Most sprites that fall off the bottom of the screen do get unloaded, but the game intentionally (as far as I can tell) clears the flag that prevents them from being loaded again. As someone who has written a few off-screen handlers I think it would be pretty easy to make them stay gone. However, I am not familiar with any hacks that change it, which I think is a matter of nobody having wanted to. Most of the people I can imagine thinking about this behavior would prefer to go really deep with weird aspects of SMW's design like this rather than change them.
Regarding 100 Rooms of Enemies: If you see a setup based on "kill all the enemies", usually that works by checking whether any enemy sprites are loaded. If you have a single-screen room and an enemy falls in a hole, it'll get unloaded and there won't be any way to load it again. So falling in a hole will usually be enough for whatever code is checking on your progress. But if the room were multiple screens wide, you'd be able to walk away, walk back, and the scrolling would make the sprite load itself again. And if you walked to a screen with no enemies, no enemies would be loaded, and that would count as "killing" everything too. In this case the loophole is closed by single-screen level design rather than by changing the fundamental mechanics. [Edit: I haven't played 100ROE and upon rereading this thread I'm realizing this might not fit what Serena described so maybe they are using a different kill detector than usual. I was going by what I've seen in kill rooms in other hacks. The hubris!]
There are a bunch of places in the above paragraphs where I could put in some weird details or edge cases. I don't think they are super pertinent but could give a fuller account if desired.
My biggest pet peeve in 2D Mario games is that when an enemy ends up in a bottomless pit, it simply despawns instead of dying. Are there any hacks where they actually die when falling into pits? Bonus points if Mario has a way to push them towards pits aside from just platform manipulation and stomping on them.