The Potential Therapeutic Effects of Botulinum Neurotoxins on Neoplastic Cells: A Comprehensive Review of In Vitro and In Vivo Studies.

A systematic review of the literature found fifteen articles on the effect of a botulinum toxin on neoplastic cell lines and eight articles on in vivo neoplasms. The reported in vitro effects rely on high doses or the mechanical disruption of cell membranes to introduce the botulinum neurotoxin into the cell cytoplasm. The potency of the botulinum neurotoxin to intoxicate non-neuronal cells (even cell lines expressing an appropriate protein receptor) is several orders of magnitude lower compared to that to intoxicate the primary neurons. The data suggest that the botulinum toxin disrupts the progression of cancer cells, with some studies reporting apoptotic effects. A majority of the data in the in vivo studies also showed similar results. No safety issues were disclosed in the in vivo studies. Limited studies have suggested similar anti-neoplastic potential for the clostridium difficile. New modes of delivery have been tested to enhance the in vivo delivery of the botulinum toxin to neoplastic cells. Careful controlled studies are necessary to demonstrate the efficacy and safety of this mode of anti-neoplastic treatment in humans.

TERT Promoter Mutation c.-124C>T Commonly Occurs in Low-Grade Fibromatosis-like Metaplastic Breast Carcinoma.

CONTEXT.­: Low-grade fibromatosis-like metaplastic carcinoma (FLMC) is a very rare subtype of triple-negative metaplastic (spindle cell) breast carcinoma. It is characterized by the proliferation of spindle cells closely resembling fibromatosis, which represents a benign fibroblastic/myofibroblastic breast proliferation. Unlike most triple-negative and basal-like breast cancers, FLMC has a very low potential for metastases, but demonstrates frequent local recurrences. OBJECTIVE.­: To genetically characterize FLMC. DESIGN.­: To this end, we analyzed 7 cases by targeted next-generation sequencing for 315 cancer-related genes and performed comparative microarray copy number analysis in 5 of these cases. RESULTS.­: All cases shared TERT alterations (6 patients with recurrent c.-124C>T TERT promoter mutation and 1 patient with copy number gain encompassing the TERT locus), had oncogenic PIK3CA/PIK3R1 mutations (activation of the PI3K/AKT/mTOR pathway), and lacked mutations in TP53. TERT was overexpressed in all FLMCs. CDKN2A/B loss or mutation was observed in 4 of 7 cases (57%). Furthermore, tumors displayed chromosomal stability, with only few copy number variations and a low tumor mutational burden. CONCLUSIONS­: We conclude that FLMCs typically show the recurrent TERT promoter mutation c.-124C>T, activation of the PI3K/AKT/mTOR pathway, low genomic instability, and wild-type TP53. In conjunction with previous data of metaplastic (spindle cell) carcinoma with and without fibromatosis-like morphology, FLMC is most likely distinguished by TERT promoter mutation. Thus, our data support the notion of a distinct subgroup within low-grade metaplastic breast cancer with spindle cell morphology and associated TERT mutations.

Apoplectic leiomyomas: does ethnicity make a difference? a clinicopathologic study.

Apoplectic leiomyomas-benign uterine leiomyomas with morphologic changes including hemorrhage, hypercellularity, mitotic activity, nuclear atypia, and even necrosis-can be difficult to distinguish from uterine leiomyosarcomas. Apoplectic leiomyomas have been associated with hormonal therapy; however, the relationship between apoplectic leiomyomas, hormones, and ethnicity has not received much attention in the literature. We evaluated the relationship of hormonal therapy and ethnicity in 869 women with uterine leiomyomas, 136 of which qualified as apoplectic leiomyomas.Apoplectic leiomyomas were observed in 23.3% (49/210) of women exposed to hormonal therapy compared to 13.2% (87/659) of women not exposed to hormonal therapy (p < 0.0001). Women taking ethinyl estradiol/norethindrone (Lo-Estrin), leuprolide, and medroxyprogesterone were significantly more likely to have apoplectic leiomyomas compared to women taking other hormonal therapies. Apoplectic leiomyomas were observed in 28.9% (44/152) of African-American women compared to 12.4% (79/639) of Caucasian women (p < 0.0001), and this difference remained statistically significant regardless of hormone use. Apoplectic leiomyomas were observed in 22.1% (77/349) of women ≤ 45 years of age compared to 11.3% (59/520) of women > 45 years of age (p < 0.0001), and this difference remained statistically significant regardless of hormone use.This is the largest study to date examining apoplectic leiomyomas in women on known hormonal therapy compared to women with uterine leiomyomas, but not on hormonal therapy. Information about hormonal therapy, ethnicity, and age can be helpful in the diagnostic interpretation of apoplectic leiomyoma.

