Converging and evolving immuno-genomic routes toward immune escape in breast cancer.

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Soemmerring's Rings Developed around IOLs, in Human Donor Eyes, Can Present Internal Transparent Areas.

Soemmerring's rings consist of a ring of lens epithelial derived cells that grow along the periphery of an aphakic lens capsule, or around an intraocular lens. These rings when visualized frontally, appear opaque, however, in some cases the cells that compose these rings are organized in the same fashion as those in normal transparent adult lenses. Thus, our purpose was to test whether any part of the adult Soemmerring's ring could be transparent and how this related to morphological factors. To study this, 16 Soemmerring's rings were extracted from donor eye globes. After imaging, they were thickly sectioned sagittally in order to analyze the degrees of transparency of different areas. All samples were also histologically analyzed using alpha smooth muscle actin, Vimentin, wheat germ agglutinin and DAPI. Our results showed that many samples had some transparent areas, mostly towards the center of their cross-section. Of the factors that we analyzed, only lens fiber organization at the bow region and an increased area of mature lens fiber cells had a significant relation to the degree of transparency at the center. Thus, we can conclude that as Soemmerring's rings mature, they can develop organized and transparent areas of lens cells.

Organoid (hypermature) ovarian teratomas: Report of four cases, one of which harbored a well-differentiated neuroendocrine tumor.

Organoid (hypermature) teratomas are highly specialized teratomas showing organ formation, most frequently from the digestive tract or bronchial wall. We present four cases of ovarian organoid teratomas, one with a distinguishable mandible with teeth, one with small intestine, one with large intestine containing a well differentiated neuroendocrine tumor and another with both large intestine and bronchial wall. These tumors have a distribution similar to conventional teratomas and usually behave benignly, although cases of malignancy have been reported.

Evolution of laparoscopic myomectomy and description of two hemostatic techniques in a large teaching gynecological center.

OBJECTIVE: To provide a description of laparoscopic myomectomy and the two hemostatic techniques performed over the last 11 years in a single reference center for gynecology and obstetrics and to evaluate the factors associated with favorable surgical outcomes. STUDY DESIGN: We retrospectively analyzed 625 who underwent laparoscopic myomectomy from January 2009 to December 2019. RESULTS: Of 625 patients, 437 (69.8%) were symptomatic. The most common symptoms were heavy uterine bleeding (33.2%). 188 patients (30.1%) were asymptomatic but were operated in 77 cases (12.3%) for rapid fibroid growth, 32 (5.1%) for uterine cavity distortion and, in 45 cases (8.6%), the myomectomy was indicated during a surgery for other medical reason due to its accessibility. In 173 cases (27.9%) intramyometrial adrenaline was injected and in 246 cases (39.7%) a temporary blockage of the uterus blood supply was performed. Only 35 (5.6%) patients presented complications, of which, 14 (40%) were hemorrhagic. These hemorrhagic complications were more frequent when intramyometrial adrenaline was used (5,8%) than after the temporary clipping of the uterine arteries and infundibulopelvic ligaments (0,8%; p < 0,001). In the multivariate logistic regression model, the only factor statistically associated with favorable surgical outcome was the use of temporary clipping of the uterine arteries at their origin and infundibulopelvic ligaments as hemostatic technique during the surgery. CONCLUSION: Laparoscopic myomectomy was generally safe with a high level of favorable outcomes. The temporary clipping of uterine arteries and infundibulopelvic ligaments presented fewer intraoperative bleedings compared with injecting intramyometrial adrenaline.

Adenoid cystic carcinoma and basaloid carcinoma of the breast: A clinicopathological study.

