Impact of Stain Normalization on Pathologist Assessment of Prostate Cancer: A Comparative Study.

In clinical routine, the quality of whole-slide images plays a key role in the pathologist's diagnosis, and suboptimal staining may be a limiting factor. The stain normalization process helps to solve this problem through the standardization of color appearance of a source image with respect to a target image with optimal chromatic features. The analysis is focused on the evaluation of the following parameters assessed by two experts on original and normalized slides: (i) perceived color quality, (ii) diagnosis for the patient, (iii) diagnostic confidence and (iv) time required for diagnosis. Results show a statistically significant increase in color quality in the normalized images for both experts (p < 0.0001). Regarding prostate cancer assessment, the average times for diagnosis are significantly lower for normalized images than original ones (first expert: 69.9 s vs. 77.9 s with p < 0.0001; second expert: 37.4 s vs. 52.7 s with p < 0.0001), and at the same time, a statistically significant increase in diagnostic confidence is proven. The improvement of poor-quality images and greater clarity of diagnostically important details in normalized slides demonstrate the potential of stain normalization in the routine practice of prostate cancer assessment.

Stain normalization in digital pathology: Clinical multi-center evaluation of image quality.

In digital pathology, the final appearance of digitized images is affected by several factors, resulting in stain color and intensity variation. Stain normalization is an innovative solution to overcome stain variability. However, the validation of color normalization tools has been assessed only from a quantitative perspective, through the computation of similarity metrics between the original and normalized images. To the best of our knowledge, no works investigate the impact of normalization on the pathologist's evaluation. The objective of this paper is to propose a multi-tissue (i.e., breast, colon, liver, lung, and prostate) and multi-center qualitative analysis of a stain normalization tool with the involvement of pathologists with different years of experience. Two qualitative studies were carried out for this purpose: (i) a first study focused on the analysis of the perceived image quality and absence of significant image artifacts after the normalization process; (ii) a second study focused on the clinical score of the normalized image with respect to the original one. The results of the first study prove the high quality of the normalized image with a low impact artifact generation, while the second study demonstrates the superiority of the normalized image with respect to the original one in clinical practice. The normalization process can help both to reduce variability due to tissue staining procedures and facilitate the pathologist in the histological examination. The experimental results obtained in this work are encouraging and can justify the use of a stain normalization tool in clinical routine.

Reoperation rate after breast conserving surgery as quality indicator in breast cancer treatment: A reappraisal.

AIM: To analyse the role of repeated breast surgery (RBS) after breast conserving surgery (BCS) as a quality indicator in a consecutive series of breast cancer patients. METHODS: Data from 1233 breast cancer patients submitted to BCS from 2015 to 2019 were reviewed. The influence of several variables on RBS rate (182/1232; 14.8%) was examined. Univariate and multivariate analyses were conducted to look for significant associations with the risk of RBS. RESULTS: Surgical workload, BCS rate and clinicopathological variables were consistent over the study period, while RBS rate decreased after the introduction of shaving of cavity margins (from 17.9% to 9.5%). Tumor persistence at RBS was higher for mastectomy vs. re-excision (87.3% vs. 37.8%; p = 0.05), inconclusive vs. positive diagnostic biopsy (48.2% vs. 69.4%; p = 0.003), ductal carcinoma in situ vs. invasive carcinoma (69.0% vs. 51.3%; p = 0.046) and lower after neoadjuvant therapy (14.3% vs. 57.8%; p = 0.044). Several clinicopathological variables were associated with the risk of RBS, but only multifocality [Odds Ratio (OR): 1.8; p = 0.009], microcalcifications (OR: 2.0, p = 0.000), neoadjuvant therapy (OR: 0.4; p = 0.014), pathological intraoperative assessment (OR: 0.6; p = 0.010) and shaving of cavity margins (OR: 0.3; p = 0.000) retained independent value at multivariate analysis. CONCLUSIONS: RBS rate can be reduced by shaving of cavity margins. Current standards for RBS should not be made more stringent due to the existence of non-actionable risk factors. The value of RBS as a quality indicator should be scrutinzed.

Activation of the MET receptor attenuates doxorubicin-induced cardiotoxicity in vivo and in vitro.

