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Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p= 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Tumor budding and CD8-positive (+) T-cells are recognized as prognostic factors in colorectal adenocarcinoma. We assessed CD8+ T-cell density and intratumoral budding in pretreatment rectal cancer biopsies to determine if they are predictive biomarkers for response to neoadjuvant therapy and survival. Pretreatment biopsies of locally advanced rectal adenocarcinoma from 117 patients were evaluated for CD8+ T-cell density using automated quantitative digital image analysis and for intratumoral budding and correlated with clinicopathological variables on postneoadjuvant surgical resection specimens, response to neoadjuvant therapy, and survival. Patients with high CD8+ T-cell density (=157 per?mm2) on biopsy were significantly more likely to exhibit complete/near complete response to neoadjuvant therapy (66% vs. 33%, p?=?0.001) and low tumor stage (0 or I) on resection (62% vs. 30%, p?=?0.001) compared with patients with low CD8+ T-cell density. High CD8+ T-cell density was an independent predictor of response to neoadjuvant therapy with a 2.63 higher likelihood of complete response (95% CI 1.04–6.65, p?=?0.04) and a 3.66 higher likelihood of complete/near complete response (95% CI 1.60–8.38, p?=?0.002). The presence of intratumoral budding on biopsy was significantly associated with a reduced likelihood of achieving complete/near complete response to neoadjuvant therapy (odds ratio 0.36, 95% CI 0.13–0.97, p?=?0.048). Patients with intratumoral budding on biopsy had a significantly reduced disease-free survival compared with patients without intratumoral budding (5-year survival 39% vs 87%, p?

Tumor budding and CD8-positive (+) T-cells are recognized as prognostic factors in colorectal adenocarcinoma. We assessed CD8+ T-cell density and intratumoral budding in pretreatment rectal cancer biopsies to determine if they are predictive biomarkers for response to neoadjuvant therapy and survival. Pretreatment biopsies of locally advanced rectal adenocarcinoma from 117 patients were evaluated for CD8+ T-cell density using automated quantitative digital image analysis and for intratumoral budding and correlated with clinicopathological variables on postneoadjuvant surgical resection specimens, response to neoadjuvant therapy, and survival. Patients with high CD8+ T-cell density (≥157 per mm2) on biopsy were significantly more likely to exhibit complete/near complete response to neoadjuvant therapy (66% vs. 33%, p= 0.001) and low tumor stage (0 or I) on resection (62% vs. 30%, p= 0.001) compared with patients with low CD8+ T-cell density. High CD8+ T-cell density was an independent predictor of response to neoadjuvant therapy with a 2.63 higher likelihood of complete response (95% CI 1.04–6.65, p= 0.04) and a 3.66 higher likelihood of complete/near complete response (95% CI 1.60–8.38, p= 0.002). The presence of intratumoral budding on biopsy was significantly associated with a reduced likelihood of achieving complete/near complete response to neoadjuvant therapy (odds ratio 0.36, 95% CI 0.13–0.97, p= 0.048). Patients with intratumoral budding on biopsy had a significantly reduced disease-free survival compared with patients without intratumoral budding (5-year survival 39% vs 87%, p< 0.001). In the multivariable model, the presence of intratumoral budding on biopsy was associated with a 3.35-fold increased risk of tumor recurrence (95% CI 1.25–8.99, p= 0.02). In conclusion, CD8+ T-cell density and intratumoral budding in pretreatment biopsies of rectal adenocarcinoma are independent predictive biomarkers of response to neoadjuvant therapy and intratumoral budding associates with patient survival. These biomarkers may be helpful in selecting patients who will respond to neoadjuvant therapy and identifying patients at risk for recurrence.

Digital pathology has made great strides in recent years culminating with the approval to market devices from the Food and Drug Administration. The pathology community is now poised to begin using these systems for diagnostic purposes. This article will discuss the preparatory steps needed to implement digital pathology as well as some implementation styles that may be sufficient for a pathology department.