Adenoid cystic carcinoma of the breast (ACCB) is a rare triple negative tumor (TNT) with an excellent prognosis in most cases. Three different histologic types are recognized: classic ACCB, solid basaloid ACCB (SB-ACCB), and ACCB with high-grade transformation. A majority of these tumors show characteristic molecular and immunohistochemical (IHC) features, with fusion of MYB and NFIB genes and overexpression of MYB, respectively. Basaloid carcinomas of the breast (BCB) are infrequently described. They resemble SB-ACCB and TNT of no special type (TNT-NST). We have studied the clinicopathological features of 17 ACCB and 9 BCB, investigating the expression of MYB by IHC and the rearrangements of MYB by fluorescence in situ hybridization (FISH). MYB was expressed by IHC in 15 ACCB and in 3 BCB. MYB FISH detected rearrangements in 11 ACCB and in 2 BCB. After a mean follow-up of 90 months, with a range of 12-204 months, 2 patients with ACCB with high-grade transformation and 1 patient with BCB developed metastases and died of disease. In summary, most ACCB have a good prognosis, but tumors with adverse histopathological features may metastasize. BCB may overlap with ACCB and TNT-NST, and their prognosis should be further studied.

Surgical Management of Vulvar Melanoma: A Case Series.

Vulvar malignant melanoma is the second most common subtype of vulvar cancer, accounting for 5-10% of all vulvar cancers. The prognosis is still very poor, although some advances have been achieved in the last years. One of the most significant changes in its management has been the development of less invasive surgical techniques that diminish the risk of postoperative morbidity and long-lasting sequelae. In this article, we review the surgical management of the pathology, based on the comment of 3 cases with vulvar melanoma treated at our institution.

Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Orbital histiocytosis with systemic involvement: A case with complex affiliations.

A 70-year-old male presented with orbital masses affecting the muscular cone. His past medical history was notable for diabetes mellitus, ischemic cardiopathy, sleep-apnea syndrome, and multiple serous effusions. The first biopsy specimen of affected orbital tissue revealed fibrohistiocytic infiltration resembling xanthogranuloma or Erdheim-Chester disease (ECD). An ulterior biopsy of affected orbital tissue showed lymphocyte emperipolesis with immunopositivity for CD68 and S100 but negative staining for CD1a marker, strongly suggesting Rosai-Dorfman disease (RDD). Afterward, pericardium and peritoneal effusions resulted in constrictive pericarditis and retroperitoneal fibrosis, respectively. The absence of distinctive clinical features made the diagnosis especially challenging. Attempts to control the disease using corticosteroids, radiation, orbital surgery, and interferon were unsuccessful. Aggressive treatments such as chemotherapy were not considered appropriate due to the general deterioration of our patient. Although the possibility of two concurrent diseases (e.g., systemic ECD and orbital RDD) cannot be discarded, we interpreted the orbital findings as likely due to RDD, and the entire condition of our patient as an extranodal RDD with atypical clinicopathological findings and outcome.

Breast density measured volumetrically in a clinical environment: cross-sectional study with photon counting technology.

OBJECTIVE: Mammographic breast density (BDen), the ratio of glandular volume (GVol) to breast volume (BVol), is the second most prevalent risk factor for breast cancer (BC). Newly developed photon counting technology allows precise and systematic measurements in clinical practice. Our objective is to see how these parameters change with age in women with and without cancer. MATERIALS AND METHODS: This retrospective study analyzed results of BDen, GVol, and BVol in 64,182 mammograms performed with photon counting technology on 32,448 consecutive women from April 2014 to December 2015. Only their first study was included. We excluded women with incomplete data or with breast implants. RESULTS: Mean age of women without BC diagnosed during the study period was 52.1 ± 9.9. BC and was found in 263 women (0.81%). Mean age was 53.0 ± 10.4. BDen, GVol, and BVol were 14%, 24%, and 2% greater in women with BC (P < 0.001 for BDen and GVol and P = 0.02 for BVol). BDen and GVol diminished following similar patterns across age in both groups, with soft slopes before and after a steep drop from 50 to 60, probably due to menopause. CONCLUSION: BDen diminishes with age in women with or without BC, but it is generally higher in women with BC. GVol could be a more robust indicator associated with BC risk than BDen. This technology can ease the way to studies of interventions to diminish BDen (or GVol) in the hope of diminishing BC incidence or predict if longitudinal changes are indicative of impending cancer.

Impaired Expression of Ectonucleotidases in Ectopic and Eutopic Endometrial Tissue Is in Favor of ATP Accumulation in the Tissue Microenvironment in Endometriosis.

