Omitting Radiotherapy after Breast-Conserving Surgery in Luminal A Breast Cancer.

BACKGROUND: Adjuvant radiotherapy is prescribed after breast-conserving surgery to reduce the risk of local recurrence. However, radiotherapy is inconvenient, costly, and associated with both short-term and long-term side effects. Clinicopathologic factors alone are of limited use in the identification of women at low risk for local recurrence in whom radiotherapy can be omitted. Molecularly defined intrinsic subtypes of breast cancer can provide additional prognostic information. METHODS: We performed a prospective cohort study involving women who were at least 55 years of age, had undergone breast-conserving surgery for T1N0 (tumor size <2 cm and node negative), grade 1 or 2, luminal A-subtype breast cancer (defined as estrogen receptor positivity of ≥1%, progesterone receptor positivity of >20%, negative human epidermal growth factor receptor 2, and Ki67 index of ≤13.25%), and had received adjuvant endocrine therapy. Patients who met the clinical eligibility criteria were registered, and Ki67 immunohistochemical analysis was performed centrally. Patients with a Ki67 index of 13.25% or less were enrolled and did not receive radiotherapy. The primary outcome was local recurrence in the ipsilateral breast. In consultation with radiation oncologists and patients with breast cancer, we determined that if the upper boundary of the two-sided 90% confidence interval for the cumulative incidence at 5 years was less than 5%, this would represent an acceptable risk of local recurrence at 5 years. RESULTS: Of 740 registered patients, 500 eligible patients were enrolled. At 5 years after enrollment, recurrence was reported in 2.3% of the patients (90% confidence interval [CI], 1.3 to 3.8; 95% CI, 1.2 to 4.1), a result that met the prespecified boundary. Breast cancer occurred in the contralateral breast in 1.9% of the patients (90% CI, 1.1 to 3.2), and recurrence of any type was observed in 2.7% (90% CI, 1.6 to 4.1). CONCLUSIONS: Among women who were at least 55 years of age and had T1N0, grade 1 or 2, luminal A breast cancer that were treated with breast-conserving surgery and endocrine therapy alone, the incidence of local recurrence at 5 years was low with the omission of radiotherapy. (Funded by the Canadian Cancer Society and the Canadian Breast Cancer Foundation; LUMINA ClinicalTrials.gov number, NCT01791829.).

Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer.

Importance: The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear. Objective: To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy. Design, Setting, and Participants: Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy. Exposure: TIL abundance in breast tissue from resected primary tumors. Main Outcomes and Measures: The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center. Results: This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6). Conclusions and Relevance: In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

Ki67 assessment protocol as an integral biomarker for avoiding radiotherapy in the LUMINA breast cancer trial.

AIMS: The LUMINA trial demonstrated a very low local recurrence rate in women ≥55 years with low-risk luminal A breast cancer (defined as grade I-II, T1N0, hormone receptor positive, HER2 negative and Ki67 index ≤13.25%) treated with breast-conserving surgery and endocrine therapy (but no other systemic therapy), supporting the safe omission of radiation in these women. Here we describe the protocol for Ki67 assessment, the companion diagnostic used to guide omission of adjuvant radiotherapy. METHODS: Ki67 immunohistochemistry was performed on full-face sections at one of three regional labs. Pathologists trained in the International Ki67 in Breast Cancer Working Group (IKWG) method demarcated tumour areas on scanned slides and scored 100 nuclei from each of at least five randomly selected 1-mm fields. For cases with high Ki67 heterogeneity, further virtual cores were selected and scored in order to confidently assign a case as luminal A (≤13.25%) or B (>13.25%). Interlaboratory variability was assessed through an annual quality assurance programme during the study period. RESULTS: From the quality assurance programme, the mean Ki67 index across all cases/labs was 13%. The observed intraclass correlation coefficient (ICC) and kappa statistics were ≥0.9 and ≥0.7, respectively, indicating a substantial level of agreement. Median scoring time was 4 min per case. The IKWG-recommended scoring method, performed directly from slides, requiring up to four scored fields, is concordant with the LUMINA scoring method (ICC ≥ 0.9). CONCLUSION: Ki67 is a practical, reproducible, and inexpensive biomarker that can identify low-risk luminal A breast cancers as potential candidates for radiation de-escalation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01791829.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

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.

