Background: The I-SPY 2 TRIAL is a neoadjuvant platform trial open to patients with locally advanced, molecular high-risk breast cancer. We are undertaking a concerted pursuit of mid-therapy response biomarkers, including evaluation of inter-regimen biopsies, to identify patients who may be candidates for treatment de-escalation. In a prior pilot study, we observed that absence of carcinoma in an inter-regimen biopsy was predictive of pathologic complete response (pCR). In this expanded study of 100 I-SPY 2 participants, we sought to confirm that finding and assess other pathologic features of the inter-regimen biopsy as predictors of tumor response to neoadjuvant therapy.

Methods: Digital H&E images of 100 inter-regimen (12 week) image-guided breast biopsies +/- ancillary immunohistochemistry (p63 +/- cytokeratin) were reviewed by 9 pathologists in the I-SPY pathology working group to record 1) tumor bed and 2) presence or absence of residual invasive carcinoma (IC) (with tumor cellularity scored as 0-100%). The data set included 393 cores (mean 3.9 (range 2-4) cores per biopsy). Interobserver percent agreement was calculated and Fisher’s exact t-test was used for association of presence/absence of IC with pCR, and tumoral HR status. Association between biopsy cellularity and RCB indices used Pearson’s correlation.

Results: In the 100 biopsy set, 84% had ≥80% inter-observer diagnostic agreement on both 1) presence of tumor bed (N= 84) and 2) presence or absence of IC (N=53 IC+ /31 IC-) and comprised the data set. There was no difference in the number of evaluable tissue cores between the IC+/IC- biopsies. The primary tumors were 63% HR+/37% HR-; and the presence of IC in the biopsy correlated with tumoral HR status (p=0.0014: 74%: HR+HER2-; 62%: TN; 60%: HR+HER2+; 10%: HR-HER2+). Of the 31 patients with IC-negative biopsies (14 HR+/17 HR-), 25 (80%) went on to pCR (9HR+/16 HR-) whereas only 7/53 (13%) of patients with IC+ biopsies had pCR (2 HR+/5 HR-). Overall, IC-negative biopsies had a calculated OR=26, Fisher p=7.5E-10 for pCR; yielding a positive predictive value (PPV) for pCR of 81% and a sensitivity of 78%; with a PPV for HR- tumors of 94% (15/16) vs. 67% (10/15) for HR+ tumors. In the 6 IC-negative biopsies from patients with non-pCR (“false-negatives”), most were HR+ (5 HR+/1HR-), and tumor bed size in the surgical specimen was smaller than that of IC+ biopsies with non-PCR: 276 mm2 (0.4-1000 mm2) vs. 1166 mm2 (1-11960 mm2). Overall, the 46/53 IC+ biopsies in patients with non-pCR (36 HR+/10 HR-) had a PPV of non-pCR of 86%, with a PPV for HR+ tumors of 94% and a PPV for HR- tumors of 66%. Tumor cellularity in the biopsy (mean 37%, [2.5-93%]) did not correlate with RCB index (p=0.57) or RCB breast-only index (p = 0.17) at surgery.

Conclusion: In this 100 biopsy set, we confirmed that the absence of residual carcinoma in inter-regimen biopsies was highly predictive of pathologic complete response, particularly for HR- tumors. The “false-negative” biopsies (IC-/non-pCR) were predominantly HR+ tumors with small residual tumor beds. Conversely, the presence of carcinoma in inter-regimen biopsies was highly predictive of non-pCR, particularly for HR+ tumors. These data demonstrate the utility, and the limitations, of the inter-regimen biopsy as one tool to identify patients who may benefit from therapeutic de-escalation.

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