases with NOTCH1 expression were greater than 15 mm in size at presentation but only 50% of NOTCH1 negative tumours were greater than 15 mm. We demonstrated a positive correlation between NOTCH1 positive AdCCs and recurrence/metastases. 63.6% of NOTCH1 positive AdCCs had either metastases or recurrence. On the contrary, only 21.5% of NOTCH1 negative AdCCs had recurrence or metastases. AdCCs with NOTCH1 positivity correlated with inferior relapse free survival (median 33 vs 129 months). CONCLUSIONS: Our study demonstrates that in patients with breast AdCC, overexpression of NOTCH1 ≥20% is associated with larger tumour size and aggressive clinical outcomes. Importantly, NOTCH1 inhibitors may have potential therapeutic effect in patients with breast AdCC.

A Comparative Evaluation of TRPS1 and GATA3 in adenoid cystic, secretory, and acinic cell carcinomas of the breast and salivary gland.

GATA3 is the most used marker to determine tumors' breast origin, but its diagnostic value in triple-negative breast cancer (TNBC) is limited. The newly identified TRPS1 is highly sensitive and specific for breast carcinoma, especially TNBC. Here, we compared the utility of TRPS1 and GATA3 expression in a subset of salivary gland-type breast tumors (including adenoid cystic, acinic cell, and secretory carcinomas [AdCC, ACC, and SC, respectively]), and we compared TRPS1 and GATA3 expression of such tumors with head and neck (H&N) and AdCC of upper respiratory tumors. TRPS1 was strongly expressed in basaloid TNBC and AdCCs with solid components, including 100 % of mixed and solid breast AdCCs. However, TRPS1 was positive in only 50 % cribriform AdCCs. Expression patterns of TRPS1 in H&N and upper respiratory AdCC were similar. TRPS1 was positive in 30 % of H&N cribriform AdCCs but was strongly expressed in mixed AdCC (67 %) and solid AdCC (100 %). In the upper respiratory AdCCs, TRPS1 was positive in 58.4 % of cribriform AdCCs and positive in 100 % of AdCCs with solid components. On the contrary, GATA3 was negative in predominant AdCCs of the breast, H&N, and upper respiratory tract. These data show that GATA3 and TRPS1 expression varies AdCCs. In addition, TRPS1 and GATA3 expression patterns were similar SC and ACC of breast and H&N. Both markers were positive in SC and negative in ACC. Therefore, TRPS1 and GATA3 cannot be used to differentiate salivary gland-type carcinomas of breast origin from those of upper respiratory or H&N origin.

Are polyploid giant cancer cells in high grade serous carcinoma of the ovary blastomere-like cancer stem cells?

Polyploid giant cancer cells, either multinucleated or mononucleated, in high grade serous carcinoma of the ovary have been previously recognized. Different theories including degenerative changes or an important step in the development of high grade serous carcinoma have been proposed. Here we investigate possible explanations for the presence of polyploid giant cancer cells in high grade serous carcinoma. We reviewed 33 cases of ovarian high grade serous carcinoma (12 stage I, 7 stage II, and 14 stage III). We counted the number of polyploid giant cancer cells in 20 consecutive 10× fields. In 11 cases where polyploid giant cancer cells were easily found, immunohistochemistry for Ki67, p53, and OCT 3/4 was performed. Patients with polyploid giant cancer cells were older than those without. Polyploid giant cancer cells were more frequent in stage I lesions (75%) than in stages II or III (57% in both) and less frequent in metastases compared with primary ovarian tumors. Mitotic figures were present in regular sized cells but were absent in polyploid giant cancer cells. OCT3/4 was negative in all cases assessed. In 8 cases, more than 70% of the mononuclear cells were positive for Ki-67, similar to the percentage of Ki-67 positive cells in polyploid giant cancer cells. p53 had a perfect correlation in regular sized cancer cells and in polyploid giant cancer cells. Polyploid giant cancer cells are neither degenerative cells nor traditional cancer stem cells but most probably represent an intermediate step between stem cells and mature tumor cells formed by endoreplication.

P53 protein overexpression in de novo acute myeloid leukemia patients with normal diploid karyotype correlates with FLT3 internal tandem duplication and worse relapse-free survival.

Although ~50% of acute myeloid leukemia (AML) patients have a normal diploid karyotype by conventional cytogenetics at diagnosis, this patient subset has a variable disease course and outcome. Aberrant overexpression of the p53 protein is usually associated with TP53 alterations and a complex karyotype, but the prevalence and impact of p53 overexpression in AML with diploid cytogenetics is unknown. We examined 100 newly diagnosed AML patients to evaluate the impact of p53 expression status quantified in bone marrow core biopsy samples using immunohistochemistry and computer-assisted image analysis. A total of 24 patients had p53 overexpression defined as 3+ staining intensity in ≥5% of cells; this finding correlated with lower platelet counts (P = .002), absence of CD34 expression in blasts (P = .009), higher bone marrow blast counts (P = .04), and a higher frequency of FLT3 internal tandem duplication (P = .007). Overexpression of p53 independently predicted for shorter leukemia-free survival in patients who underwent allogeneic stem cell transplantation by univariate (P = .021) and multivariate analyses (P = .004). There was no correlation between MDM2 and p53 protein expression in this cohort. We conclude that p53 expression evaluated by immunohistochemistry in bone marrow biopsy specimens at the time of AML diagnosis may indicate distinct clinical characteristics in patients with normal diploid cytogenetics and is a potentially valuable tool that can enhance risk-stratification.