Supplemental Digital Content is available in the text.Despite their association with DNA mismatch repair (MMR) protein deficiency, colonic adenocarcinomas with mucinous, signet ring cell, or medullary differentiation have not been associated with improved survival compared with conventional adenocarcinomas in most studies. Recent studies indicate that increased T-cell infiltration in the tumor microenvironment has a favorable prognostic effect in colonic adenocarcinoma. However, the prognostic effect of tumor-associated T cells has not been evaluated in histologic subtypes of colonic adenocarcinoma. We evaluated CD8-positive T-cell density in 259 patients with colonic adenocarcinoma, including 113 patients with tumors demonstrating mucinous, signet ring cell, or medullary differentiation, using a validated automated quantitative digital image analysis platform and correlated CD8-positive T-cell density with histopathologic variables, MMR status, molecular alterations, and survival. CD8-positive T-cell densities were significantly higher for MMR protein-deficient tumors (P<0.001), BRAFV600E mutant tumors (P=0.004), and tumors with medullary differentiation (P<0.001) but did not correlate with mucinous or signet ring cell histology (P>0.05 for both). In the multivariable model of factors predicting disease-free survival, increased CD8-positive T-cell density was associated with improved survival both in the entire cohort (hazard ratio=0.34, 95% confidence interval, 0.15-0.75, P=0.008) and in an analysis of patients with tumors with mucinous, signet ring cell, or medullary differentiation (hazard ratio=0.06, 95% confidence interval, 0.01-0.54, P=0.01). The prognostic effect of CD8-positive T-cell density was independent of tumor stage, MMR status, KRASmutation, and BRAFmutation. Venous invasion was the only other variable independently associated with survival in both the entire cohort and in patients with tumors with mucinous, signet ring cell, or medullary differentiation. In summary, our results indicate that the prognostic value of MMR protein deficiency is most likely attributed to increased tumor-associated CD8-positive T cells and that automated quantitative CD8 T-cell analysis is a better biomarker of patient survival, particularly in patients with tumors demonstrating mucinous, signet ring cell, or medullary differentiation.

Supplemental Digital Content is available in the text.SATB2 is a sensitive immunohistochemistry marker of colorectal carcinoma and non-neoplastic colorectal epithelium that is complementary to CDX2. However, its expression is affected by molecular alterations. Inflammatory bowel disease–associated neoplasia demonstrates molecular alterations that are different from those in sporadic colorectal neoplasia. Given these differences, we examined SATB2 expression in 73 cases of inflammatory bowel disease–associated neoplasia including 37 dysplasia cases and 36 carcinomas and compared the expression patterns with 50 cases of nondysplastic colorectal mucosa in patients with active inflammatory bowel disease, 40 sporadic colonic polyps (20 conventional adenomas and 20 sessile serrated lesions/polyps), and 343 sporadic colorectal adenocarcinomas to assess SATB2 immunohistochemistry as a biomarker of inflammatory bowel disease–associated neoplasia. Loss of SATB2 expression was only identified in colorectal dysplasia arising in inflammatory bowel disease (15/37, 41%) and was not seen in nondysplastic colorectal mucosa with active inflammatory bowel disease or sporadic colonic polyps (P<0.001). Loss of SATB2 expression was identified in both endoscopically visible dysplasia (11/28, 39%) and invisible (4/9, 44%) dysplasia. Loss of SATB2 expression was identified in 67% (24/36) of inflammatory bowel disease–associated carcinomas and was significantly more frequent compared with sporadic colorectal carcinomas (47/343, 14%, P<0.001). There was no difference in positive CDX2 expression between inflammatory bowel disease–associated colorectal carcinoma and sporadic colorectal carcinoma (89% vs. 85%, P=1.0). In conclusion, loss of SATB2 expression is common in inflammatory bowel disease–associated colorectal dysplasia and adenocarcinoma and may be a helpful ancillary biomarker when evaluating for inflammatory bowel disease–associated dysplasia.

Supplemental digital content is available in the text.

Objectives:Anastomotic reconstruction following intestinal resection in Crohn’s disease (CD) may employ side-to-side anastomosis (STSA; anti-peristaltic orientation) or end-to-end anastomosis (ETEA). Our aim was to determine the impact of these two anastomotic techniques on long-term clinical status in postoperative CD patients.Methods:We performed a comparative effectiveness study of prospectively collected observational data from consented CD patients undergoing their first or second ileocolonic bowel resection and re-anastomosis between 2008 and 2012, in order to assess the association between anastomosis type and 2-year postoperative quality of life (QoL), healthcare utilization, disease clinical or endoscopic recurrence, use of medications, and need for repeat resection.Results:One hundred and twenty eight postoperative CD patients (60 STSA and 68 ETEA) were evaluated. At 2 years postoperatively, STSA patients had higher rates of emergency department visits (33.3% vs. 14.7%; P=0.01), hospitalizations (30% vs. 11.8%; P=0.01), and abdominal computed tomography scans (50% vs. 13.2%; P<0.001) with lower QoL (mean short inflammatory bowel disease questionnaire 47.9 vs. 53.4; P=0.007). There was no difference among the two groups in the 30 day surgical complications and 2-year patterns of disease activity, CD medication requirement, endoscopic recurrence, and need for new surgical management (all P > 0.05).Conclusions:At 2 years postoperatively, CD patients with ETEA demonstrated better QoL and less healthcare utilization compared with STSA, despite having similar patterns of disease recurrence and CD treatment. These findings suggest that surgical reconstruction of the bowel as an intact tube (ETEA) contribute to improved functional and clinical status in patients with CD.

