Ultrasound appearance and targeted therapy of symptomatic neural lesions in the breast and chest wall after breast cancer surgery.

AIM: To evaluate the feasibility of ultrasound (US) in identification of nerve lesions after breast cancer surgery in patients with neuropathic pain and assess the effect of a targeted US-guided therapy. MATERIAL AND METHODS: Patients with neuropathic pain after breast cancer surgery underwent US examination. Nerve lesions identified by US were treated by a US-guided application of a mixture of local anesthetics and corticoids. The patients reported pain relief on a 100-point scale (0% = no effect, 100% = complete relief) and its duration in the next 18 months. RESULTS: We performed 17 interventions in 11 women. A neuroma was observed in 2 patients, edema of the nerve in 5 patients, and scarring across the nerve in 4 patients. The affected nerves were the intercostobrachial nerve (5 patients), the long thoracic nerve (4), cutaneous branch of the pectoral nerve (1), and both the intercostobrachial and the long thoracic nerve (1). After 15 (88%) interventions, the patients reported relief (55±32%) with a median duration of 3 months (0.5-18 months). CONCLUSION: In patients after breast cancer surgery, ultrasound can reliably identify small painful neural lesions which can be efficiently treated by ultrasound-guided intervention.

Uptake and Effectiveness of Risk-Reducing Surgeries in Unaffected Female BRCA1 and BRCA2 Carriers: A Single Institution Experience in the Czech Republic.

Unnafected female carriers of BRCA1 and BRCA2 pathogenic/likely pathogenic variants (P/LPVs) are at higher risk of breast cancer (BC) and ovarian cancer (OC). In the retrospective single-institution study in the Czech Republic, we analyzed the rate, longitudinal trends, and effectiveness of prophylactic risk-reducing mastectomy (RRM) and risk-reducing salpingo-oophorectomy (RRSO) on the incidence of BC and OC in BRCA1/2 carriers diagnosed between years (y) 2000 to 2020. The study included 496 healthy female BRCA1/2 carriers. The median follow-up was 6.0 years. RRM was performed in 156 (31.5%, mean age 39.3 y, range 22-61 y) and RRSO in 234 (47.2%, mean age 43.2 y, range 28-64 y) BRCA1/2 carriers. A statistically significant increase of RRM (from 12% to 29%) and RRSO (from 31% to 42%) was observed when comparing periods 2005-2012 and 2013-2020 (p < 0.001). BC developed in 15.9% of BRCA1/2 carriers without RRM vs. 0.6% of BRCA1/2 carriers after RRM (HR 20.18, 95% CI 2.78- 146.02; p < 0.001). OC was diagnosed in 4.3% vs. 0% of BRCA1/2 carriers without vs. after RRSO (HR not defined due to 0% occurrence in the RRSO group, p < 0.001). Study results demonstrate a significant increase in the rate of prophylactic surgeries in BRCA1/2 healthy carriers after 2013 and the effectiveness of RRM and RRSO on the incidence of BC and OC in these populations.

Bilateral Prophylactic Nipple-Sparing Mastectomy: Analysis of the Risk-Reducing Effect in BRCA1/2 Mutation Carriers.

BACKGROUND: Mutations in the BRCA1 or BRCA2 genes increase the lifetime risk of developing breast cancer to 68-72% by the age of 80. One of the modalities to manage the risk is a prophylactic mastectomy. Bilateral nipple-sparing mastectomy specifically offers the most favorable esthetic outcomes but the evidence for its oncological safety remains limited. Thus, we aimed to compare the occurrence of breast cancer between nipple-sparing mastectomy and surveillance groups of BRCA1 or BRCA 2 mutations carriers. MATERIALS AND METHODS: BRCA1 or BRCA2-positive patients undergoing bilateral prophylactic nipple-sparing mastectomy at our department were identified. Only those unaffected by breast cancer were eligible. Each patient was pair-matched with a BRCA1 or BRCA2-positive patient of equal age from the surveillance group. Breast cancer incidence in both groups was recorded and the results were compared. RESULTS: None of 105 patients who underwent NSM between 2009 and 2019 at a single institution with a mean follow-up time of 50 months developed breast cancer over this time period. One patient in this group died of an unrelated cause. Nine patients from 105 in the match-paired surveillance group were diagnosed with breast cancer during a mean follow-up time of 58.3 months, however, none of them died. CONCLUSION: To the best of our knowledge, this is the largest single-center study of risk-reducing bilateral NSM in healthy BRCA1 or BRCA2 mutation carriers. Based on our results and those of other series, we conclude that NSM in its current form appears to be at least equally as safe as other types of mastectomy for preventing breast cancer in BRCA1 or BRCA2 mutation carriers. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Review of tumor infiltrating lymphocytes assessment in breast cancer in routine diagnostic practice.