Co-expression of p16 and p53 characterizes aggressive subtypes of ductal intraepithelial neoplasia.

In the USA alone, approximately 61,000 new diagnoses of ductal intraepithelial neoplasia 1c-3 (DIN) are made each year. Around 10-20 % of the patients develop a recurrence, about 50 % of which are invasive. Prior studies have shown that invasive breast carcinomas positive for p16 or p53 have a higher frequency of recurrence and a more aggressive course; however, the co-expression of these markers across the entire spectrum of DIN and its potential correlation with grade of the lesions has not been studied previously. Immunohistochemical staining for p16 and p53 was evaluated on 262 DIN lesions from 211 cases diagnosed between 1991 and 2008. The lesions ranged from DIN1b (atypical intraductal hyperplasia) to DIN3 (DCIS, grade 3) and included 45 cases with associated invasive carcinoma. Frequency of staining for both p16 and p53 increased with increasing grade of DIN. Strong co-expression was found exclusively in higher grade DIN lesions (DIN2 and DIN3) particularly those associated with periductal stromal fibrosis and lymphocytic infiltrate. Strong co-expression was seen in 8 of 12 DIN3 lesions (67 %) associated with invasive carcinoma. In conclusion, co-expression of p16 and p53 increases with advancing grade of DIN and is maximal in high grade DIN lesions associated with invasive carcinoma, indicating a more aggressive phenotype. A distinctive variant of DIN with periductal fibrosis and lymphocytic infiltrate invariably falls into the high-grade category, based on either morphology or marker expression. Co-expression of p16/p53 may be of help in distinguishing between high-grade and low-grade DIN lesions.

Distribution pattern of Ki67 immunoreactivity in ductal intraepithelial neoplasia: Correlation with lesion grade and potential utility.

BACKGROUND: In this study, the pattern of distribution of the nuclei immunoreactive with Ki67 was examined in DIN1c (DCIS, grade 1/low grade), DIN2 (DCIS, grade 2/intermediate grade), and DIN3 (DCIS, grade 3/high grade). The lesions were evaluated to determine if distinctive patterns could be identified in correlation with lesion grade. METHODS: Fifty seven (n=57) consecutive DIN cases were investigated. Of these, 15 qualified as DIN1c, 28 as DIN2 and 14 as DIN3. The patterns of distribution were recorded for each case as either basal/peripheral or haphazard within the epithelial proliferation. RESULTS: There was a statistically significant difference between the DIN1c, DIN2 and DIN3 in terms of basal/peripheral versus haphazard distribution of Ki67 immunostaining (Chi-square test, P<0.0001). Basal/peripheral staining pattern was dominant among the DIN1c cases, while haphazard staining pattern was the dominant distribution among the DIN3 cases. One half of the DIN2 cases showed basal/peripheral staining pattern, while the other half showed a haphazard staining pattern. CONCLUSION: High grade DIN lesions show haphazard Ki67 staining while low grade DIN lesions show basal/peripheral Ki67 staining in the proliferating epithelial cells. This feature could be practical in separating DIN lesions into low grade (basal/peripheral-Ki67) and high grade (haphazard-Ki67) eliminating the grade 2/intermediate category.

Expression of C-KIT, CD24, CD44s, and COX2 in benign and non-invasive apocrine lesions of the breast.