OBJECTIVES:Peripheral blood eosinophilia (PBE) in inflammatory bowel disease (IBD) is associated with ulcerative colitis (UC) and active disease. Little data exist on the long-term impact of PBE on disease course. We aimed to investigate the multi-year patterns of PBE and its impact on disease severity in a large IBD cohort.METHODS:We performed a registry analysis of a consented, prospective, natural history IBD cohort at a tertiary center from 2009 to 2014. Demographics, comorbidities, disease activity, healthcare utilization, and time to hospitalization or surgical resection of patients who displayed PBE were compared to patients without PBE.RESULTS:Of the 2,066 IBD patients, 19.2% developed PBE. PBE was significantly associated with UC (P<0.001), extensive colitis (P<0.001), and shorter disease duration (P=0.03). Over six years, PBE patients had more active disease (Harvey–Bradshaw Index P=0.001; ulcerative colitis activity index P<0.001), concurrent C-reactive protein elevation (P<0.001), healthcare utilization (hospitalization P<0.001, IBD surgery P<0.001), and more aggressive medical therapy (prednisone P<0.001, anti-TNF P<0.001). Patients with PBE had a significantly reduced time to hospitalization in both UC (P<0.001) and Crohn’s disease (CD) (P<0.001) and reduced time to colectomy in UC (P=0.003). On multivariable modeling, PBE remained significantly associated with hospitalization and surgery in both CD and UC. New diagnosis of UC with PBE was associated with increased steroid (P=0.007) and anti-TNF (P=0.001) requirement.CONCLUSION:This multi-year study of a large IBD cohort suggests that peripheral blood eosinophilia represents a biomarker of a distinct IBD subgroup, with a unique inflammatory signature, and at risk for worse clinical outcomes.

Artificial intelligence/machine learning tools are being created for use in pathology. Some examples related to lung pathology include acid-fast stain evaluation, programmed death ligand-1 (PDL-1) interpretation, evaluating histologic patterns of non–small-cell lung carcinoma, evaluating histologic features in mesothelioma associated with adverse outcomes, predicting response to anti-PDL-1 therapy from hematoxylin and eosin–stained slides, evaluation of tumor microenvironment, evaluating patterns of interstitial lung disease, nondestructive methods for tissue evaluation, and others. There are still some frameworks (regulatory, workflow, and payment) that need to be established for these tools to be integrated into pathology.

Morphologic and molecular data for staging of multifocal lung squamous cell carcinomas (LSCC) are limited. In this study, whole exome sequencing (WES) was used as the gold standard to determine if multifocal LSCC represented separate primary cancers (SPLC) or intrapulmonary metastases (IPM). Genomic profiles were compared to the comprehensive morphologic assessment.

Recently, several technological advances have been introduced to mobile phones leading some people to refer to them as “smartphones.” These changes have led to widespread consumer adoption. A similar adoption has occurred within the medical field and this revolution is changing the practice of medicine, including pathology. Several mobile applications have been published for dermatology, orthopedics, ophthalmology, neurosurgery, and clinical pathology. The applications are wide ranging, including mobile technology to increase patient engagement, self-monitoring by patients, clinical algorithm calculation, facilitation between experts to resource-poor environments. These advances have been received with mixed reviews. For anatomic pathology, mobile technology applications can be broken into 4 broad categories: (a) educational uses, (b) microscope with mobile phone, (c) mobile phone as microscope/acquisition device, and (d) miscellaneous. Using a mobile phone as an acquisition device paired with a microscope seems to be the most interesting current application because of the need for expert consultation with resource-poor environments. However, several emerging uses for mobile technology may become more prominent as the technology matures including image analysis, alternative light sources, and increased opportunities for clinician and patient engagement. The flexibility represented by mobile technology represents a burgeoning field in pathology informatics.

