Purpose: PREDIX HER2 is a randomized Phase II trial that compared neoadjuvant docetaxel, trastuzumab, and pertuzumab (THP) with trastuzumab emtansine (T-DM1) for HER2-positive breast cancer. Rates of pathologic complete response (pCR) did not differ between the two groups. Here, we present the survival outcomes from PREDIX HER2 and investigate metabolic response and tumor-infiltrating lymphocytes (TIL) as prognostic factors. Patients and Methods: In total, 202 patients with HER2-positive breast cancer were enrolled and 197 patients received six cycles of either THP or T-DM1. Secondary endpoints included event-free survival (EFS), recurrence-free survival (RFS), and overall survival (OS). Assessment with PET/CT was performed at baseline, after two and six treatment cycles. TILs were assessed manually at baseline biopsies, while image-based evaluation of TILs [digital TILs (DTIL)] was performed in digitized full-face sections. Results: After a median follow-up of 5.21 years, there was no difference between the two treatment groups in terms of EFS [HR = 1.26; 95% confidence interval (CI), 0.54–2.91], RFS (HR = 0.69; 95% CI, 0.24–1.93), or OS (HR = 0.52; 95% CI, 0.09–2.82). Higher SUVmax at cycle 2 (C2) predicted lower pCR (ORadj = 0.65; 95% CI, 0.48–0.87; P = 0.005) and worse EFS (HRadj = 1.27; 95% CI, 1.12–1.41; P < 0.001). Baseline TILs and DTILs provided additional prognostic information to clinical parameters and C2 SUVmax. Conclusions: Long-term outcomes following neoadjuvant T-DM1 were similar to neoadjuvant THP. SUVmax after two cycles of neoadjuvant therapy for HER2-positive breast cancer may be an independent predictor of both short- and long-term outcomes. Combined assessment with TILs may facilitate early selection of poor responders for alternative treatment strategies.

Ki67 is an important biomarker with prognostic and potential predictive value in breast cancer. However, the lack of standardization hinders its clinical applicability. In this study, we aimed to investigate the reproducibility among pathologists following the guidelines of the International Ki67 in Breast Cancer Working Group (IKWG) for Ki67 scoring and to evaluate the prognostic potential of this platform in an independent cohort. Four algorithms were independently built by four pathologists based on our study cohort using an open-source digital image analysis (DIA) platform (QuPath) following the detailed guideline of the IKWG. The algorithms were applied on an ER+ breast cancer study cohort of 157 patients with 15 years of follow-up. The reference Ki67 score was obtained by a DIA algorithm trained on a subset of the study cohort. Intraclass correlation coefficient (ICC) was used to measure reproducibility. High interobserver reliability was reached with an ICC of 0.938 (CI: 0.920–0.952) among the algorithms and the reference standard. Comparing each machine-read score against relapse-free survival, the hazard ratios were similar (2.593–4.165) and showed independent prognostic potential (p ≤ 0.018, for all comparisons). In conclusion, we demonstrate high reproducibility and independent prognostic potential using the IKWG DIA instructions to score Ki67 in breast cancer. A prospective study is needed to assess the clinical utility of the IKWG DIA Ki67 instructions.

Background: Pseudoangiomatous stromal hyperplasia (PASH) is a benign lesion of myofibroblasts that is composed of a network of slit-like channels that resemble vascular spaces. The aims of this study were to document the frequency of PASH in core needle biopsy specimens (CNBS) of the breast, to describe which histopathologic findings coexist with PASH and to examine any endothelial cell differentiation. Materials and Methods: We reevaluated hematoxylin and eosin–stained sections of all CNBS that were obtained during a 1-year period. First, we performed CD34 and CD31 immunostainings to highlight the areas of PASH, then performed D2-40/podoplanin (lymphatic endothelial marker) and Fli-1 (vascular endothelial cell marker) immunostains. Results: The total number of CNBS was 412. Areas of PASH were noted in 37 of the 412 cases (9%), with a mean age of 38.5 years. The lesions that were described in association with PASH were “benign breast parenchyma with stromal fibrosis” (17/37; 46%), “fibroepithelial tumors” (17/37; 46%), “columnar cell changes (CCC)” (2/37; 5%), and “invasive carcinoma” (1/37; 3%). There were 2 cases of CCC within the foci of PASH (direct contact with PASH), and 8 additional cases of CCC that coexisted in the same specimen but were not in direct contact. There was no staining for D2-40 or Fli-1 within PASH foci. Conclusion: PASH lesions occurred with a frequency of 9% in CNBS and were mostly in association with benign breast lesions in premenopausal women. CCC was determined as an accompanying epithelial lesion within or near PASH areas. No obvious immunopositivity compatible with endothelial cell differentiation was revealed.

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

Breast cancer (BC) represents a heterogeneous ecosystem and elucidation of tumor microenvironment components remains essential. Our study aimed to depict the composition and prognostic correlates of immune infiltrate in early BC, at a multiplex and spatial resolution. Pretreatment tumor biopsies from patients enrolled in the EORTC 10994/BIG 1-00 randomized phase III neoadjuvant trial (NCT00017095) were used; the CNN11 classifier for H&E-based digital TILs (dTILs) quantification and multiplex immunofluorescence were applied, coupled with machine learning (ML)-based spatial features. dTILs were higher in the triple-negative (TN) subtype, and associated with pathological complete response (pCR) in the whole cohort. Total CD4+ and intra-tumoral CD8+ T-cells expression was associated with pCR. Higher immune-tumor cell colocalization was observed in TN tumors of patients achieving pCR. Immune cell subsets were enriched in TP53-mutated tumors. Our results indicate the feasibility of ML-based algorithms for immune infiltrate characterization and the prognostic implications of its abundance and tumor-host interactions.

Prevalences of diverticulum and low-grade mucinous neoplasm (LGMN) were reported as 0.04-2% and less than 1% in the appendix. In this study, the frequency of diverticulum in LGMN cases, the relationship between diverticula and periappendicular mucin, and the possible role of diverticula in pseudomyxoma peritonei pathogenesis were researched. Through systematic review and targeted search, 38 LGMN and 96 diverticula were identified, frequencies and relationship between diverticulum and LGMN were analysed. Diverticulum and LGMN were determined in 4.8% and 1.04%, respectively, of 1922 appendectomy materials specifically grossed by the same pathologist. The prevalence of diverticulum was higher in our study than literature. The difference may be due to detailed macroscopic examination. Diverticulum was detected in 60% of LGMN cases. The rate of diverticulum was found to be significantly higher in LGMNs than non-neoplastic diverticulum (p < 0.001). Periappendicular mucin deposition was significantly more frequent in LGMN cases with diverticulum than in other groups (p < 0.05). Follow-ups were available in 18 patients; none of them had mucin deposition in the peritoneal cavity. We detected that periappendicular mucin was highly associated with diverticula in LGMN cases. Periappendicular acellular mucin deposition may not give rise to pseudomyxoma peritonei. We may think that mucin could move out of the appendix through the diverticulum rather than neoplastic spread in some of these cases.