Benign apocrine metaplasia (AM) of the adult breast is a very common, but enigmatic lesion. It has been speculated that AM might be a precursor of malignancy or an indicator of a susceptibility of the breast tissue to develop neoplasia, mainly based on comparing the frequency of AM in breast cancer and non-breast cancer patients [1]. Studies using comparative genomic hybridization have supported this by showing similar molecular alterations in benign and malignant apocrine lesions [2]. Few studies, however, have compared expression of biomarkers involved in tumor progression in AM and progressively more advanced atypical apocrine lesions. The expression of C-KIT, COX2, CD24, and CD44s was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded material of 9 AM, 20 apocrine ductal intraepithelial neoplasia (DIN1c-3) and 40 atypical apocrine lesions (not qualifying for DIN1c-3) and compared to expression of the same biomarkers in adjacent normal ductal epithelium. Of the 66 apocrine lesions, 62 (94 %) did not express C-KIT compared to 4/63 (6 %) of the normal glands (Fisher's exact, p < 0.001). COX2 was expressed in a significantly higher proportion of apocrine lesions than of normal glands (49 vs. 14 %, p < 0.001), and the number of apocrine lesions positive for CD24 was found to be higher with increasing aggressiveness of the lesions (Spearman, p < 0.001). In conclusion, benign and non-invasive proliferative apocrine lesions of the breast display immuno-phenotypical characteristics previously ascribed mainly to malignant transformation. This could lend support to the theory that AM is an early step towards malignant transformation, albeit associated with slow progression to carcinoma.

Cytokeratin 7-negative mammary Paget's disease: A diagnostic pitfall.

Pathologists should be aware of the existence of a rare CK7-negative variant of breast carcinoma in general, and of Paget's disease in particular. Cytokeratin 7-negative Paget's disease and CK7-negative ductal intraepithelial neoplasia (ductal carcinoma in situ) present a major diagnostic challenge for pathologists since there is limited awareness of their existence. When there is classic Paget's morphology on H&E sections, GATA3 positivity should resolve any doubts about the diagnosis in the setting of a CK7-negative neoplastic cell population.

A targetable androgen receptor-positive breast cancer subtype hidden among the triple-negative cancers.

CONTEXT: Triple-negative breast cancer (TNBC) is a subgroup of breast cancers that by definition lack expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). A diverse group of tumors, TNBC shares some morphologic and molecular features with basal-like breast cancer, a category of breast cancer defined by gene expression profiling. More likely to occur in young women and African Americans, TNBCs may exhibit aggressive behavior and are associated with poor prognosis despite their initial response to conventional chemotherapy. Because hormonal or HER2-targeted therapies are ineffective for these tumors, the main therapeutic option is systemic chemotherapy. Therefore, identification of new targets for therapy is urgently needed for this group. OBJECTIVE: To review and present recent literature along with our own experience regarding the clinical and morphologic characteristics and the prevalence of androgen receptor (AR) expression in TNBC, and to discuss the potential use of AR as a therapeutic target for AR(+) TNBC. DATA SOURCES: Data sources are published articles from peer-reviewed journals in PubMed (US National Library of Medicine). CONCLUSIONS: AR is the most commonly expressed hormone receptor among all breast carcinomas, with a prevalence of 25% to 75% among TNBCs. Therefore, we strongly support the routine assessment of AR in TNBC, and preferably in all breast carcinomas.

Androgen receptor (AR) expression in 400 breast carcinomas: is routine AR assessment justified?

BACKGROUND: Triple negative breast carcinomas (TNBC) do not benefit from hormonal or Herceptin therapies. In search of novel therapeutic targets for TNBC, interest is escalating in a subset of these tumors that are androgen receptor (AR) positive with potential benefit from anti-androgen therapy. Against this background, the frequency of AR expression alone and in combination with other markers and morphologic features was assessed to identify TNBC subtypes for targeted therapy. METHODS: 400 consecutive invasive mammary carcinomas with known estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and HER2 status were selected for study. The frequency of AR positivity alone or in combination with other markers was recorded with specific attention to the morphology of AR+ TNBCs. Ki67 was evaluated in selected group of cases. ASCO/CAP guidelines were used for interpretation of the various biomarkers. RESULTS: Of the 400 tumors, 32 (8%) carcinomas were quadruple negative (ER-, PR-, AR-, Her2-), while 50 tumors (12.5%) were triple negative (ER-, PR-, Her2-); 18 (36%) of the triple negative tumors were AR positive and 10 (55%) of these were classic apocrine carcinomas. Fourteen cases, all apocrine carcinomas, were AR and Her2 positive. All 32 QN carcinomas were poorly differentiated and they had the highest Ki67 labeling index. CONCLUSION: The relatively high proportion of AR+ tumors (36%) among the 50 triple negative carcinomas is an important finding in support of routine assessment of AR in at least all TNBCs and apocrine carcinomas as a potential target for therapy.

