Table 3.

Breast cancer studies; Characteristics, Prognosis/Prediction, Heterogeneity.

Author	Journal	Year	Tissue Type	Disease Setting	Treatment	BC Type	Pts No	Assay	Panel	Scanning	Software	ROIs Number, Area (per Patient)	Tissue Segmentation	Prognosis/ Prediction	Spatial Heterogeneity
Fluorescent Multiplex Immunohistochemistry
Angelis [62]	CCR	2019	WTS	Neo-adjuvant	Lapatinib trastuzumab	HER2+ BC	29	mIF	CD4, CD8, CD20, FoxP3, CD68, CK, DAPI	Vectra	InForm	5, 2 mm${}^2$	(CK+/DAPI+) (CK-/DAPI+)	Higher Stromal CD4, Tumoral CD4,CD20: Higher pCR	CD8 enriched In tumoral, CD4, CD8, CD20 Positive correlation
Brown [80]	CCR	2014	WTS	Neo-adjuvant	Taxane, anthracycline trastuzumab	all	87	mIF	CD3, CD8, CD20, CK, DAPI	PM-2000 hardware (HistoRx)	AQUA	At least 3 CK enriched	(CD3+/DAPI+) (CD3-/DAPI-)	Higher stromal CD4, CD8, CD20: Higher pCR	TNBC Higher CD20, CD8 densities
Graeser [61]	JIC	2020	WTS	Neo-adjuvant	Paclitaxel, gemcitabine, carboplatin	TNBC	66	mIF	CD4, CD8, CD73, PD1, PD-L1, CK7, DAPI	Vectra/Polaris	InForm	Whole slide	(CK7+/DAPI+) (CK7-/DAPI+)	On treatment CD4+PD1+: Higher pCR	NA
Griguolo [60]	Npj precision oncology	2021	WTS	Neo-adjuvant	Lapatinib, trastuzumab	HER2+ BC	65	MCISSS	CD3, CD4, CD8 Foxp3, Ki67, panCK, hematoxylin	NanoZoomer 2.0HT (Hamamatsu Photonics, Japan)	VISIOPHARM	Whole slide	(CK+/HTX+) (CK-/HTX-)	On treatment TumoralCD8+ higher pCR	Ki67+CD3+ Higher densities closer to cancer cells, enriched in HR-
Kearney [63]	SABCS	2021	WTS	Neo-adjuvant	Anthracycline Trastuzumab pertuzumab	HR+/ HER2+ BC	28	mIF	CD3, CD8, CD68, FOXP3, Pan-CK, DAPI	NA	HALO	NA	CK+ CK-	Higher CD3+CD8-FoxP3- higher pCR	NA
Yam [71]	CCR	2021	WTS	Neo-adjuvant	doxorubicin, cyclo-phosphamide, paclitaxel	TNBC	102	mIF	PDL1, PD1, CD3, CD68, Pan-CK, DAPI	Vectra 3.0	InForm	NA	CK+ CK-	Higher CD3+/CD68+ ratio, CD3-cancer proximity higher pCR	PD-L1 expression: tumor (more common) and stromal compartments, CD3+ PD1+ rare population
Janiszewska [83]	JCIinsight	2021	WTS	Neo-adjuvant	Trastuzumab emtansine, pertuzumab	HER2+ BC	20	mIF	20	NA	NA	NA	CK+ CK-	pCR: higher CD8, RD:GZM+ mf closer to cancer, immune cells less proximal to vimentin+ cells	NA
Egelston [67]	JCIinsight	2019	WTS	Adjuvant	doxorubicin, cyclophospamide hamide, paclitaxel	TNBC	25	mIF	CD8, CD103, CD69, pan-CK, DAPI	Vectra 3.0	InForm	multiple	(CK+/DAPI+) (CK-/DAPI+)	Higher Tumoral CD8+CD103+: Better RFS	CD8+CD103+ Enriched closer to cancer cells
Millar [84]	Cancers	2020	TMA	Adjuvant	CMF, anthracycline	all	485	mIF	CD3, CD8, CD20, CD68, Fox P3, PD-1, PD-L1, PanCK, DAPI	Vectra/Polaris	InForm	1, 780 μm${}^2$	(CK+/DAPI+) (CK-/DAPI+)	Combined Stromal lower CD8, CD20: Shorter OS	NA