SRY-box transcription factor 10 is a highly specific biomarker of basal-like breast cancer.

AIMS: Basal-like breast cancer is an aggressive molecular subtype associated with younger age and early relapse. Most cases lack expression of oestrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2, limiting targeted therapeutic options. Basal-like breast cancer is defined by the expression of genes in the outer/basally located epithelial layer of mammary glands, including those encoding cytokeratin (CK) 5 and CK14, and epidermal growth factor receptor (EGFR). SRY-box transcription factor 10 (SOX10), for which there is a readily available immunohistochemical stain, is expressed in a subset of breast cancers, particularly triple-negative carcinomas. In this study, we sought to: (i) assess the association between SOX10 expression and intrinsic molecular subtypes as defined by Prediction Analysis of Microarray 50 (PAM50) gene expression; and (ii) compare the performance of SOX10 with that of other surrogate markers of the basal-like subtype, including CK5, EGFR, nestin, and inositol polyphosphate 4-phosphatase type II (INPP4B). METHODS AND RESULTS: SOX10 immunostaining was performed on tissue microarrays constructed from a contemporary series enriched for ER-negative and weakly ER-positive cancers that had also undergone PAM50 gene profiling. A total of 211 cases were informative for both SOX10 immunohistochemistry and PAM50 subtype, including 103 basal-like cancers. Staining for SOX10 was positive in 73 of 103 basal-like cancers and in only two of 108 cancers of other subtypes (P < 0.001), resulting in a sensitivity of 70.9% and a specificity of 98.1%. SOX10 was more specific than the other tested basal markers, and the results were independent of ER status. CONCLUSIONS: SOX10 is a moderately sensitive, but highly specific, immunohistochemical biomarker for the basal-like intrinsic subtype of breast cancer, which, unlike other commonly used immunohistochemical biomarkers, is independent of hormone receptor status.

A clinical calculator to predict disease outcomes in women with triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by substantial risks of early disease recurrence and mortality. We constructed and validated clinical calculators for predicting recurrence-free survival (RFS) and overall survival (OS) for TNBC. METHODS: Data from 605 women with centrally confirmed TNBC who underwent primary breast cancer surgery at Mayo Clinic during 1985-2012 were used to train risk models. Variables included age, menopausal status, tumor size, nodal status, Nottingham grade, surgery type, adjuvant radiation therapy, adjuvant chemotherapy, Ki67, stromal tumor-infiltrating lymphocytes (sTIL) score, and neutrophil-to-lymphocyte ratio (NLR). Final models were internally validated for calibration and discrimination using ten-fold cross-validation and compared with their base-model counterparts which include only tumor size and nodal status. Independent external validation was performed using data from 478 patients diagnosed with stage II/III invasive TNBC during 1986-1992 in the British Columbia Breast Cancer Outcomes Unit database. RESULTS: Final RFS and OS models were well calibrated and associated with C-indices of 0.72 and 0.73, as compared with 0.64 and 0.62 of the base models (p < 0.001). In external validation, the discriminant ability of the final models was comparable to the base models (C-index: 0.59-0.61). The RFS model demonstrated greater accuracy than the base model both overall and within patient subgroups, but the advantages of the OS model were less profound. CONCLUSIONS: This TNBC clinical calculator can be used to predict patient outcomes and may aid physician's communication with TNBC patients regarding their long-term disease outlook and planning treatment strategies.

The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Detection of CSF1 rearrangements deleting the 3' UTR in tenosynovial giant cell tumors.

Tenosynovial giant cell tumors (TGCTs) are characterized by rearrangements of CSF1, thought to drive overexpression of macrophage colony-stimulating factor (CSF1), thereby promoting tumor growth and recruitment of non-neoplastic mononuclear and multinucleated inflammatory cells. While fusions to collagen promoters have been described, the mechanism of CSF1 overexpression has been unclear in a majority of cases. Two cohorts of TGCT were investigated for CSF1 rearrangements using fluorescence in situ hybridization (FISH) and either RNA-seq or DNA-seq with Sanger validation. The study comprised 39 patients, including 13 localized TGCT, 21 diffuse TGCT, and five of unspecified type. CSF1 rearrangements were identified by FISH in 30 cases: 13 translocations, 17 3' deletions. Sequencing confirmed CSF1 breakpoints in 28 cases; in all 28 the breakpoint was found to be downstream of exon 5, replacing or deleting a long 3' UTR containing known miRNA and AU-rich element negative regulatory sequences. We also confirmed the presence of CBL exon 8-9 mutations in six of 21 cases. In conclusion, TGCT in our large cohort were characterized by variable alterations, all of which led to truncation of the 3' end of CSF1, instead of the COL6A3-CSF1 fusions previously reported in some TGCTs. The diversity of fusion partners but consistent integrity of CSF1 functional domains encoded by exons 1-5 support a hypothesis that CSF1 overexpression results from transcription of a truncated form of CSF1 lacking 3' negative regulatory sequences. The presence of CBL mutations affecting the linker and RING finger domain suggests an alternative mechanism for increased CSF1/CSF1R signaling in some cases.