Chronic lymphocytic leukemia with proliferation centers in bone marrow is associated with younger age at initial presentation, complex karyotype, and TP53 disruption.

The presence of expanded proliferation centers (PCs) in lymph nodes involved by chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma has been associated with adverse clinical outcomes, but the frequency and significance of PCs in bone marrow (BM) remain unclear. The study group included 36 patients with BM involvement by CLL in which PCs were present. We compared this group with 110 randomly selected BM samples involved by CLL without morphologically discernable PCs. Patients with PCs in BM were younger (median age, 53 years [range,18-71 years] versus 58 years [range, 31-82 years]; P = .007), more frequently experienced B symptoms (27.8% versus 8.2%, P = .0076), more often had Rai stage IV disease (30.6% versus 17.3%, P = .02) and higher serum lactate dehydrogenase (P = .0037) and ß2-microglobulin (P = .0001) levels, and lower hemoglobin (P = .026) and platelet counts (P = .0422). TP53 alterations were more common in patients with PCs in BM (45.4% versus 18.7%; P = .0049), as was a complex karyotype (26.4% versus 9%; P = .019). There were no significant differences in the frequency of ZAP70 or CD38 positivity or IGHV mutation status. The median time to first treatment was shorter in patients with PCs in BM (7 months versus 19 months, P = .047), and the frequency of Richter syndrome was higher (14% versus 4%, P = .041). Patients with PCs in BM had significantly shorter overall survival compared with the control group (median, 249.3 months versus undefined; P = .0241). These data suggest that identification of PCs in BM samples involved by CLL is associated with adverse prognostic features.

Characteristics and clinical implications of reactive germinal centers in the bone marrow.

Reactive germinal centers (GCs) in the bone marrow (BM) have been described in patients with autoimmune disorders, infections, malignancies, and following certain drug therapies, or as an isolated finding without obvious underlying disease. In this study, we describe the clinical conditions in which reactive GCs occur in BM samples, and their topography and accompanying laboratory and ancillary findings in the setting of a cancer center. We identified 32 BM specimens with reactive GCs with an estimated frequency less than 0.02% over a 12-year period. Fifteen (46.9%) BM specimens had concurrent hematolymphoid neoplasms: most often a variety of small B-cell lymphomas, but also myelodysplastic syndromes. One (3.1%) case was involved by metastatic melanoma. Isolated reactive GCs were observed in 16 (50%) patients. Most BM specimens (n = 25; 78.1%) showed only one reactive GC with a size ranging from 20 to 500 µm, and most GCs (29/32) were nonparatrabecular. GCs were positive for CD10 and BCL6, and were negative for BCL2. CD3 and CD5 demonstrated T cells surrounding the GC and CD21, and CD23 highlighted follicular dendritic cells. Reactive GCs are uncommon and can be seen in association with hematolymphoid and other types of neoplasms or as an isolated finding. Reactive GCs are usually located in a nonparatrabecular distribution. A panel of immunohistochemical stains is useful to confirm the nonneoplastic nature of these GCs to avoid misdiagnosis as lymphoma or as histologic evidence of transformation in a patient with small B-cell lymphoma in the bone marrow.

Myeloid neoplasms with concurrent BCR-ABL1 and CBFB rearrangements: A series of 10 cases of a clinically aggressive neoplasm.

Chronic myeloid leukemia (CML) is defined by the presence of t(9;22)(q34;q11.2)/BCR-ABL1. Additional chromosomal abnormalities confer an adverse prognosis and are particularly common in the blast phase of CML (CML-BP). CBFB rearrangement, particularly CBFB-MYH11 fusion resulting from inv(16)(p13.1q22) or t(16;16)(p13.1;q22), is an acute myeloid leukemia (AML)-defining alteration that is associated with a favorable outcome. The co-occurrence of BCR-ABL1 and CBFB rearrangement is extremely rare, and the significance of this finding remains unclear. We identified 10 patients with myeloid neoplasms harboring BCR-ABL1 and CBFB rearrangement. The study group included six men and four women with a median age of 51 years (range, 20-71 years). The sequence of molecular alterations could be determined in nine cases: BCR-ABL1 preceded CBFB rearrangement in seven, CBFB rearrangement preceded BCR-ABL1 in one, and both alterations were discovered simultaneously in one patient. BCR-ABL1 encoded for p210 kD in all cases in which BCR-ABL1 preceded CBFB rearrangement; a p190 kD was identified in the other three cases. Two patients were treated with the FLAG-IDA regimen (fludarabine, cytarabine, idarubicin, and G-CSF) and tyrosine kinase inhibitors (TKI); seven with other cytarabine-based regimens and TKIs, and one with ponatinib alone. At last follow up (median, 16 months; range 2-85), 7 of 10 patients had died. The co-existence of BCR-ABL1 and CBFB rearrangement is associated with poor outcome and a clinical course similar to that of CML-BP, and unlike de novo AML with CBFB rearrangement, suggesting that high-intensity chemotherapy with TKI should be considered in these patients.