Endometriosis is a prevalent disease defined by the presence of endometrial tissue outside the uterus. Adenosine triphosphate (ATP), as a proinflammatory molecule, promotes and helps maintain the inflammatory state of endometriosis. Moreover, ATP has a direct influence on the two main symptoms of endometriosis: infertility and pain. Purinergic signaling, the group of biological responses to extracellular nucleotides such as ATP and nucleosides such as adenosine, is involved in the biology of reproduction and is impaired in pathologies with an inflammatory component such as endometriosis. We have previously demonstrated that ectonucleotidases, the enzymes regulating extracellular ATP levels, are active in non-pathological endometria, with hormone-dependent changes in expression throughout the cycle. In the present study we have focused on the expression of ectonucleotidases by means of immunohistochemistry and in situ activity in eutopic and ectopic endometrial tissue of women with endometriosis, and we compared the results with endometria of women without the disease. We have demonstrated that the axis CD39-CD73 is altered in endometriosis, with loss of CD39 and CD73 expression in deep infiltrating endometriosis, the most severe, and most recurring, endometriosis subtype. Our results indicate that this altered expression of ectonucleotidases in endometriosis boosts ATP accumulation in the tissue microenvironment. An important finding is the identification of the nucleotide pyrophophatase/phosphodiesterase 3 (NPP3) as a new histopathological marker of the disease since we have demonstrated its expression in the stroma only in endometriosis, in both eutopic and ectopic tissue. Therefore, targeting the proteins directly involved in ATP breakdown could be an appropriate approach to consider in the treatment of endometriosis.

Changes in breast density during hormone treatment with transdermal estrogens alone or in combination with progesterone.

A retrospective observational study to assess whether hormonal treatment (HT) with transdermal estrogens alone or in combination with micronized progesterone increases breast density and to compare these changes to those of a control group of 4120 patients were not given HT. We included 150 patients whose baseline breast density was assessed with photon-counting spectral mammography and 1 year after hormone treatment. The reduction in breast density was compared using an analysis of covariance. The difference in breast density between mammographies in the HT group was -0.40 ± 5.5 and -0.85 ± 4.2 in the control group. The changes in density according to the type of HT, we found that women on treatment with estrogen alone presented a difference of 0.44 ± 5.8, and -1.35 ± 5 (p = 0.13) in women on combined treatment. After adjusting changes in density for age and average number of days between mammographies, we observed a difference of -0.36 95% confidence intervals (CI) [-1.04 to -0.31] in the women on HT and -0.71 95% CI [-1.65 to -0.21] in the control group. No increased breast density was observed in women on HT treatment, nor did we observe an increase according to HT type. The difference in breast density loss was smaller in the HT group versus the control group.

Epigenetic changes found in uterine decidual and placental tissues can also be found in the breast cancer microenvironment of the same unique patient: description and potential interpretations.

Microenvironmental properties are thought to be responsible for feto-maternal tolerance. Speculatively, ectopic expression of placental gene programs might also be related to cancer cells' ability to escape from immune vigilance mechanisms during carcinogenesis and cancer progression. Recently, we published the first human genomic evidence of similar immune related gene expression profiles in both placenta (placenta and decidual tissue) and cancer (both primary and metastatic) in the same patient with lymph-node positive breast carcinoma during pregnancy. Here we report the first epigenomic analysis of these tissue samples and describe their main findings, with respect to immune related genes regulation (over or under expressed) in cancer cells with regards placental tissues. We confirm significant similarities, and hierarchical clustering (both unsupervised and supervised), in CpG island methylation patterns between decidual/placental and cancer microenvironments, which cannot be easily explained by simple models or unique pathways. Several different cell types are probably involved in these complex immune regulation mechanisms. Cancers may somehow "hijack" gene programs evolved over millions of years to allow for feto-maternal tolerance in placental mammals in order to escape from immune vigilance and spread locally or to distant sites.

Labral reconstruction with tendon allograft: histological findings show revascularization at 8 weeks from implantation.