Correction to: Mismatch repair protein loss in breast cancer: clinicopathological associations in a large British Columbia cohort.

In the original publication of the article, the funding statement was published incompletely. The corrected funding statement should read as below.

Mismatch repair protein loss in breast cancer: clinicopathological associations in a large British Columbia cohort.

PURPOSE: Alterations to mismatch repair (MMR) pathways are a known cause of cancer, particularly colorectal and endometrial carcinomas. Recently, checkpoint inhibitors have been approved for use in MMR-deficient cancers of any type (Prasad et al. in JAMA Oncol 4:157-158, 2018). Functional studies in breast cancer have shown associations between MMR loss, resistance to aromatase inhibitors and sensitivity to palbociclib (Haricharan et al. in Cancer Discov 7:1168-1183, 2017). Herein, we investigate the clinical meaning of MMR deficiency in breast cancer by immunohistochemical assessment of MSH2, MSH6, MLH1 and PMS2 on a large series of breast cancers linked to detailed biomarker and long-term outcome data. METHODS: Cases were classified as MMR intact when all four markers expressed nuclear reactivity, but MMR-deficient when at least one of the four biomarkers displayed loss of nuclear staining in the presence of positive internal stromal controls on the tissue microarray core. RESULTS: Among the 1635 cases with interpretable staining, we identified 31 (1.9%) as MMR-deficient. In our cohort, MMR deficiency was present across all major breast cancer subtypes, and was associated with high-grade, low-progesterone receptor expression and high tumor-infiltrating lymphocyte counts. MMR deficiency is significantly associated with inferior overall (HR 2.29, 95% CI 1.02-5.17, p = 0.040) and disease-specific survival (HR 2.71, 95% CI 1.00-7.35, p = 0.042) in the 431 estrogen receptor-positive patients who were uniformly treated with tamoxifen as their sole adjuvant systemic therapy. CONCLUSION: Overall, this study supports the concept that breast cancer patients with MMR deficiency as assessed by immunohistochemistry may be good candidates for alternative treatment approaches such as immune checkpoint or CDK4 inhibitors.

Differential expression and prognostic relevance of autophagy-related markers ATG4B, GABARAP, and LC3B in breast cancer.

PURPOSE: Previous studies indicate that breast cancer molecular subtypes differ with respect to their dependency on autophagy, but our knowledge of the differential expression and prognostic significance of autophagy-related biomarkers in breast cancer is limited. METHODS: Immunohistochemistry (IHC) was performed on tissue microarrays from a large population of 3992 breast cancer patients divided into training and validation cohorts. Consensus staining scores were used to evaluate the expression levels of autophagy proteins LC3B, ATG4B, and GABARAP and determine the associations with clinicopathological variables and molecular biomarkers. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models. RESULTS: We found subtype-specific expression differences for ATG4B, with its expression lowest in basal-like breast cancer and highest in Luminal A, but there were no significant associations with patient prognosis. LC3B and GABARAP levels were highest in basal-like breast cancers, and high levels were associated with worse outcomes across all subtypes (DSS; GABARAP: HR 1.43, LC3B puncta: HR 1.43). High ATG4B levels were associated with ER, PR, and BCL2 positivity, while high LC3B and GABARAP levels were associated with ER, PR, and BCL2 negativity, as well as EGFR, HER2, HER3, CA-IX, PD-L1 positivity, and high Ki67 index (p < 0.05 for all associations). Exploratory multi-marker analysis indicated that the combination of ATG4B and GABARAP with LC3B could be useful for further stratifying patient outcomes. CONCLUSIONS: ATG4B levels varied across breast cancer subtypes but did not show prognostic significance. High LC3B expression and high GABARAP expression were both associated with poor prognosis and with clinicopathological characteristics of aggressive disease phenotypes in all breast cancer subtypes.