BACKGROUND AND PURPOSE: Doxorubicin anti-cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its effective use is compromised by low biodistribution. In this study, we have investigated whether the activation of the HGF receptor-encoded by the Met gene-by an agonist monoclonal antibody (mAb) could protect against doxorubicin-induced cardiotoxicity. EXPERIMENTAL APPROACH: The mAb (5 mg·kg-1 ) was injected in vivo into C57BL/6J mice, before doxorubicin (three doses of 7 mg·kg-1 ). Cardiac functions were evaluated through MRI after treatment termination. Heart histological staining and mRNA levels of genes associated with heart failure (Acta1 and Nppa), inflammation (IL-6), and fibrosis (Ctgf, Col1a2, Timp1, and Mmp9) were assessed. MAb (100 nM) was administered in vitro to H9c2 cardiomyoblasts before addition of doxorubicin (25 µM). DDR and apoptosis markers were evaluated by quantitative western blotting, flow cytometry, and immunofluorescence. Stattic was used for pharmacological inactivation of STAT3. KEY RESULTS: In vivo, administration of the mAb alleviated doxorubicin-induced cardiac dysfunction and fibrosis. In vitro, mAb mimicked the response to HGF by (a) inhibiting histone H2AX phosphorylation at S139, (b) quenching the expression of the DNA repair enzyme PARP1, and (c) reducing the proteolytic activation of caspase 3. The MET-driven cardioprotection involved, at least in vitro, the phosphorylation of STAT3. CONCLUSION AND IMPLICATIONS: The MET agonist mAb provides a new tool for cardioprotection against anthracycline cardiotoxicity.

Cold Formalin Fixation Guarantees DNA Integrity in Formalin Fixed Paraffin Embedded Tissues: Premises for a Better Quality of Diagnostic and Experimental Pathology With a Specific Impact on Breast Cancer.

Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (stdFFPE) and at 4°C (coldFFPE), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study, n = 21), cohort B (collected at time of the study, n = 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples (mean values: 0.69 vs. 0.36, p < 0.0001) and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.

Open partial horizontal laryngectomy using CO2 fiber laser.

BACKGROUND: The application of CO2 fiber laser technology to ENT surgery has led to new interesting scenarios, both in endoscopic and in open surgical approaches. METHODS: The current video shows three examples of open partial horizontal laryngectomies (OPHLs) performed using CO2 fiber laser for resection procedures. RESULTS: CO2 fiber laser helped the surgeon in improving the accuracy of resection and the quality of surgical margins on specimen. The low thermal damage on tissues resulted in minor postoperative edema and a fast recovery of laryngeal function. CONCLUSIONS: In our experience, the application of CO2 fiber laser showed some very useful features for performing OPHLs: a high cutting precision with very low tissue damage, the possibility of delivering energy without touching the organ, a modulable power for the various surgical steps, a very good maneuverability of the fine fiber holder during the procedure allowing the surgeon to "draw" the resection with a great accuracy.

Arytenoid Fixation in Laryngeal Cancer: Radiological Pictures and Clinical Correlations with Respect to Conservative Treatments.

Background: The aim of this retrospective study was to identify different radiological features in intermediate⁻advanced laryngeal cancer (LC) associated with arytenoid fixation, in order to differentiate cases still safely amenable to conservative treatment by partial laryngectomy or chemoradiotherapy. Methods: 29 consecutive patients who underwent open partial horizontal laryngectomies (OPHLs), induction chemotherapy followed by radiotherapy in the case of >50% response (IC + RT) or total laryngectomy were classified as: pattern I (supraglottic LC fixing the arytenoid due to weight effect), pattern II (glottic LC involving the posterior paraglottic space and spreading toward the crico-arytenoid joint and infraglottic extension <10 mm), pattern III (glottic-infraglottic LC involving the crico-arytenoid joint and infraglottic extension >10 mm) and pattern IV (transglottic and infraglottic LC with massive crico-arytenoid unit involvement, reaching the hypopharyngeal submucosa). All glottic cancers treated with surgery were studied by a cross sectional approach. Results: A substantial agreement between the work-up and the pathology results has been obtained in each of the subcategories. Three-year disease-free survivals, local control and freedom from laryngectomy were significantly better in pattern II compared to pattern III⁻IV. Conclusions: LC showing fixed arytenoid due to weight effect or posterior paraglottic space involvement with infraglottic extension <10 mm assessed at the true vocal cord midline are still safely manageable by OPHL or IC + RT.

FOXA1 and AR in invasive breast cancer: new findings on their co-expression and impact on prognosis in ER-positive patients.