Evaluation of tumor infiltrating lymphocytes (TIL) is gaining importance in many cancers not only because of their prognostic, but also predictive significance. One of the tumors in which the evaluation of TIL is of prognostic importance and has potential predictive impact on the modification of treatment procedures is breast cancer, especially its so-called triple negative, and HER2 positive variants.The aim of this review is to provide an overview of the issue of TIL evaluation in breast cancer, focusing not only on the clinical significance of this evaluation, but especially on the methodological aspects of evaluation and standardized reporting of the results.

Germline CHEK2 Gene Mutations in Hereditary Breast Cancer Predisposition - Mutation Types and their Biological and Clinical Relevance.

BACKGROUND: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. PATIENTS AND METHODS: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. RESULTS: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. CONCLUSION: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVČR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.

Estrogen Receptor Status Oppositely Modifies Breast Cancer Prognosis in BRCA1/BRCA2 Mutation Carriers Versus Non-Carriers.

Breast cancer (BC) prognosis in BRCA1 and BRCA2 mutation carriers has been reported contradictorily, and the significance of variables influencing prognosis in sporadic BC is not established in BC patients with hereditary BRCA1/BRCA2 mutations. In this retrospective cohort study, we analyzed the effect of clinicopathological characteristics on BC prognosis (disease-free survival [DFS] and disease-specific survival [DSS]) in hereditary BRCA1/BRCA2 mutation carriers. We enrolled 234 BRCA1/BRCA2 mutation carriers and 899 non-carriers, of whom 191 carriers and 680 non-carriers, with complete data, were available for survival analyses. We found that patients with ER-positive tumors developed disease recurrence 2.3-times more likely when they carried a BRCA1/BRCA2 mutation (23/60; 38.3% ER-positive carriers vs. 74/445; 16.6% ER-positive non-carriers; p < 0.001). ER-positive mutation carriers also had a 3.4-times higher risk of death due to BC compared with ER-positive non-carriers (13/60; 21.7% vs. 28/445; 6.3%; p < 0.001). Moreover, prognosis in ER-negative BRCA1/BRCA2 mutation carriers was comparable with that in ER-positive non-carriers. Our study demonstrates that ER-positivity worsens BC prognosis in BRCA1/BRCA2 mutation carriers, while prognosis for carriers with ER-negative tumors (including early-onset) is significantly better and comparable with that in ER-positive, older BC non-carriers. These observations indicate that BRCA1/BRCA2 mutation carriers with ER-positive BC represent high-risk patients.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

The PALB2 gene is a strong candidate for clinical testing in BRCA1- and BRCA2-negative hereditary breast cancer.