Evaluation of glucocorticoid-induced TNF receptor (GITR) expression in breast cancer and across multiple tumor types.

Glucocorticoid-induced TNF receptor (GITR) is an emerging immunotherapy target that is expressed at high levels on regulatory T cells. Agonistic anti-GITR antibodies have anti-tumor activity in cancer mouse models, and recent phase 1 trials have demonstrated their safe pharmacological profile. However, there is limited knowledge on the relationship between GITR expression and the tumor microenvironment. GITR protein expression was assayed by immunohistochemistry on 3992 breast cancer surgical excision specimens assembled into tissue microarrays and scored visually by a pathologist for GITR expression on tumor-infiltrating lymphocytes and on carcinoma cells. GITR expression by the malignant cells was further surveyed in gastrointestinal stromal tumor (N = 713), lung carcinoma (N = 705), pancreatic cancer (N = 486), ovarian cancer (N = 445), bladder cancer (N = 88), prostate cancer (N = 88), testicular cancer (N = 76), melanoma (N = 75), renal cell carcinoma (N = 68),  epithelioid sarcoma (N = 53), and neuroendocrine tumors (N = 41). In breast cancer, GITR expression on tumor-infiltrating lymphocytes (12.4%) correlated with other immune response biomarkers (PD-L1+ on tumor cells, and PD-1+, LAG-3+, TIM-3+ lymphocytes; p < 0.001), and T-cell markers (CD8+, FOXP3+; p < 0.001). GITR+ carcinoma cells were observed in 6.0% of breast cancer cases and correlated with worse relapse-free survival (p = 0.015). Among the additional tumor types examined, cancers with GITR+ malignant cells included bladder cancer (5.7%), primary (but not metastatic) melanoma (4.5%), and ovarian cancer (3.2%); no expression was identified among examined sarcomas. To our knowledge, this is the first immunohistochemistry study to report the frequency and pattern of GITR expression in a large breast cancer cohort, or to report membranous GITR expression on malignant cells. The co-infiltration of GITR with other immune biomarkers and T-cell markers supports a potential role for anti-GITR agents in combination immunotherapies. In addition, GITR expression on carcinoma cells could imply the existence of a novel cancer immune evasion strategy worthy of further investigation.

Emerging targets in cancer immunotherapy.

The first generation of immune checkpoint inhibitors (anti-CTLA-4 and anti-PD-1/PD-L1) targeted natural immune homeostasis pathways, co-opted by cancers, to drive anti-tumor immune responses. These agents led to unprecedented results in patients with previously incurable metastatic disease and may become first-line therapies for some advanced cancers. However, these agents are efficacious in only a minority of patients. Newer strategies are becoming available that target additional immunomodulatory mechanisms to activate patients' own anti-tumor immune responses. Herein, we present a succinct summary of emerging immune targets with reported pre-clinical efficacy that have progressed to active investigation in clinical trials. These emerging targets include co-inhibitory and co-stimulatory markers of the innate and adaptive immune system. In this review, we discuss: 1) T lymphocyte markers: Lymphocyte Activation Gene 3 [LAG-3], T-cell Immunoglobulin- and Mucin-domain-containing molecule 3 [TIM-3], V-domain containing Ig Suppressor of T cell Activation [VISTA], T cell ImmunoGlobulin and ITIM domain [TIGIT], B7-H3, Inducible T-cell Co-stimulator [ICOS/ICOS-L], CD27/CD70, and Glucocorticoid-Induced TNF Receptor [GITR]; 2) macrophage markers: CD47/Signal-Regulatory Protein alpha [SIRPα] and Indoleamine-2,3-Dioxygenase [IDO]; and 3) natural killer cell markers: CD94/NKG2A and the Killer Immunoglobulin-like receptor [KIR] family. Finally, we briefly highlight combination strategies and potential biomarkers of response and resistance to these cancer immunotherapies.

Ki67 reproducibility using digital image analysis: an inter-platform and inter-operator study.