This description shows the histological findings of a peroneus brevis tendon allograft used for labral reconstruction, implanted 8 weeks before being retrieved due to a postoperative complication unrelated to the graft. As far as we have knowledge this is the first description about revascularization of an allograft used for hip labral reconstruction. The histological report of the removed peroneus brevis tendon allograft shows evidence of vascular ingrowth represented by small vessels with a thin muscular wall in all layers of the graft and cellular migration mainly represented by mature fibroblasts.

Placental immune editing switch (PIES): learning about immunomodulatory pathways from a unique case report.

The hypothesis of this work is that, in order to escape the natural immune surveillance mechanisms, cancer cells and the surrounding microenvironment might express ectopically genes that are physiologically present in the placenta to mediate fetal immune-tolerance. These natural "placental immune-editing switch" mechanisms (PIES) may represent the result of millions of years of mammalian evolution developed to allow materno-fetal tolerance. Here, we introduce genes of the immune regulatory pathways that are either similarly over- or under-expressed in tumor vs normal tissue. Our analysis was carried out in primary breast cancer with metastatic homolateral axillary lymph nodes as well as placenta tissue (both uterine decidual tissue and term placenta tissue) from a pregnant woman. Gene expression profiling of paired non-self and self tissues (i.e. placenta/uterus; breast cancer/normal breast tissue; metastatic lymphnode/normal lymphnode tissue) was performed using the PanCancer Immune gene panel, a 770 Nanostring gene expression panel. Our findings reveal overlapping in specific immune gene expression in placenta and cancer tissue, suggesting that these genes might play an important role in maintaining immune tolerance both physiologically (in the placenta) and pathologically (in the cancer setting).

Fatal gastrointestinal toxicity with ipilimumab after BRAF/MEK inhibitor combination in a melanoma patient achieving pathological complete response.

Approximately 50% of metastatic melanoma patients harbor BRAF mutations. Several treatment options including the combination of BRAF and MEK inhibitors (BRAF/MEKi) and immunotherapy (mainly anti CTLA-4 and anti PD-1 antibodies), have been shown to improve survival in these patients. Although preclinical data support the synergistic effect of both modalities in combination, data confirming the activity and tolerability of these combinations are not yet available in the clinical setting. Herein, we report the case of a melanoma patient treated with sequential BRAF/MEKi (dabrafenib plus trametinib) followed by the anti CTLA-4 antibody ipilimumab who achieved a pathological complete response. Unfortunately, the patient died due to fatal gastrointestinal (GI) toxicity. Analysis of the BRAFV600E mutation in circulating tumoral DNA (ctDNA) from peripheral blood samples and serial tumor tissue biopsies throughout treatment demonstrated a good correlation with clinical evolution.

Breast sarcoma. A case report and review of literature.

INTRODUCTION: Breast sarcomas are rare with an annual incidence of 4.6 cases/1,000,000 women. They can appear as primary forms or secondary to radiation therapy or chronic lymphedema. PRESENTATION OF CASE: A 41 year old woman attended our hospital after having noticed an increase in the size of her fibroadenoma. The examination revealed a 7cm retroareolar nodule. Breast sonography described a hypoechoic bilobulated lesion and MRI showed a large size polinodular image, suggesting a Phyllodes tumor. A core needle biopsy was performed with a histological result of low-grade fusiform cells sarcoma on Phyllodes tumor so we proceeded to surgical treatment with a mastectomy. After two years and a half she noticed a tough nodule over the mastectomy scar, which was resected with a histological result of fusiform cells sarcoma. Considering the diagnosis of recurrence of the disease, surgery was undertaken. DISCUSSION: Breast sarcoma is a rare but aggressive entity. Core biopsy is the procedure of choice for the diagnosis. Lymphatic spread is uncommon so nodal status in breast sarcoma is less informative. Staging study differs from other breast tumors and chest computed tomography is helpful since lungs are the predominant metastatic sites. The use of radiotherapy or chemotherapy is controversial and will depend on the risk of tumor recurrence. CONCLUSION: Surgery represents the only potentially curative therapy for breast sarcoma. Tumor size and adequate resection margin are the most important prognostic factors. Approximately 80% of recurrences appear in the first two years.

Histopathology Findings of Corneal Buttons in Congenital Aniridia Patients.