Reproducibility of current classifications of endometrial endometrioid glandular proliferations: further evidence supporting a simplified classification.

AIMS: To compare the reproducibility of the current (2003) World Health Organization (WHO), endometrial intraepithelial neoplasia (EIN) and European Working Group (EWG) classifications of endometrial endometrioid proliferations. METHODS AND RESULTS: Nine expert gynaecological pathologists from Europe and North America reviewed 198 endometrial biopsy/curettage specimens originally diagnosed as low-grade lesions. All observers were asked to classify the cases by using the categories described in each scheme: six for WHO, four for EIN, and three for EWG. The results were evaluated by kappa statistics for more than two observations. The analysis was repeated using only two major categories (benign versus atypical/carcinoma). Both the WHO and EIN classifications showed poor interobserver agreement (κ = 0.337 and κ = 0.419, respectively), whereas the EWG classification showed moderate agreement (κ = 0.530). Full agreement between pathologists occurred in only 28% for the WHO classification, 39% for the EIN classification, and 59% for the EWG classification. With only two diagnostic categories, kappa values increased in all classifications, but only the EWG classification reached a substantial level of agreement (κ = 0.621); similarly, full agreement among all pathologists increased to 70% for the WHO classification, 69% for the EIN classification, and 72% for the EWG classification. CONCLUSIONS: A two-tier classification of endometrial endometrioid proliferative lesions improves reproducibility, and should be considered for the diagnosis of endometrial biopsy/curettage specimens.

Intramedullary metastasis in breast cancer--a comprehensive literature review.

BACKGROUND: Intramedullary breast cancer metastasis (IMBCM) is considered rare but its true incidence is unknown. Previous reviews of this subject are few and provided limited information. OBJECTIVE: To evaluate the precise location(s) of IMBCM, its concurrence rate with brain metastasis, its frequency compared to other cancers, intervals between breast cancer diagnosis and detection of IMBCM and between detection of IMBCM and death, frequency of various clinical symptoms, neuroimaging, cerebrospinal fluid (CSF) and biomarker data, treatment strategies and survival issues. STUDY DESIGN: All relevant literature from 1900 to present was identified through Yale search Engine including but not limited to Medline/Pub Med, Ovid and Erasmus. RESULTS: A total of 36 publications were identified describing 85 patients with IMBCM. Breast cancer was the second highest source of intramedullary metastasis after lung (26.5% versus 45%). Cervical and thoracic cord was equally affected. Presence of thoracic cord lesions (P=0.039), concurrent brain metastasis, bladder dysfunction and Brown-Sequard syndrome were associated with a more unfavorable prognosis. Treatment strategies which included surgery suggested prolonged survival. Patients with breast metastasis to the spinal cord had longer survival than lung metastasis (P=0.05). The data on CSF and tumor markers was too limited to be conclusive. CONCLUSIONS: Introduction of Magnetic Resonance Imaging has significantly increased the detection rate of IMBCM. Factors associated with better prognosis are presented. Definition of the true incidence of IMBCM would require a prospective clinical and neuroimaging study.

Basal cell adenocarcinoma arising in salivary gland metaplasia of the breast: a novel salivary gland-type tumor developing in the breast.

A variety of salivary gland-type lesions occur in the breast. Three cases of a novel mammary carcinoma arising in a background of salivary gland metaplasia and morphologically similar to basal cell adenocarcinoma of the salivary gland are presented. The clinical presentation, morphologic features, treatment, and follow-up of these cases are discussed.

Myxoid leiomyosarcoma of the uterus.