BACKGROUND: The role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive (ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status. METHODS: Immunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis. RESULTS: Expression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P < 0.001) and ER mRNA (r = 0.7326; P < 0.001). In ER+ BC, FOXA1 was associated with a good prognosis independently of AR expression in the three subgroups analyzed (FOXA1+/AR+; FOXA1+/AR-; FOXA1-/AR-). Multivariate analyses confirmed that FOXA1 may provide more information than AR in Disease-Free Interval (DFI) of ER+ BC patients. CONCLUSION: Our results suggest that in BC the expression of FOXA1 is directly related to the expression of AR. Despite that, FOXA1 is found as superior predicting marker of recurrences compared to AR in ER+ BC patients.

Is Regression after Neoadjuvant Chemotherapy for Locally Advanced Breast Cancer Different in Sentinel and Non-sentinel Nodes?

Tumor draining sentinel lymph nodes (SLNs) are the sites of selective changes as compared to non-SLNs. They show features of tumor-reactive lymphadenopathy, including increased total number of functional blood vessels, but a relative immunosuppressed status has also been described in them. We explored the hypothesis of a selective regression or non-regression in SLNs versus non-SLNs in 142 patients with 110 estrogen receptor-positive and 32 estrogen receptor-negative tumors undergoing both SLN biopsy and axillary lymph node dissection after neoadjuvant therapy by assessing the tumoral (metastatic) and regression statuses of SLNs and non-SLNs separately. Of the 89 cases with signs of nodal regression, 22 cases (25%) were in favor of a selective non-regression in SLNs, 18 cases (20%) were supportive of a selective and more pronounced regression in the SLNs and the remaining showed equal degrees of regression or non-regression in SLNs and non-SLNs. The results indicate that there is no obvious difference in the degree of regressive histological changes shown by SLNs and NSLNs. Therefore, this phenomenon may not be a major contributor to the higher false negative rate of SLN biopsy after neoadjuvant treatment.

The impact of malignant nipple discharge cytology (NDc) in surgical management of breast cancer patients.

BACKGROUND: The role of nipple discharge cytology (NDc) in the surgical management of breast cancer patients is unclear. We aimed: (i) to evaluate the effect of malignant NDc on the surgical approach to the nipple-areola complex, and (ii) to verify the association between malignant NDc and nipple malignancy. METHODS: We retrospectively analyzed a case series of 139 patients with NDc who underwent breast surgery. The clinical and histological findings, types of surgery with emphasis on nipple-areola complex amputation, immunohistochemical phenotypes of the carcinomas and measurements of the tumor-nipple distance were recorded. Additionally, in patients who showed HER2-positive lesions on definitive surgery, we evaluated the HER2 immunocytochemistry of the NDc smears. RESULTS: Thirty-two malignant and 107 benign/borderline NDc diagnoses were identified. All 32 malignant-NDc cases were histologically confirmed as malignant. Thirty borderline/benign-NDc cases were histologically diagnosed as malignant (sensitivity 58%). The majority of the patients with malignant NDc were treated with nipple-areola complex amputations in both the mastectomy and conservative surgery groups (P<0.001, χ251.77). Nipple involvement was strongly associated with HER2-positive ductal carcinoma in-situ (P<0.001, χ211.98). HER2 immunocytochemistry on the NDc revealed a 100% correlation with the immunocytochemistry performed on the surgical tissues. CONCLUSIONS: Malignant NDc influenced surgical management. The association of malignant NDc with nipple involvement is highly related to ductal carcinoma in-situ with HER2 overexpression. In case of HER2 positive NDc, nipple-areola complex involvement is more likely than in HER2 negative cases.

Primary tumor location predicts the site of local relapse after nipple-areola complex (NAC) sparing mastectomy.