Garaud [79]	JCIinsight	2019	WTS	Adjuvant	Chemotherapy	HER2+ BC, TNBC	249	mIF	CD4, CD8, CD20, FoxP3, CD68, CK, DAPI	Vectra/Polaris	InForm	NA	NA	Higher CD20: Favorable prognosis	Description of Lymphocytic aggregates
Costa [81]	Cancer Cell	2018	NA	NA	NA	TNBC	NA	mIF	CD25, FAP, PDL2, OX40L, DAPI	HistoFluor microscope	manual	NA	NA	NA	FAP+PDL2+ OX40L+: T cell exhaustion
Wortman [73]	Npj Breast Cancer	2021	WTS	Adjuvant	Chemotherapy	TNBC	36	mIF	CD3, CD4, CD8, FOXP3, CD20, DAPI, PanCK	Vectra 3.0	InForm	NA	NA	Higher tumoral Distribution CD3,CD20: Better RFS	CD3, CD20: Description of Aggregates
Mani [72]	Breast cancer research	2016	WTS	NA	Surgery	NA	31	mIF	CK, DAPI, CD3, CD8, CD20	NA	AQUA	6–50	NA	NA	Approximately 70% of spatial Heterogeneity of B cells: Within same tissue section
O’Meara [85]	SABCS	2021	NA	NA	NA	all	132	mIF	CD8, FoxP3, PD1, PDL1, CK	Vectra	NA	NA	NA	NA	HR+ Immune mark.: Correlates with Grade, Recurrence score
Shimada [86]	SABCS	2021	NA	NA	NA	HR+ BC	5	mIF	9-21 proteins	CyteFinder microscope	MCMICRO	NA	Tumor Stroma Immune cells	NA	Description Of cold, hot Excluded TIME
Noel [66]	JCI	2021	WTS	Adjuvant	Chemotherapy, HER2 targeted	HER2+ BC, TNBC	48	mIF	CD4, CD20, PD-1, ICOS, Ki-67, Foxp3	Vectra/Polaris	NA	NA	NA	Active TLS: Favorable prognosis	Active TLS: Combined CD4+PD1+, CD20+ki67+, GZMB+CD8+
Bedard [68]	SABCS	2021	NA	Metastatic	INT230-6 (cisplatin and vinblastine) pembrolizumab	TNBC	3	mIF	CD4, CD8, FoxP3	NA	NA	NA	NA	NA	Metastasis Lower CD4,CD8 Higher FoxP3
Zhu [87]	J.Immun. Cancer	2019	WTS	Metastatic	NA	all	5	mIF	CD8, FoxP3, CD68, CD20, CK, DAPI	NA	NA	NA	NA	NA	Metastasis Higher CD68
He [69]	Plos One	2020	WTS	Metastatic	Chemotherapy	TNBC	10	mIF	CD4, CD8, FOXP3, CD20, CD33, PD1, CK, DAPI	Vectra	InForm	NA	CK+ CK-	Higher PD1+CD8+ PD1+CD4+ Better outcome	Metastasis Lower Str. CD8+, PD1+CD8+
Ultivue InSituPlex
Ahmed [70]	CCR	2020	WTS	Neo-adjuvant	nab-paclitaxel, doxorubicin, cyclophosphamide, durvalumab	TNBC	45	InSitu Plex	CD8,CD68,PDL1, CK, Sox10, Hoechst	PM2000 microscope (HistoRx)	AQUA	NA	CK+, CK- CD68-, CK- CD68+	pCR vs RD: higher baseline PDL1 expression and similar CD68 densities	PD-L1 expression: on both stromal and tumoral compartments
GeoMx Digital Spatial Profiling
Carter [75]	SABCS	2020	WTS	Neo-adjuvant	taxane	HR+/ HER2- BC	39	DSP	58 proteins	GeoMx	NA	6, 600 $\mathsf{\mu }$m${}^2$	(CK+/SYTO13+) (CK-/SYTO13+)	After NAT: LAG3 expression: Residual disease	Most immune Markers: Stromal enrichment