BACKGROUND: Several reports indicate that inherited mutations in the PALB2 gene predispose to breast cancer. However, there is little agreement about the clinical relevance and usefulness of mutation screening in this gene. We analyzed the prevalence and spectrum of germline mutations in PALB2 to estimate their contribution to hereditary breast and/or ovarian cancer in the Czech Republic. METHODS: The entire PALB2 coding region was sequenced in 409 breast/ovarian cancer patients negative for BRCA1 and BRCA2 mutations. Testing for large genomic rearrangements (LGR) was performed by multiplex ligation-dependent probe amplification (MLPA) analysis. RESULTS: We have identified 13 different pathogenic alterations including 10 truncating mutations and three LGRs in 16 of 409 patients (3.9%), whereas one truncating mutation was found in a group of 1,226 controls (0.08%; P = 2.6 × 10(-9)). Three novel LGRs included deletions involving exons 7-8 and 9-10, respectively, and a duplication spanning exons 9-11. Five frameshift and two nonsense mutations were novel, whereas three truncating mutations were described previously. The only recurrent mutation was the c.172_175delTTGT detected in four unrelated breast cancer individuals. CONCLUSIONS: Our analyses demonstrated the significant role of the PALB2 gene in breast cancer susceptibility. The highest frequency of PALB2 mutations (comparable with that previously reported for BRCA2) was found in a subgroup of patients with hereditary breast cancer (HBC) (13/235; 5.5%). IMPACT: Our results show that mutation analysis of the PALB2 gene, including the analysis of LGRs, is primarily indicated in patients with HBC in case of their BRCA1 and BRCA2 negativity.

[Oxidative stress after anthracycline therapy in patients with solid tumors]. / Oxidacní stres u nemocných po lécbe antracyklíny pro solidní nádorová onemocnení

BACKGROUND: Anthracyclines are regarded as some of the most potent oxidative stress inductors. Despite the fact that oxidative stress induction by anthracyclines is believed to be the key factor in anthracycline-related cardiotoxicity, the precise timeline of oxidative stress changes after anthracycline treatment remains unknown. The aim of the present study is to assess the level of oxidative stress after anthracycline therapy in patients with solid tumors. PATIENTS: The study population consists of 128 adult patients (14 males, 114 females, mean age 56 ± 10 years) receiving anthracycline chemotherapy for solid tumors. The control group consists of 38 patients (4 males, 34 females, mean age 59 ± 11 years) receiving anthracycline-free chemotherapy for solid tumors. METHODS: The main activities of antioxidant enzymes (catalase, glutathione peroxidase-1, superoxide dismutase, and paraoxonase-1) and concentrations of conjugated dienes, surrogate markers of oxidative stress level, were established at the baseline and after anthracycline therapy (median 45; IQR 27-69 days after the end of anthracycline therapy) in all patients. By comparing the activities of antioxidant enzymes and the concentrations of conjugated dienes, before and after therapy, changes in oxidative stress level within the time period were established for both study groups. Differences between the study groups, with regard to changes in the activities of antioxidant enzymes and the concentrations of conjugated dienes, were also evaluated. CONCLUSIONS: An increase in oxidative stress was observed after the end of anthracycline therapy in patients with solid tumors. However, our study shows that this persistent elevation of oxidative stress after the end of anthracycline therapy is probably not caused by anthracyclines.

Screening for genomic rearrangements in BRCA1 and BRCA2 genes in Czech high-risk breast/ovarian cancer patients: high proportion of population specific alterations in BRCA1 gene.

Large genomic rearrangements (LGR) represent substantial proportion of pathogenic mutations in the BRCA1 gene, whereas the frequency of rearrangements in the BRCA2 gene is low in many populations. We screened for LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification (MLPA) in 521 unrelated patients negative for BRCA1/2 point mutations selected from 655 Czech high-risk breast and/or ovarian cancer patients. Besides long range PCR, a chromosome 17-specific oligonucleotide-based array comparative genomic hybridization (aCGH) was used for accurate location of deletions. We identified 14 patients carrying 8 different LGRs in BRCA1 that accounted for 12.3% of all pathogenic BRCA1 mutations. No LGRs were detected in the BRCA2 gene. In a subgroup of 239 patients from high-risk families, we found 12 LGRs (5.0%), whereas two LGRs were revealed in a subgroup of 282 non-familial cancer cases (0.7%). Five LGRs (deletion of exons 1-17, 5-10, 13-19, 18-22 and 21-24) were novel; two LGRs (deletion of exons 5-14 and 21-22) belong to the already described Czech-specific mutations; one LGR (deletion of exons 1-2) was reported from several countries. The deletions of exons 1-17 and 5-14, identified each in four families, represented Czech founder mutations. The present study indicates that screening for LGRs in BRCA1 should include patients from breast or ovarian cancer families as well as high-risk patients with non-familial cancer, in particular cases with early-onset breast or ovarian cancer. On the contrary, our analyses do not support the need to screen for LGRs in the BRCA2 gene. Implementation of chromosome-specific aCGH could markedly facilitate the design of primers for amplification and sequence analysis of junction fragments, especially in deletions overlapping gene boundaries.

