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.

Exploring chromosomal abnormalities and genetic changes in uterine smooth muscle tumors.

Smooth muscle tumors of the uterus are a diagnostically challenging group of tumors. Molecular surrogate markers reliably distinguishing between benign and malignant tumors are not available. Therefore, the diagnosis is based on morphologic criteria. The aim was to investigate a well-characterized group of challenging uterine smooth muscle tumors consisting of 20 leiomyomas, 13 leiomyomas with bizarre nuclei, and 14 leiomyosarcomas for copy number alterations, MED12 mutations and FH deletions to search for potential diagnostically useful surrogate markers. MED12 mutations were detected in 47, 15, and 25% of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas, respectively. MED12 mutations in leiomyomas with bizarre nuclei were detected outside the hotspot region. FH-deletions were seen in 27, 30.8, and 25% of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas, respectively. By using copy number alteration profiling a clear separation of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas could not be observed. Copy number alterations revealed clear genetic similarities between leiomyomas with bizarre nuclei and leiomyosarcomas. Leiomyosarcomas showed a similar pattern of gains and losses as leiomyomas with bizarre nuclei, with additional copy number alterations and more homozygous losses and high-level amplifications compared to leiomyomas with bizarre nuclei. In conclusion, this study demonstrates that known FH-deletions, a recurrent molecular change in leiomyomas, occur in morphologically challenging variants of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas. Although MED12 mutations are common in leiomyomas, they infrequently occur in leiomyomas with bizarre nuclei and leiomyosarcomas. The genetic similarities between leiomyomas with bizarre nuclei and leiomyosarcomas raise the intriguing possibility that uterine leiomyomas with bizarre nuclei and leiomyosarcomas are closely related and challenge the traditional concept that leiomyoma with bizarre nuclei is a tumor with just marked 'degenerative' cellular changes. These findings support the hypothesis that tumor progression within uterine smooth muscle tumors might occur.

Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF). METHODS: We used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen's kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas. RESULTS: We found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66-69 % of the variance was attributable to differences between FOVs in the same section and 30-33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20. CONCLUSIONS: T and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.

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.

Brain metastasis in breast cancer: a comprehensive literature review.

This comprehensive review provides information on epidemiology, size, grade, cerebral localization, clinical symptoms, treatments, and factors associated with longer survival in 14,599 patients with brain metastasis from breast cancer; the molecular features of breast cancers most likely to develop brain metastases and the potential use of these predictive molecular alterations for patient management and future therapeutic targets are also addressed. The review covers the data from 106 articles representing this subject in the era of modern neuroimaging (past 35 years). The incidence of brain metastasis from breast cancer (24 % in this review) is increasing due to advances in both imaging technologies leading to earlier detection of the brain metastases and introduction of novel therapies resulting in longer survival from the primary breast cancer. The mean age at the time of breast cancer and brain metastasis diagnoses was 50.3 and 48.8 years respectively. Axillary node metastasis was noted in 32.8 % of the patients who developed brain metastasis. The median time intervals between the diagnosis of breast cancer to identification of brain metastasis and from identification of brain metastasis to death were 34 and 15 months, respectively. The most common symptoms experienced in patients with brain metastasis consisted of headache (35 %), vomiting (26 %), nausea (23 %), hemiparesis (22 %), visual changes (13 %) and seizures (12 %). A majority of the patients had multiple metastases (54.2 %). Cerebellum and frontal lobes were the most common sites of metastasis (33 and 16 %, respectively). Of the primary tumors for which biomarkers were recorded, 37 % were estrogen receptor (ER)+, 41 % ER-, 36 % progesterone receptor (PR)+, 34 % PR-, 35 % human epithelial growth factor receptor 2 (HER2)+, 41 % HER2-, 27 % triple negative and 18 % triple positive (TP). Treatment in most patients consisted of a multimodality approach often with two or more of the following: whole brain radiation therapy (52 %), chemotherapy (51 %), stereotactic radiosurgery (20 %), surgical resection (14 %), trastuzumab (39 %) for HER2 positive tumors, and hormonal therapy (34 %) for ER and/or PR positive tumors. Factors that had an impact on prognosis included grade and size of the tumor, multiple metastases, presence of extra-cranial metastasis, triple negative or HER2+ biomarker status, and high Karnovsky score. Novel therapies such as application of agents to reduce tumor angiogenesis or alter permeability of the blood brain barrier are being explored with preliminary results suggesting a potential to improve survival after brain metastasis. Other potential therapies based on genetic alterations in the tumor and the microenvironment in the brain are being investigated; these are briefly discussed.