McNamara [77]	Nature Cancer	2018	WTS	Neo-adjuvant	Trastuzumab lapatinib	HER2+ BC	28	DSP	40 proteins	GeoMx	NA	4, 464–666 $\mathsf{\mu }$m${}^2$	CK+ CK-	On treatment Expression CD45, CD56 Higher pCR	Compartment enrichment Tumoral CD56, B7H4 PDL1, IDO Stromal CD3, CD4, CD8, CD68
Carter [76]	SABCS	2020	TMA	adjuvant	Chemotherapy	TNBC	167	DSP	58 proteins	GeoMx	NA	1, 600 $\mathsf{\mu }$m${}^2$	(CK+/SYTO13+) (CK-/SYTO13+)	Stromal LAG3 Higher PFS	Compartment Enrichment Tumoral CD27, HLADR, IDO1 PDL1 Stromal PDL1
Stewart [74]	Scientific reports	2020	WTS	Adjuvant	Chemotherapy	TNBC	10	DSP	39 proteins	GeoMx	NA	6, 300 $\mathsf{\mu }$m${}^2$	CK+ CK-	No relapse: Tumoral HLA-DR, IDO1, B2M Stromal CD45, CD4, PD-1	Most immune Markers Stromal
Kulasinghe [88]	Frontiers In Oncology	2022	TMA	Adjuvant	Chemotherapy	TNBC	24	DSP	68 proteins	GeoMx	NA	1, NA	CK+ CK-	Responders Tumoral higher GZMA, STING, Fibronectin Lower CD80 Stromal Lower 4-1BB	NA
Carter [89]	CCR	2021	TMA	NA	Chemotherapy untreated	TNBC	184	DSP	58 proteins	GeoMx	GeoMx software	1, 600 $\mathsf{\mu }$m${}^2$	CK+, CK-/SYTO13+	NA	PD-L1+: immunologically hotter (mf, T cells, checkpoints (IDO-1), antigen presentation, STING)
Leon-Ferre [90]	SABCS	2021	TMA	NA	Surgery	TNBC	111	DSP	58 proteins	GeoMx	NA	1, NA	CK+ CK-	NA	LAR vs non-LAR TNBC Lower Stromal CD45, CD14, IDO1 Tumoral CD45, B7H3
Schlam [78]	JTM	2021	WTS	Metastatic	Chemotherapy HER2 targeted	HER2+ BC	8	DSP	70 proteins	GeoMx	NA	2	CK+ CK-	NA	Metastasis Lower CD3, CD8 Tim-3, CD27, 4-1BB
Mass Spectrometry Immunohistochemistry
Bianchini [91]	SABCS	2021	WTS	Neo-adjuvant	atezolizumab, carboplatin, nab-paclitaxel	TNBC	243	IMC	43 proteins	TOF	NA	NA	NA	Higher pCR: density APC PDL1+IDO+, spatial correlation of CK+ and CD8+PD1+, CD8+GZMB+, CD20+	NA
Keren [28]	Cell	2018	WTS	NA	Surgery	TNBC	41	MIBI- TOF	36 proteins	TOF	NA	1, 800 μm${}^2$	NA	Compartmentalized pattern longer OS	Hot tumors Were divided Into “Mixed”, “Compartmentalized”

Abbreviations: APC: antigen presenting cell, BC: breast cancer, RD: residual disease, CK: cytokeratin, DSP: digital spatial profiling, HR: hormone receptor, IMC: imaging mass cytometry, MIBI-TOF: multiplex ion beam imaging with time of flight, mIF: multiplex immunofluorescence, MCISSS: Multiplexed Immunohistochemical Consecutive Staining on Single Slide, OS: overall survival, pCR: pathologic complete response, PFS: progression-free survival, RFS: relapse-free survival, TLS: tertiary lymphoid structure, TMA: tissue microarray, TNBC: triple negative breast cancer, TIME: Tumor immune microenvironment, WTS: whole tissue section.