PURPOSE: To assess the oncological safety of nipple-areola complex (NAC) sparing mastectomy in breast cancer patients. METHODS: From 2010 to 2015, 518 breast cancer patients were submitted to NAC sparing mastectomy. Breast MRI and intraoperative assessment of the subareolar (SD) and proximal (ND) nipple ducts were performed to predict NAC involvement. Significant associations between pre- and postoperative variables with SD/ND involvement and with the risk of local recurrence were retrospectively investigated. RESULTS: SD/ND were involved in 26.1% of the cases. Final pathology of SD/ND was predicted by tumor-NAC distance at MRI and intraoperative pathology with 75 and 93% accuracy, respectively. NAC involvement was more frequent in case of positive ND than positive SD (68.3 vs. 38.3%; p = 0.003). Fourteen (2.7%) local relapses developed over a mean follow-up of 33 months. Ki-67 ≥25% (p = 0.002) and high tumor grade (p = 0.027) correlated with local recurrence. Most relapses developed in the subcutaneous tissue of the quadrant where the primary tumor was located (12/14; 85.7%). No local relapses occurred in patients who received post-mastectomy radiotherapy as compared to patients who did not, although they had a higher rate of positive surgical margins (40.5 vs. 16.2%; p = 0.000). CONCLUSIONS: NAC involvement can be predicted by MRI and intraoperative pathology of ND/SD. Local recurrences after NAC sparing mastectomy almost invariably develop in the same quadrant where the primary tumor was located and in highly proliferative tumors.

Predictive Diagnostic Pathology in the Target Therapy Era in Breast Cancer.

Treatment strategies in oncology are nowadays largely based on the "target therapy model", which allows to personalize the cure of each patient depending on distinctive host and disease features. As a general concept "targeted drugs" are effective only when the tumor exhibits the "target", which in breast cancer pathology may correspond to the expression of estrogen receptors and/or of HER2. These biomarkers are evaluated on breast cancer tissues by companion diagnostic tests, however, evidence suggests that the first step in breast cancer predictive pathology is still represented by morphology. For instance, histological types, such as tubular and cribriform carcinomas, define patients who may not need any treatments other than surgical excision. Neoadjuvant studies have shown that patients affected by lobular carcinomas are not likely to have any beneficial effects from chemotherapy. The second step in prediction is represented by immunophenotyping. If the immunohistochemical evaluation of four markers (estrogen and progesterone receptors, HER2 and Ki67) remains the best practice for breast cancer predictive pathology, molecular pathology has certainly reshaped the way we approach breast cancer diagnosis. The aim of this review is to discuss current knowledge in predictive pathology for the management of breast cancer patients, focusing on the benefits and drawbacks of traditional tools and of novel improvements of molecular biology.

Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study.

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco's Modified Eagle's Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

BACKGROUND: The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS: A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS: HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION: The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Gastric Cancer After Restrictive Bariatric Surgery: A Clinical Pitfall.

Although vertical banded gastroplasty is rarely performed at present, most bariatric surgery departments continue to follow up patients who underwent this procedure in the past few decades. In view of this, it is advisable for bariatric and general surgeons to know how to diagnose the very rare event of the development of a gastric cancer after this restrictive procedure. In this report, 2 cases of gastric cancer occurring years after vertical banded gastroplasty are presented, and clinical presentation and diagnostic difficulties are discussed.

High-throughput molecular analysis from leftover of fine needle aspiration cytology of mammographically detected breast cancer.

We investigated whether residual material from diagnostic smears of fine needle aspirations (FNAs) of mammographically detected breast lesions can be successfully used to extract RNA for reliable gene expression analysis. Twenty-eight patients underwent FNA of breast lesions under ultrasonographic guidance. After smearing slides for cytology, residual cells were rinsed with TRIzol to recover RNA. RNA yield ranged from 0.78 to 88.40 µg per sample. FNA leftovers from 23 nonpalpable breast cancers were selected for gene expression profiling using oligonucleotide microarrays. Clusters generated by global expression profiles partitioned samples in well-distinguished subgroups that overlapped with clusters obtained using "biologic scores" (cytohistologic variables) and differed from clusters based on "technical scores" (RNA/complementary RNA/microarray quality). Microarray profiling used to measure the grade of differentiation and estrogen receptor and ERBB2/HER2 status reflected the results obtained by histology and immunohistochemistry. Given that proliferative status in the FNA material is not always assessable, we designed and performed on FNA leftover a multiprobe genomic signature for proliferation genes that strongly correlated with the Ki67 index examined on histologic material. These findings show that cells residual to cytologic smears of FNA are suitable for obtaining high-quality RNA for high-throughput analysis even when taken from small nonpalpable breast lesions.

Reliability of whole sentinel lymph node analysis by one-step nucleic acid amplification for intraoperative diagnosis of breast cancer metastases.

OBJECTIVE: To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. BACKGROUND: Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. METHODS: Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. RESULTS: Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. CONCLUSIONS: Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.