PURPOSE: To evaluate the corneal button of primary penetrating keratoplasty of patients diagnosed with congenital aniridia. The study took place at the Instituto Universitario Barraquer and the Centro de Oftalmología Barraquer. METHODS: A retrospective analysis of cases diagnosed with congenital aniridia was carried out. We analyzed 13 corneal buttons of 11 eyes with congenital aniridia. We only included those patients who underwent penetrating keratoplasty for the first time. The corneal buttons were analyzed for histological characteristics of the presence of vascularization, the presence or not of Bowman's layer, the thickness of the stroma and Descemet's membrane, and endothelium layer alterations. RESULTS: We found alterations in the epithelium and stroma in all patients, although this loss of architecture was not seen in Descemet's membrane and the endothelial population. CONCLUSION: Patients with advanced congenital aniridic keratopathy may be good candidates for deep or superficial anterior lamellar keratoplasty for the preservation of normal endothelium and Descemet's membrane, along with limbal stem cell transplantation, to address epithelial and stromal pathology.

Endoresection technique with/without brachytherapy for management of high posterior choroidal melanoma: extended follow-up results.

PURPOSE: To evaluate the long-term efficacy and safety of endoresection for high posterior choroidal melanoma. METHODS: Retrospective nonrandomized interventional case series. Forty-one patients had endoresection as primary treatment for posterior choroidal melanoma. Of these, 21 patients had adjuvant brachytherapy. The inclusion criteria were tumor thickness ≥8 mm, base diameter <15 mm, and posterior tumors not extending anterior to the equator. Main outcomes measures were enucleation rate, visual outcome, surgical complications, local recurrence, metastasis, and mortality. RESULTS: Mean follow-up time was 102.5 months. Mean preoperative best-corrected visual acuity was 20/100. Mean tumor thickness was 9.8 mm (range, 7.7-13.5 mm; standard deviation, 1.7 mm), mean base diameter was 9.9 mm (range, 5-15 mm; standard deviation, 2.8 mm). At the latest visit, 36 patients (87.8%) still retained the eye. Mean postoperative best-corrected visual acuity was 20/1,625. Retinal detachment was the main postoperative complication (28.9%). At completion of follow-up, 12% of patients had phthisis bulbi, and 3 developed chronic hypotony. Five patients (12.2%) had local tumor recurrence; none of them had received brachytherapy as initial treatment. At 5 years of follow-up, 3 patients (7.3%) had liver metastasis. On Kaplan-Meier analysis at 10 years, all-cause mortality was 7.3% and specific mortality because of melanoma was 2.4%. CONCLUSION: Endoresection of high posterior melanomas was not associated with a higher risk of metastasis, death, or local recurrence than other reported techniques used to treat similar melanomas.

Endometrial stromal tumors: immunohistochemical and molecular analysis of potential targets of tyrosine kinase inhibitors.

BACKGROUND: The systemic treatment of malignant endometrial stromal tumors (EST) is not well established. A few reports describe objective responses to imatinib, which suggest a novel therapeutic strategy for these tumors. Due to these facts, we aimed to perform a retrospective analysis of possible molecular targets of tyrosine kinase inhibitors (TKI) in EST: KIT, PDGFRA and EGFR. METHODS: 52 endometrial stromal sarcomas and 13 undifferentiated endometrial sarcomas were examined and reviewed. Mutational analysis were performed for exons 9, 11, 13, and 17 of the KIT gene, exons 12 and 18 of the PDGFRA gene and exons 18, 19, 20 and 21 of the EGFR gene. The incidence and distribution of the KIT, PDGFRA, and EGFR expression were examined by immunohistochemistry, and EGFR amplification was assessed by fluorescence in situ hybridization. RESULTS: No mutations in KIT, PDGFRA and EGFR genes were detected. Overexpression of KIT, PDGFRA, EGFR, was detected in 2 (3%), 23 (35.4%), 7 (10.8%) cases respectively, whereas amplification of EGFR gene was not found. CONCLUSIONS: Absence of significant expression, amplification and activating mutations on these tyrosine kinase receptors suggest that it is unlikely that EST can benefit from therapies such as TKI on the systemic setting.