Optimizing pathologist workflow can be difficult because it is affected by many variables. Surgical pathologists must complete many tasks that culminate in a final pathology report. Several software systems can be used to enhance/improve pathologist workflow. These include voice recognition software, pre–sign-out quality assurance, image utilization, and computerized provider order entry. Recent changes in the diagnostic coding and the more prominent role of centralized electronic health records represent potential areas for increased ways to enhance/improve the workflow for surgical pathologists. Additional unforeseen changes to the pathologist workflow may accompany the introduction of whole-slide imaging technology to the routine diagnostic work.

Recent advances in hardware and computing power contained within mobile devices have made it possible to use these devices to improve and enhance pathologist workflow. This article discusses the possible uses ranging from basic functions to intermediate functions to advanced functions. Barriers to implementation are also discussed.

Optimizing pathologist workflow can be difficult because it is affected by many variables. Surgical pathologists must complete many tasks that culminate in a final pathology report. Several software systems can be used to enhance/improve pathologist workflow. These include voice recognition software, pre–sign-out quality assurance, image utilization, and computerized provider order entry. Recent changes in the diagnostic coding and the more prominent role of centralized electronic health records represent potential areas for increased ways to enhance/improve the workflow for surgical pathologists. Additional unforeseen changes to the pathologist workflow may accompany the introduction of whole-slide imaging technology to the routine diagnostic work.

The underlying molecular alterations in chronic idiopathic inflammatory bowel disease–associated intestinal adenocarcinoma remain largely unknown. Somatic IDHmutations are often seen in gliomas and myeloid leukemia but have also been recently reported in a subset of other neoplasms. We analyzed a series of intestinal adenocarcinomas with (n=23) and without (n=39) associated chronic idiopathic inflammatory bowel disease treated at our institution for IDH1and IDH2mutations and correlated the clinicopathologic findings with mutation status. Compared with intestinal adenocarcinomas not associated with inflammatory bowel disease, adenocarcinomas associated with inflammatory bowel disease more frequently demonstrated IDHmutations (13 vs. 0, P=0.047). All IDHmutations were identified in IDH1and resulted in substitution of arginine by cysteine at position 132 (p.R132C, c.394C>T). IDH1mutations were frequently (66) associated with concurrent KRASmutations (p.G12D, c.35G>A). IDH1-mutated intestinal adenocarcinomas were seen in the setting of both Crohn disease and ulcerative colitis and were located in both the ileum and colon. Compared with IDH1-negative inflammatory bowel disease–associated adenocarcinoma, IDH1-positive adenocarcinomas more frequently demonstrated tubuloglandular histology (100 vs. 25, P=0.032) and were more frequently associated with precursor lesions exhibiting serrated morphology (66 vs. 6, P=0.034). IDH1mutations were also identified in the precursor dysplastic lesions associated with IDH1-positive adenocarcinomas. In conclusion, we demonstrate that IDH1mutations are occasionally identified in inflammatory bowel disease–associated intestinal adenocarcinoma but not in intestinal adenocarcinoma not associated with inflammatory bowel disease. In addition, IDH1-mutated intestinal adenocarcinoma is associated with a characteristic low-grade tubuloglandular histology and often harbors concurrent KRASmutations. Identification of patients with IDH1-mutated intestinal adenocarcinoma may become clinically important as new therapies emerge that target tumors that harbor IDHmutations.

ABSTRACTThe pathogenesis of Candida glabratainfections is poorly understood. We studied the pathogenesis of intra-abdominal candidiasis (IAC) in mice that were infected intraperitoneally with C. glabrataand sterile feces. C. glabrataBG2 (5 × 108CFU) caused a 100% mortality rate. Sublethal inocula of BG2 (1 × 108or 1 × 107CFU) caused peritonitis that progressed to abscesses. Three clinical C. glabratastrains (5 × 108CFU) caused 80 to 100% mortality rates, while a fourth (strain 346) caused a 29% mortality rate. Following sublethal inocula (1 × 107CFU), the intra-abscess burdens of virulent strain 356 were ~1 log greater than those of strain 346. A C. glabrata?plb1-2mutant (phospholipase B genes disrupted) killed mice as well as BG2 did. When sublethal inocula were used, however, the ?plb1-2mutant was associated with more rapid abscess resolution and lower intra-abscess burdens; these findings were reversed by PLB1and PLB2reinsertion. The ?plb1-2mutant was also more susceptible than BG2 to killing by human neutrophils in vitro. BG2 and the ?plb1-2mutant were indistinguishable during hematogenously disseminated candidiasis. C. albicansSC5314 was more virulent than C. glabrataBG2 during IAC, causing a 100% mortality rate following a challenge with 5 × 107CFU. In contrast, a sublethal inoculum (1 × 107CFU) of BG2 caused less neutrophil infiltration and greater burdens in peritoneal fluid than SC5314 did and abscesses that persisted longer and contained greater burdens. In conclusion, a mouse model of C. glabrataIAC mimics disease in humans and distinguishes the relative virulence of clinical and gene disruption strains. C. glabratadiffered from C. albicansduring IAC by being less lethal and eliciting dampened neutrophil responses but resulting in more persistent peritonitis and abscesses.