AIMS: Recent guidelines have suggested the presence of tumour cell necrosis (TCN), atypia and mitotic index as major features in the distinction of myxoid leiomyosarcomas (MLMSs) from myxoid leiomyomas. The aim of this study was to focus on an invasive growth pattern as a significant feature in this distinction in the absence of TCN. METHODS AND RESULTS: Twelve uterine smooth muscle tumours with myxoid change in ≥60% of the lesion were interpreted as MLMS on the basis of the presence of focal mild atypia as well as one or more of the following features: (i) infiltrative growth pattern; (ii) vascular invasion; (iii) mitotic index of ≥5 mitotic figures (m.f.)/50 high-power fields (HPFs); and (iv) a combination of at least focal severe atypia and at least 2-4 m.f./50 HPFs. Unequivocal TCN was not evident in any of these tumours. The various morphological features were correlated with outcome. With follow-ups ranging from 19 to 113 months (mean 60 months), five of the 12 women developed recurrences, and two of them died. Nine of the 12 tumours had an infiltrative growth pattern, and all five recurrent tumours were from this group. CONCLUSION: In the absence of TCN, an infiltrative margin is a major factor related to the potential for aggressive behaviour of MLMS.

Chromosomal aberrations as detected by array comparative genomic hybridization in early low-grade intraepithelial neoplasias of the breast.

AIMS: Low-grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs concomitantly with lobular intraepithelial neoplasia (LIN). We aimed to elucidate chromosomal aberrations in these early neoplastic breast lesions with the use of array comparative genomic hybridization analysis. METHODS AND RESULTS: Laser capture microdissection of 12 archival formalin-fixed, paraffin-embedded specimens harbouring foci of both DIN1a and LIN was performed. All analysed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss of 16q, noted in seven DIN1a (70% of those successfully examined) and 10 LIN (91%) cases. The next most common alteration was a gain on 1q, noted in four DIN1a (40%) and seven LIN (64%) cases. CONCLUSIONS: The results show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and DIN1a. These aberrations are known to be common in low-grade invasive (ductal and lobular) carcinomas as well as in more advanced (conventional) types of low-grade ductal intraepithelial neoplasia (DIN) (low-grade ductal carcinoma in situ). Our results raise the possibility of similar molecular-genetic pathways in coexisting LIN and low-grade flat DIN.

Diminished number or complete loss of myoepithelial cells associated with metaplastic and neoplastic apocrine lesions of the breast.

The presence of myoepithelial (ME) cells is considered an important feature in the vast majority of benign breast lesions. Recently, a case showing the absence of myoepithelium in a mammary duct with apocrine metaplasia was reported. To investigate the status of ME cells associated with apocrine metaplasia, the distribution of ME cells in 59 metaplastic and intraductal proliferative apocrine lesions was evaluated using immunohistochemical expression of p63 and Calponin. p63 showed a diminished number of ME cells and increased intermyoepithelial nuclear distance in ducts with all variants of apocrine metaplasia and proliferation compared with normal glands. In the majority of cases, Calponin showed a continuous ME layer. In 6 cases, including an apocrine papilloma, there were definitive ME gaps confirmed by both markers, in the absence of atypia and with preservation of the basement membrane. In all cases, there was frequent heterogeneity in the distribution of ME cells in ducts harboring apocrine cells and even in various papillae within papillary lesions. In summary, benign and noninvasive apocrine lesions can show reduction and occasional complete loss of ME cells. This observation is particularly important when evaluating apocrine papillary proliferations, in which the absence of ME cells may lead to overdiagnosis of atypia and/or malignancy. The observation suggests that at least 2 ME markers should be used when evaluating apocrine lesions, and that a malignant diagnosis should be based on features of the proliferating cells until more data become available on the significance, if any, of the absence of ME cells in apocrine lesions.

Skip lesion of DIN (DCIS) in the nipple in a case of breast cancer.

The authors present a case of breast cancer with a skip lesion of DIN 1 (ductal carcinoma in situ [DCIS] grade 1) in the nipple, leaving the base of the nipple free of disease. During the surgical procedure of nipple-sparing mastectomy (NSM) a frozen section evaluation of the base of the nipple and areola complex is often carried out to ascertain absence of any intraepithelial (in situ) or invasive neoplasms that could lead to nipple recurrences if left behind. This case illustrates a situation where a frozen section would have given a false-negative prediction of nipple involvement if the patient had been selected for NSM. This case illustrates the need to adhere to strict criteria for selecting patients eligible for NSM and to consider the use of additional preoperative and intraoperative measurements and methods to refine the selection of candidates for NSM and enhance the oncological safety of the procedure.