Clinicopathologic and genetic features of primary bronchopulmonary mucoepidermoid carcinoma: the MD Anderson Cancer Center experience and comprehensive review of the literature.

Primary bronchopulmonary mucoepidermoid carcinoma (BPMEC) is a rare tumor. The fusion protein MECT1-MAML2 has been implicated as a causative genetic event in salivary and BPMECs. Several studies have shown the impact of MECT1-MAML2 on the diagnosis and prognosis of salivary gland mucoepidermoid carcinoma; however, few studies have been published regarding MECT1-MAML2 in the context of primary BPMEC. We describe the clinicopathologic, genetic, and outcome data of 16 patients with BPMEC. Clinicopathologic features were recorded from the electronic medical records. All tumors were reviewed by two expert pulmonary pathologists and graded according to previously established criteria. The presence of MECT1-MAML2 was evaluated with reverse transcription polymerase chain reaction using RNA extracted from formalin-fixed paraffin-embedded tumor tissue. Patients included 9 women and 7 men with a median age of 50 years (range, 7 to 82 years). Tumors exhibited low (n = 14, 88%), and high (n = 2, 12%) grade histologic features. Eight of nine tested tumors (89%) were positive for MECT1-MAML2. The median follow-up time was 40.8 months (range, 1.8-120). Median overall survival for patients with high-grade tumors was 12 months, which was significantly (p = 0.002) shorter than that for patients with low-grade tumors (survival undefined). We also provide a comprehensive review of literature of cases of primary bronchopulmonary mucoepidermoid carcinoma and summarize our findings in this context. MECT1-MAML2 fusion transcript is a driver genetic event in the pathogenesis of primary BPMEC. Histologic grade continues to play a pivotal role in the survival of patients with primary bronchopulmonary mucoepidermoid carcinoma.

Herpes simplex infection simulating Richter transformation: a series of four cases and review of the literature.

AIMS: Richter transformation (RT) occurs in 5-10% of patients with chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), and is associated with a poor outcome. The histological manifestations of RT are variable, and include an increase in the proportion of prolymphocytes, immunoblasts, and other pleomorphic cells, along with an increased proliferation rate and often necrosis. Rarely, superimposed herpes simplex virus (HSV) infection involving lymph nodes in patients with CLL/SLL may mimic RT clinically and histologically. In this study, we describe four cases of CLL/SLL with superimposed HSV lymphadenitis, and we review the literature. METHODS AND RESULTS: We describe the clinicopathological and immunophenotypic findings of four cases of CLL/SLL with superimposed HSV infection. The patients presented with B-type symptoms, and laboratory and imaging findings suggested progression of lymphoma. Histologically, there were geographical areas of necrosis and increased numbers of prolymphocytes and immunoblasts, in some areas forming sheets, with increased mitotic figures and a high proliferation (Ki67) rate. However, the necrotic regions were associated with acute inflammatory cells and larger cells, mostly necrotic, showing viral cytopathic effects. Therapy with antiviral agents resulted in improvement of clinical symptoms and laboratory and imaging findings. Additionally, we identified 11 cases of CLL/SLL with superimposed HSV infection described in the English-language literature, and integrated the cases that we report here into this wider context. CONCLUSIONS: Herpes simplex virus infection of lymph nodes in patients with CLL/SLL results in clinicopathological and radiological findings that may mimic RT. It is essential to refrain from misclassifying these cases as RT, as patients can respond remarkably well to antiviral therapy.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma.

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression "on" or "off" according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1×10(8), 1×10(9), or 1×10(10) viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1×10(9) vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma.

Safety profile of gutless adenovirus vectors delivered into the normal brain parenchyma: implications for a glioma phase 1 clinical trial.

Adenoviral vectors (Ads) have been evaluated in clinical trials for glioma. However, systemic immunity against the vectors can hamper therapeutic efficacy. We demonstrated that combined immunostimulation and cytotoxic gene therapy provides long-term survival in preclinical glioma models. Because helper-dependent high-capacity Ads (HC-Ads) elicit sustained transgene expression, in the presence of antiadenoviral immunity, we engineered HC-Ads encoding conditional cytotoxic herpes simplex type 1 thymidine kinase and immunostimulatory cytokine Fms-like tyrosine kinase ligand-3 under the control of the TetOn system. Escalating doses of combined HC-Ads (1×10(8), 1×10(9), and 1×10(10) viral particles [VP]) were delivered into the rat brain. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points after vector delivery. Histopathological analysis did not reveal any evidence of toxicity or long-term inflammation at the lower doses tested. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data indicate that doses of up to 1×10(9) VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads.

A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.