Recently, a monoclonal antibody to cytotoxic T-lymphocyte–associated antigen 4, ipilimumab, was approved for the treatment of metastatic melanoma. One of the most common side effects associated with this therapy is diarrhea and colitis. We report 3 cases of perforating colitis induced by ipilimumab requiring colectomy. The histologic findings of mucosal biopsies have been previously described. Herein, we describe novel associated histologic findings (pseudopolyp formation, fissuring ulcers, dilated crypts, and lack of intraepithelial lymphocytosis and epithelial apoptosis) of segmental resections in patients who required subtotal colectomy after perforation due to the severity of their ipilimumab-induced colitis. Although steroid therapy is the standard treatment for ipilimumab-induced colitis, surgery may be necessary. In the setting of progressive or worsening diarrhea after steroid therapy in patients with colitis, bowel perforation should be considered.

We evaluated a consecutive series of signet ring cell colorectal carcinomas in an attempt to correlate the histopathologic pattern of infiltration with molecular alterations and prognosis. Of the 4760 primary colorectal carcinomas surgically resected between the years 2002 and 2012, 53 (1) were composed of >50 signet ring cells. Of the 53 signet ring cell carcinomas, 40 (75) were composed of >50 extracellular mucin with signet ring cells floating within pools of mucin and were subclassified as mucin-rich signet ring cell carcinomas. Thirteen (25) carcinomas were characterized by diffusely infiltrating carcinomas with minimal to no extracellular mucin and were subclassified as mucin-poor signet ring cell carcinomas. All 13 mucin-poor signet ring cell carcinomas were either stage III or IV, whereas many cases of mucin-rich signet ring cell carcinoma were stage I or II (17 cases) (P=0.005). Compared with mucin-rich tumors, mucin-poor signet ring cell carcinomas more frequently demonstrated adverse histologic features such as lymphatic invasion (1313, 100 vs. 2240, 55; P=0.002), venous invasion (613, 46 vs. 340, 8; P=0.004), and perineural invasion (1113, 85 vs. 940, 23; P=0.0001). Twenty-three of 53 (43) signet ring cell carcinomas demonstrated high levels of microsatellite instability (MSI-H). Twenty-two of 23 (96) MSI-H signet ring cell carcinomas were mucin rich; only 1 MSI-H signet ring carcinoma was mucin poor (P=0.0033). Mucin-poor signet ring cell carcinoma had significantly reduced overall and recurrence-free survival compared with mucin-rich signet ring cell carcinomas (P=0.0035 and 0.0001, respectively), even when adjusting for tumor stage. Mucin-poor signet ring cell carcinoma had a higher propensity for peritoneal dissemination (513, 38) compared with mucin-rich signet ring cell carcinoma (540, 12.5), although this was not statistically significant (P=0.052). Finally, MSI-H and microsatellite-stable signet ring cell carcinomas had similar overall and recurrence-free survival (P=0.2266 and 0.1055, respectively), even when adjusting for tumor stage. In conclusion, we identified a unique subset of signet ring cell colorectal carcinoma with diffuse infiltration and minimal to no extracellular mucin (mucin-poor signet ring cell carcinoma), which lacks MSI-H and has a dismal prognosis with an aggressive clinical course often with peritoneal dissemination. Further, our results confirm that MSI does not affect survival in colorectal signet ring cell carcinomas.

We present depth-resolved spatial-domain low-coherence quantitative phase microscopy, a simple approach that utilizes coherence gating to construct a depth-resolved structural feature vector quantifying sub-resolution axial structural changes at different optical depths within the sample. We show that this feature vector is independent of sample thickness variation, and identifies nanoscale structural changes in clinically prepared samples. We present numerical simulations and experimental validation to demonstrate the feasibility of the approach. We also perform experiments using unstained cells to investigate the nanoscale structural changes in regulated cell proliferation through cell cycle and chromatin decondensation induced by histone acetylation.