4-years results of weekly trastuzumab and paclitaxel in the treatment of women with HER2/neu overexpressing advanced breast cancer: single institution prospective study.

Background. - Trastuzumab is known as an active agent in HER2/neu overexpressing advanced breast cancer. In the prospective study we investigated efficacy, safety and toxicity of trastuzumab and paclitaxel in advanced breast cancer progressing on previous therapy. Patients and methods. - Seventeen patients with histologically confirmed disease were accrued. Inclusion criteria were as follows: Karnofsky performance status >/= 60 %, age < 75, pretreatment with at least two regimens; HER2/neu by immunohistochemistry 3+ or 2+, adequate organ function. Trastuzumab was given 4 mg/kg i.v. as a loading dose followed by 2mg/kg i.v. weekly. Paclitaxel was given 80 mg/m2 i.v. weekly. Both drugs were given until disease progression or unacceptable toxicity. We assessed the response rate (RR), the time to progression (TTP), the overall survival (OS) and toxicity. Results. RR in the intent to treat population was 59 % (10 out of 17), including 2 complete responses. In the median follow up of 4,3 years median TTP was 9 month and median OS 23 month. In total 710 cycles including 528 full dose cycles of trastuzumab and paclitaxel were administered. One patient developed hypersensitivity reaction after the first trastuzumab infusion and discontinued from study. Trastuzumab infusion related pyretic reaction was observed in 6 patients. Left ventricular ejection fraction decline occurred in 2 patients (grade 2 and grade 3). Five patients experienced grade 3 neuropathy. Hematological toxicity was very modest: 1 episode of grade 4 neutropenia and grade 3 anemia. Other grade 3/4 toxicity: 4 episodes of grade 3 infection without neutropenia, grade 3 elevation of liver function tests in 1 patient, 1 episode of grade 3 hyperglycemia, and 1 episode of grade 3 weight gain. Other grade 3 or 4 toxicity was not detected. Conclusion. - Trastuzumab and paclitaxel have shown activity and good tolerability in HER-2/neu overexpressing advanced breast cancer patients.

Assessment of in vitro drug resistance of human breast cancer cells subcultured from biopsy specimens.

UNLABELLED: The low cellular yield of a breast cancer sample represents a limiting factor for in vitro chemosensitivity/chemoresistance testing. The use of in vitro serially cultured cells can help overcome this obstacle. MATERIALS AND METHODS: In vitro drug resistance of cells cultured from mammary carcinomas by the 3T3 feeder-layer technique was tested by the MTT assay. Out of the 33 tested cultures, 9 were derived from cells obtained from true-cut biopsies of primary tumours, with sample volume less than 0.03 cm3. The cultures were treated with 6 anticancer drugs at 6-8 concentrations. The chemoresistance of cultured cells was monitored by the surviving cell fraction as a function of the drug concentration. RESULTS: The average time to obtain a result was 28 days. The volume of an original sample had no effect on the in vitro resistance of a culture, suggesting minimal alteration of in vitro chemosensitivity of cells by their cultivation. Histopathological grade, estrogen receptor status or expression of the c-erb-B2 protein of the original tumours did not significantly correlate with the resistance of cultures. Individual drugs displayed distinct in vitro effectiveness. Paclitaxel and cisplatin were the most potent drugs. Gemcitabine, vinorelbine and mafosfamide were the least potent drugs. Doxorubicin and gemcitabine most frequently failed to completely metabolically inhibit 100% of cultured cells at any concentration. CONCLUSION: Combination of the optimised feeder-layer cultivation technique and the MTT test permits extensive drug resistance testing from very small breast cancer samples.