Mutation Profiling of Usual Ductal Hyperplasia of the Breast Reveals Activating Mutations Predominantly at Different Levels of the PI3K/AKT/mTOR Pathway.

Usual ductal hyperplasia (UDH) of the breast is generally regarded as a nonneoplastic proliferation, albeit loss of heterozygosity has long been reported in a part of these lesions. To gain deeper insights into the molecular drivers of these lesions, an extended mutation profiling was performed. The coding regions of 409 cancer-related genes were investigated by next-generation sequencing in 16 cases of UDH, nine unassociated with neoplasia (classic) and seven arising within papillomas. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) activation was investigated by phosphorylated AKT, mTOR, and S6 immunohistochemistry. Of 16 lesions, 10 (63%) were mutated; 56% of classic lesions were unassociated with neoplasia, and 71% of lesions arose in papillomas. Fourteen missense mutations were detected: PIK3CA [6 (43%) of 14], AKT1 [2 (14%) of 14], as well as GNAS, MTOR, PIK3R1, LPHN3, LRP1B, and IGF2R [each 1 (7%) of 14]. Phosphorylated mTOR was seen in 83% and phosphorylated S6 in 86% of evaluable lesions (phospho-AKT staining was technically uninterpretable). In conclusion, UDH displays mutations of the phosphatidylinositol 3-kinase/AKT/mTOR axis at different levels, with PIK3R1, MTOR, and GNAS mutations not previously described. Specifically, oncogenic G-protein activation represents a yet unrecognized route to proliferation in UDH. On the basis of evidence of activating mutations, loss of heterozygosity, and a mass forming proliferation, we propose that UDH is most appropriately viewed as an early neoplastic intraductal proliferation.

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.

Brain metastasis from ovarian cancer: a systematic review.

To review the existing literature on brain metastasis (BM) from ovarian cancer and to assess the frequency, anatomical, clinical and paraclinical information and factors associated with prognosis. Ovarian cancer is a rare cause of brain metastasis with a recently reported increasing prevalence. Progressive neurologic disability and poor prognosis is common. A comprehensive review on this subject has not been published previously. This systematic literature search used the Pubmed and Yale library. A total of 66 publications were found, 57 of which were used representing 591 patients with BM from ovarian cancer. The median age of the patients was 54.3 years (range 20-81). A majority of patients (57.3 %) had multiple brain lesions. The location of the lesion was cerebellar (30 %), frontal (20 %), parietal (18 %) and occipital (11 %). Extracranial metastasis was present in 49.8 % of cases involving liver (20.7 %), lung (20.4 %), lymph nodes (12.6 %), bones (6.6 %) and pelvic organs (4.3 %). The most common symptoms were weakness (16 %), seizures (11 %), altered mentality (11 %) visual disturbances (9 %) and dizziness (8 %). The interval from diagnosis of breast cancer to BM ranged from 0 to 133 months (median 24 months) and median survival was 8.2 months. Local radiation, surgical resection, stereotactic radiosurgery and medical therapy were used. Factors that significantly increased the survival were younger age at the time of ovarian cancer diagnosis and brain metastasis diagnosis, lower grade of the primary tumor, higher KPS score and multimodality treatment for the brain metastases. Ovarian cancer is a rare cause of brain metastasis. Development of brain metastasis among older patients and lower KPS score correlate with less favorable prognosis. The more prolonged survival after using multimodality treatment for brain metastasis is important due to potential impact on management of brain metastasis in future.

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.