Ki67 expression has been a valuable prognostic variable in breast cancer, but has not seen broad adoption due to lack of standardization between institutions. Automation could represent a solution. Here we investigate the reproducibility of Ki67 measurement between three image analysis platforms with supervised classifiers performed by the same operator, by multiple operators, and finally we compare their accuracy in prognostic potential. Two breast cancer patient cohorts were used for this study. The standardization was done with the 30 cases of ER+ breast cancer that were used in phase 3 of International Ki67 in Breast Cancer Working Group initiatives where blocks were centrally cut and stained for Ki67. The outcome cohort was from 149 breast cancer cases from the Yale Pathology archives. A tissue microarray was built from representative tissue blocks with median follow-up of 120 months. The Mib-1 antibody (Dako) was used to detect Ki67 (dilution 1:100). HALO (IndicaLab), QuantCenter (3DHistech), and QuPath (open source software) digital image analysis (DIA) platforms were used to evaluate Ki67 expression. Intraclass correlation coefficient (ICC) was used to measure reproducibility. Between-DIA platform reproducibility was excellent (ICC: 0.933, CI: 0.879-0.966). Excellent reproducibility was found between all DIA platforms and the reference standard Ki67 values of Spectrum Webscope (QuPath-Spectrum Webscope ICC: 0.970, CI: 0.936-0.986; HALO-Spectrum Webscope ICC: 0.968, CI: 0.933-0.985; QuantCenter-Spectrum Webscope ICC: 0.964, CI: 0.919-0.983). All platforms showed excellent intra-DIA reproducibility (QuPath ICC: 0.992, CI: 0.986-0.996; HALO ICC: 0.972, CI: 0.924-0.988; QuantCenter ICC: 0.978, CI: 0.932-0.991). Comparing each DIA against outcome, the hazard ratios were similar. The inter-operator reproducibility was particularly high (ICC: 0.962-0.995). Our results showed outstanding reproducibility both within and between-DIA platforms, including one freely available DIA platform (QuPath). We also found the platforms essentially indistinguishable with respect to prediction of breast cancer patient outcome. Results justify multi-institutional DIA studies to assess clinical utility.

Basal biomarkers nestin and INPP4B predict gemcitabine benefit in metastatic breast cancer: Samples from the phase III SBG0102 clinical trial.

In a formal prospective-retrospective analysis of the phase III SBG0102 clinical trial randomizing metastatic breast cancer patients to gemcitabine-docetaxel or to single agent docetaxel, patients with basal-like tumors by PAM50 gene expression had significantly better overall survival in the gemcitabine arm. By immunohistochemistry (IHC), triple negative status was not predictive, but more specific biomarkers have since become available defining basal-like by nestin positivity or loss of inositol-polyphosphate-4-phosphate (INPP4B). Here, we evaluate their capacity to identify which patients benefit from gemcitabine in the metastatic setting. Nestin and INPP4B staining and interpretation followed published methods. A prespecified statistical plan evaluated the primary hypothesis that patients with basal-like breast cancer, defined as "nestin+ or INPP4B-", would have superior overall survival on gemcitabine-docetaxel when compared to docetaxel. Interaction tests, Kaplan-Meier curves and forest plots were used to assess prognostic and predictive capacities of biomarkers relative to treatment. Among 239 cases evaluable for our study, 36 (15%) had been classified as basal-like by PAM50. "Nestin+ or INPP4B-" was observed in 41 (17%) of the total cases and was significantly associated with PAM50 basal-like subtype. Within an estimated median follow-up of 13 years, patients assigned as IHC basal "nestin+ or INPP4B-" had significantly better overall survival on gemcitabine-docetaxel versus docetaxel monotherapy (HR = 0.31, 95%CI: 0.16-0.60), whereas no differences were observed for other patients (HR = 0.99), p-interaction < 0.01. In the metastatic setting, women with IHC basal breast cancers defined as "nestin+ or INPP4B-" have superior overall survival when randomized to gemcitabine-containing chemotherapy compared to docetaxel alone. These findings need to be validated using larger prospective-retrospective phase III clinical trials series.

Expression of lymphocyte immunoregulatory biomarkers in bone and soft-tissue sarcomas.

Despite advances in our understanding of the underlying molecular drivers of sarcomas, few treatments are available with proven benefit for advanced metastatic sarcomas. Immunotherapy has value in this setting for some types of cancers, but sarcomas, with their multiplicity of rare types, have not been characterized in detail for their expression of targetable immune biomarkers. This study provides the most systematic evaluation to date of tumor-infiltrating lymphocytes and immune checkpoint biomarker expression in sarcomas. We examined by morphology and immunohistochemistry 1072 sarcoma specimens representing 22 types, in addition to 236 benign bone and soft-tissue tumors. Genomically-complex sarcoma types-those driven by mutations and/or copy-number alterations-had much higher numbers of tumor-infiltrating lymphocytes than translocation-associated sarcomas. Prior exposure to radiotherapy was associated with increased immune infiltrates. Higher lymphocytic infiltration was associated with better overall survival among the non-translocation-associated sarcomas. Expression of PD-1 and CD56 were associated with worse overall survival. LAG-3 and TIM-3, two emerging immune checkpoints, were frequently expressed in most sarcoma types. Indeed, most cases positive for PD-(L)1 coexpressed one or both of these novel biomarkers, providing a potential rationale in support for trials targeting LAG-3 and/or TIM-3 in conjunction with PD-1 inhibition.

An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer.

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

AIMS: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. METHODS AND RESULTS: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799-0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74-0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. CONCLUSIONS: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Indoleamine Dioxygenase Inhibitors: Clinical Rationale and Current Development.

PURPOSE OF REVIEW: This review focuses on the recent clinical development of indolamine-2,3-dioxygenase-1 (IDO-1) inhibitors. RECENT FINDINGS: IDO-1 alters tryptophan metabolism in a manner enhancing T-regulatory cell activity, but pre-clinical data show that its role in tumorigenesis is context-dependent on host and tumor interaction, highlighting some challenges in understanding the molecular oncology of this enzymatic drug target. Because results from phase I/II trials of IDO-1 inhibitor monotherapy have been disappointing, current clinical trials employ IDO-1 inhibitors in combination strategies with other immunotherapy agents or with chemotherapy ± radiation. Combinations with anti-PD-1/PD-L1 antibodies are already showing promise, and related strategies are under active evaluation. While further research is needed to elucidate the precise role of IDO-1 in tumor development, its mechanisms of action appear sufficiently distinct from other immunotherapy targets to warrant inclusion in combination immunotherapy regimens, an approach where multiple clinical trials are currently underway.

The Prosigna gene expression assay and responsiveness to adjuvant cyclophosphamide-based chemotherapy in premenopausal high-risk patients with breast cancer.

BACKGROUND: The PAM50-based (Prosigna) risk of recurrence (ROR) score and intrinsic subtypes are prognostic for women with high-risk breast cancer. We investigate the predictive ability of Prosigna regarding the effectiveness of cyclophosphamide-based adjuvant chemotherapy in premenopausal patients with high-risk breast cancer. METHODS: Prosigna assays were performed on the NanoString platform in tumors from participants in Danish Breast Cancer Group (DBCG) 77B, a four-arm trial that randomized premenopausal women with high-risk early breast cancer to no systemic treatment, levamisole, oral cyclophosphamide (C) or cyclophosphamide, methotrexate and fluorouracil (CMF). RESULTS: In total, this retrospective analysis included 460 women (40% of the 1146 randomized patients). The continuous Prosigna ROR score was prognostic in the no systemic treatment group (unadjusted P < 0.001 for disease-free survival (DFS), P = 0.001 for overall survival (OS)). No statistically significant interaction of continuous ROR score and treatment on DFS and OS was found. A highly significant association was observed between intrinsic subtypes and C/CMF treatment for DFS (Pinteraction = 0.003 unadjusted, P = 0.001 adjusted) and OS (Pinteraction = 0.04). In the adjusted analysis treatment with C/CMF was associated with a reduced risk of DFS events in patients with basal-like (hazard ratio (HR) 0.14; 95% CI 0.06; 0.32) and luminal B (HR 0.48; 95% CI 0.27; 0.84) subtypes but not in patients with Human epidermal growth factor receptor-enriched (HR 1.05; 95% CI 0.56; 1.95) or luminal A (HR 0.61; 95% CI 0.32; 1.16) subtypes. CONCLUSION: The Prosigna ROR score and intrinsic subtypes were prognostic in high-risk premenopausal patients with breast cancer, and intrinsic subtypes identify high-risk patients with or without major benefit from adjuvant C/CMF treatment.